<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>1</sup>Division of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Japan<sup> </sup></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>1</sup>Division of Risk Assessment, Center for Biological Safety and Research, National Institute of Health Sciences, Kawasaki, Japan</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>2</sup>KIST Europe Forschungsgesellschaft mbH, Saarbrücken, Germany</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>3</sup>University of Wisconsin-Madison at U.S. Environmental Protection Agency, Duluth, Minnesota, United States</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>5</sup>European Commission, Joint Research Centre (JRC), Ispra, Italy</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>6</sup>Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>8</sup>Oak Ridge National Laboratory, Oak Ridge, United States</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>9</sup>Brunel University London, London, United Kingdom</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>10</sup>King’s College London, London, United Kingdom</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>11</sup>Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>12</sup>U.S. Army Engineer Research and Development Center (ERDC), Vicksburg, United States</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>13</sup>Independent Scientist & Consultant, North Macedonia </span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>13</sup>GenOmics, Bioinformatics, and Translational Research Center, RTI International, Washington DC, United States</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>14</sup>GenOmics, Bioinformatics, and Translational Research Center, RTI International, Washington DC, United States</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>14</sup>Safer Medicines Trust, Kingsbridge, United Kingdom</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>15</sup>Safer Medicines Trust, Kingsbridge, United Kingdom</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>17</sup>Finnish Safety and Chemicals Agency, Finland</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>18</sup>Finnish Safety and Chemicals Agency, Finland</span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:10.5pt"><span style="color:#000000"><sup>18</sup>Institute of Clinical and Experimental Research (IREC), UCLouvain, Brussels, Belgium</span></span></span></p>
<td>Under development: Not open for comment. Do not cite</td>
<td>Under Development</td>
<td>1.96</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">Coronavirus disease-19 (COVID-19) is circulating all over the world. To understand and find a way of the COVID-19 treatment, the signaling pathway and therapeutic mechanism of COVID-19 should be investigated. The pathogenesis of COVID-19 includes molecular networks such as the binding of the membrane proteins, signaling pathways, and RNA replication. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a new type of coronavirus causing COVID-19, infects the cells via the binding of the membrane proteins of human cells and is internalized by the cells. The viral genome is replicated by RNA-dependent RNA polymerase (RdRp), followed by the packaging and releasing of the viral particles. These steps can be the main targets for the therapeutics of COVID-19. The AOP379 "Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation" consists of the molecular initiating event (MIE) as “Binding to ACE2” (KE1739), key events (KEs) as “SARS-CoV-2 cell entry” (KE1738), “Interferon-I antiviral response, antagonized by SARS-CoV-2” (KE1901), "Increased SARS-CoV-2 production" (KE1847), “Diminished protective oxidative stress response" (KE1869) and "Coagulation" (KE1845), and adverse outcome (AO) as "Thrombosis and Disseminated Intravascular Coagulation" (KE1846).</span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">1. Support for Biological Plausibility of KERs</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Biological Plausibility of the MIE => KE1 is high.<br />
Rationale: Binding of SARS-CoV-2 to ACE2 cell-surface receptor initiates infection of SARS-CoV-2 where SARS-CoV-2 cell entry is essential (Zhou P et al., 2020, Benton DJ et al., 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Biological Plausibility of the KE1 => KE2 is moderate.<br />
Rationale: The expression of type I interferons (IFN-I) is antagonized by SARS-CoV-2 (Benton DJ et al., 2020). Individual viral proteins interact with and block host proteins in the IFN-I pathway or IFN-I stimulated genes.</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Biological Plausibility of the KE3 => KE4 is moderate.<br />
Rationale: The fixation of SARS-CoV-2 in ACE2 receptor results in excessive production of pro-inflammatory and pro-oxidant agents (Ramdani and Bachari, 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Biological Plausibility of the KE5 => AO is high.<br />
Rationale: Extreme aggravation of blood coagulation induces multiple thrombi in the microvasculature, which leads to consumption coagulopathy followed by disseminated intravascular disease.</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">2. Support for Essentiality of KEs</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Rationale for Essentiality of KEs in the AOP is Moderate.</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the MIE => KE1 is moderate.<br />
Rationale: The SARS-CoV-2 binding to ACE2 induce fusion of the virus and cell membranes to release the virus genome into the cell. (Zhou P et al., 2020, Benton DJ et al., 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE1 => KE2 is moderate.<br />
Rationale: SARS-CoV-2-infection induces IFN-I pathway reduction (Sui C et al., 2022, Blanco-Melo, 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE2 => KE3 is moderate.<br />
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE3 => KE4 is moderate.<br />
Rationale: As SARS-CoV-2 is attached to ACE2, ACE2 is not available within the renin-angiotensin system to convert Ang II to angiotensin-(1,7) resulting in Ang II to accumulate. Ang II stimulates membrane-bound NADPH oxidase, which in turn generate ROS and oxidative stress (Janardhan et al., 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE4 => KE5 is moderate.<br />
Rationale: The presence of ROS assists in the transformation of a circulating, non-oxidized, circular-shaped beta2-glycoprotein 1 into an oxidized J-shape, which binds to antiphospholipid antibodies such as anticardiolipin, lupus anticoagulant, and anti-beta2-GP1 antibodies (Janardhan et al., 2020). Domain V of beta2gP1 binds with the phospholipid layer of platelets or endothelial cells via Annexin (Janardhan et al., 2020).</span></span></span></span></span></p>
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE5 => KE4 is moderate.<br />
<p style="text-align:left"><span style="font-size:10.5pt"><span style="font-family:游明朝,serif"><span style="font-size:12pt"><span style="font-family:"Yu Gothic",sans-serif"><span style="color:black">Empirical Support of the KE5 => AO is high.<br />
Rationale: Extreme aggravation of blood coagulation induces multiple thrombi in the microvasculature, which leads to consumption coagulopathy followed by disseminated intravascular disease.</span></span></span></span></span></p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p>The AOP379 focuses on the coronavirus-induced thrombosis and disseminated intravascular coagulation, which may contribute to the development of therapeutics of COVID-19 and long COVID syndrome. The understanding of the mechanism of the coronavirus-induced vascular adverse outcome may predict adverse responses of COVID-19 vaccines.</p>
</div>
<div id="references">
<h2>References</h2>
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</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/1739">Event: 1739: Binding to ACE2</a></h4>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/374">Aop:374 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on brain cells (neuronal and non-neuronal) leads to neuroinflammation resulting in encephalitis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/381">Aop:381 - Binding of viral S-glycoprotein to ACE2 receptor leading to dysgeusia</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/385">Aop:385 - Viral spike protein interaction with ACE2 leads to microvascular dysfunction, via ACE2 dysregulation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/395">Aop:395 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on pericytes leads to disseminated intravascular coagulation resulting in cerebrovascular disease (stroke)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/426">Aop:426 - SARS-CoV-2 spike protein binding to ACE2 receptors expressed on pericytes leads to endothelial cell dysfunction, microvascular injury and myocardial infarction. </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/427">Aop:427 - ACE2 downregulation following SARS-CoV-2 infection triggers dysregulation of RAAS and can lead to heart failure.</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/422">Aop:422 - Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/428">Aop:428 - Binding of S-protein to ACE2 in enterocytes induces ACE2 dysregulation leading to gut dysbiosis </a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/430">Aop:430 - Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Receptor recognition is an essential determinant of molecular level in this AOP. ACE2 was reported as an entry receptor for SARS-CoV-2. The viral entry process is mediated by the envelope-embedded surface-located spike (S) glycoprotein. Jun Lan and Walls, A.C et al (Nature 581, 215–220; Cell 180, 281–292) demonstrated a critical initial step of infection at the molecular level from the interaction of ACE2 and S protein. ACE2 has shown that receptor binding affinity to S protein is nM range. To elucidate the interaction between the SARS-CoV-2 RBD and ACE2 at a higher resolution, they also determined the structure of the SARS-CoV-2 RBD–ACE2 complex using X-ray crystallography.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">The expression and distribution of the ACE2 in human body may indicate the potential infection of SARS-CoV-2. Through the developed single-cell RNA sequencing (scRNA-Seq) technique and single-cell transcriptomes based on the public database, researchers analyzed the ACE2 RNA expression profile at single-cell resolution. High ACE2 expression was identified in type II alveolar cells (Zou, X. et al.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Front. Med.2020)</span></span></p>
<p><span style="font-size:12px"><span style="font-family:"Times New Roman",serif">SARS-CoV-2 belongs to the Coronaviridae family, which includes evolutionary related enveloped (+) strand RNA viruses of vertebrates, such as seasonal common coronaviruses, SARS-CoV and CoV-NL63, SARS-CoV (Kim Young Jun et al)</span></span></p>
<p>The KE is applicable to broad species/life stage/sex. The binding of ACE2 occurs in the cells which express ACE2. </p>
<h4>Key Event Description</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Angiotensin-converting enzyme 2 (<a href="https://www.genecards.org/cgi-bin/carddisp.pl?gene=ACE2">ACE2</a>) is an enzyme that can be found either attached to the membrane of the cells (mACE2) in many tissues and in a soluble form form (sACE2). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">A table on ACE2 expression levels according to tissues <em>(Kim et al.)</em></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 mean expression</span></strong></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Standard deviation of expression</span></strong></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.905</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="color:#0070c0">ACE2 receptors in the brain (endothelial, neuronal and glial cells):</span></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2. </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">In the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020). Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>ACE2 receptors in the intestines</em></strong></span></span></span></p>
<p dir="ltr" style="text-align:justify"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><span style="background-color:transparent">The highest levels of ACE2 are found at the luminal surface of the enterocytes, the differentiated epithelial cells in the small intestine, lower levels in the crypt cells and in the colon (Liang et al, 2020; Hashimoto et al., 2012, Fairweather et al. 2012; Kowalczuk et al. 2008). </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><strong><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif"><em>In vitro</em> methods supporting interaction between ACE2 and SARS-CoV-2 spike protein</span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Several reports using surface plasmon resonance (SPR) or biolayer interferometry binding (BLI) approaches. to study the interaction between recombinant ACE2 and S proteins have determined a dissociation constant (Kd) for SARS-CoV S and SARS-CoV-2 S as follow,</span></span></span></span></span></p>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 protein </span></strong></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">34.6 nM</span></span></span></p>
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<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Pseudo typed vesicular stomatitis virus expressing SARS-CoV-2 S (VSV-SARS-S2) expression system can be used efficiently infects cell lines, with Calu-3 human lung adenocarcinoma epithelial cell line, CaCo-2 human colorectal adenocarcinoma colon epithelial cell line and Vero African grey monkey kidney epithelial cell line being the most permissive (Hoffmann et al., 2020; Ou et al., 2020). It can be measured using a wide variety of assays targeting different biological phases of infection and altered cell membrane permeability and cell organelle signaling pathway. Other assay measured alteration in the levels of permissive cell lines all express ACE2 or hACE2-expressing 293T cell (e.g. pNUO1-hACE2, pFUSE-hIgG1-Fc2), as previously demonstrated by indirect immunofluorescence (IF) or by immunoblotting are associated with ELISA(W Tai et al., nature 2020). To prioritize the identified potential KEs for selection and to select a KE to serve as a case study, further in-silico data that ACE2 binds to SARS-CoV-2 S is necessary for virus entry. The above analysis outlined can be used evidence-based assessment of molecular evidence as a MIE.</span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
<td><a href="/aops/320">Aop:320 - Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortality</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
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<td><a href="/aops/394">Aop:394 - SARS-CoV-2 infection of olfactory epithelium leading to impaired olfactory function (short-term anosmia)</a></td>
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<td><a href="/aops/395">Aop:395 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on pericytes leads to disseminated intravascular coagulation resulting in cerebrovascular disease (stroke)</a></td>
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<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
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<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
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<td><a href="/aops/426">Aop:426 - SARS-CoV-2 spike protein binding to ACE2 receptors expressed on pericytes leads to endothelial cell dysfunction, microvascular injury and myocardial infarction. </a></td>
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<td><a href="/aops/422">Aop:422 - Binding of SARS-CoV-2 to ACE2 in enterocytes leads to intestinal barrier disruption</a></td>
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<td><a href="/aops/430">Aop:430 - Binding of SARS-CoV-2 to ACE2 leads to viral infection proliferation</a></td>
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<td><a href="/aops/468">Aop:468 - Binding of SARS-CoV-2 to ACE2 leads to hyperinflammation (via cell death)</a></td>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 vertebrates (Lam et al., 2020)</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">NRP1 in human & rodents (but also present in monkey and other vertebrates </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lu and Meng, 2015)</span></span></p>
<p><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The ability for SARS-CoV-2 to use multiple host pathways for viral entry, means that it is critical to map which viral entry pathway is prevalent in specific cell types. This is key for understanding coronavirus biology, but also use informed decisions to select cells for cell-based genetic and small-molecule screens and to interpret data. In fact, a combination of protease inhibitors that block both TRMPSS2 and cathepsin L is the most efficient combination to block coronavirus infection </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. In accordance, SARS-CoV-2 entry processes are highly dependent on endocytosis and endocytic maturation in cells that do not express TMPRSS2, such as VeroE6 or 293T cells </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012, Hoffmann et al., 2020a)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Treatment of SARS-CoV-2 with trypsin enables viral cell surface entry, even when TMPRSS2 is absent. Moreover, TMPRSS2 is more efficient to promote viral entry than cathepsins </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lamers et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Therefore it is critical to map which cells contain the different types of proteases.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AT2 and clara cells of lungs</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">sinusoidal endothelium, and hepatocyte of the liver, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">endocrine cells of the prostate, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">goblet cells , and enterocytes of the small intestine, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">intercalated cells, and the proximal tubular of the kidney.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Ciliated, secretory and suprabasal of nasal</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">spermatogonial stem cells of testes</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">cyto tropoblast and peri vascular cells of placenta</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The nasal epithelium expresses various combinations of factors that, in principle, could facilitate SARS-CoV-2 infection, but it also expresses robust basal levels of RFs, which may act as a strong protective barrier in this tissue.</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">There is a shift in TMPRSS2 regulation during nasal epithelium differentiation in young individuals that is not occurring in old individuals (Lin et al., 1999, Lucas et al., 2008, Singh et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Only a small minority of human respiratory and intestinal cells have genes that express both ACE2 and TMPRSS2. Amongst the ones that do, three main cell types were identified: A) lung cells called type II pneumocytes (which help maintain air sacs, known as alveoli); B) intestinal cells called enterocytes, which help the body absorb nutrients; and C) goblet cells in the nasal passage, which secrete mucus (Ziegler et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The clinical manifestations of COVID‐19 include not only complications from acute myocardial injury, elevated liver enzymes, and acute kidney injury in patients presenting to hospitals, but also gastrointestinal symptoms in community patients experiencing milder forms of the disease (Madjid et al., 2020, Pan et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">All life stages</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of isoforms 1 (NRP1) and 2 (NRP2) does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules. Expressed in colon and 234 other tissues with Low tissue specificity (UniProtKB). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Androgen receptors (ARs) play a key role in the transcription of TMPRSS2 (Fig. 1). This may explain the predominance of males to COVID-19 infection, fatality, and severity because males tend to have a higher expression and activation of ARs than females, which is due to the presence of dihydrotestosterone (DHT).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Regulation of COVID-19 severity and fatality by sex hormones. Females have aromatase, the enzyme that converts androgen substrates into estrogen. On the other hand, males have steroid 5α reductase, the enzyme that is responsible for the conversion of testosterone into dihydrotestosterone (DHT). In case of males, DHT activates androgen receptor (AR) that binds to the androgen response element (ARE) present in the promoter of TMPRSS2 gene, leading to its transcription. This ultimately results into enhanced processing of viral spike protein for greater entry and spread of SARS-CoV-2 into host cells. On the other hand,in females, estrogen activates estrogen receptor (ER), which binds to the estrogen response element (ERE) present in the promoter of eNOS gene to drive its transcription and catalyze the formation of nitric oxide (NO) from L-arginine. This NO is involved in vasodilation as well as inhibition of viral replication. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">For more information difference of NRP1 expression between male and female see <a href="https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue"><span style="color:blue">https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue</span></a><span style="color:blue">.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of NRP1 protein in gastric cancer was not related to gender, age. The expression of NRP1 protein in gastric cancer is closely correlated to clinical stage, tumor size, TNM stage, differentiation, and lymph node metastasis (Cao et al., 2020).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signalling to induce analgesia had same results on both male and female rodents </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Moutal et al., 2020)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<h4>Key Event Description</h4>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Coronavirus is recognized by the binding of S protein on the viral surface and angiotensin-converting enzyme 2 (ACE2) receptor on the cellular membrane, followed by viral entry via processing of S protein by transmembrane serine protease 2 (TMPRSS2) <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b).</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> ACE2 is expressed on epithelial cells of the lung and intestine, and also can be found in the heart, kidney, adipose, and male and female reproductive tissues </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 is an enveloped virus characterized by displaying spike proteins at the viral surface (Juraszek et al., 2021). Spike is critical for viral entry (Hoffmann et al., 2020b) and is the primary target of vaccines and therapeutic strategies, as this protein is the immunodominant target for antibodies (Yuan et al., 2020, Ju et al., 2020, Robbiani et al., 2020, Premkumar et al., 2020, Liu et al., 2020). Spike is composed of S1 and S2 subdomains. S1 contains the N-terminal (NTD) and receptor-binding (RBD) domains, and the S2 contains the fusion peptide (FP), heptad repeat 1 (HR1) and HR2, the transmembrane (TM) and cytoplasmic domains (CD) (Lan et al., 2020). S1 leads to the recognition of the angiotensin-converting enzyme 2 (ACE2) receptor and S2 is involved in membrane fusion (Hoffmann et al., 2020b, Letko et al., 2020, Shang et al., 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">Upon binding to ACE2, the spike protein needs to be activated (or primed) through proteolytic cleavage (by a host protease) to allow membrane fusion. Fusion is a key step in viral entry as it is the way to release SARS-CoV-2 genetic material inside the cell. Cleavage happens between its spike’s S1 and S2 domains, liberating S2 that inserts its N-terminal domain into a host cell membrane and mediates membrane fusion </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Millet and Whittaker, 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">.</span></span></span> Many proteases were identified to activate coronaviruses including furin, cathepsin L, trypsin-like serine proteases TMPRSS2, TMPRSS4, TMPRSS11, and human airway trypsin-like protease (HATs). These may operate at four different stages of the<a href="https://www.wikipathways.org/index.php/Pathway:WP4846"> virus infection cycle</a>: (a) pro-protein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d ) lysosomal proteases (e.g., cathepsin L) after virus endocytosis in virus-targeting cells (Li, 2016).<span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> SARS-CoV-2 lipidic envelope may fuse with two distinct membrane types, depending on the host protease(s) responsible for cleaving the spike protein: (i) cell surface following activation by serine proteases such as TMPRSS2 and furin </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">; or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Yang and Shen, 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. These flexibility for host cell factors mediating viral entry, highlights that the availability of factors existing in a cell type dictates the mechanism of viral entry </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. When TMPRSS2 (or other serine proteases such as TMPRSS4 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Zang et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> or human airway trypsin-like protease [HAT]</span></span></span> <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Bestle et al., 2020a)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">) is expressed, fusion of the virus with the cell surface membrane is preferred </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Shirato et al., 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">, while in their absence, the virus can penetrate the cell by endocytosis </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. A third factor has also been shown to facilitate SARS-CoV-2 entry in cells that have ACE2 and even promote, although to very low levels, SARS-CoV-2 entry in cells that lack ACE2 and TMPRSS2 which is the neuropilin-1 (NRP-1) </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Cantuti-Castelvetri et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 (transmembrane serine protease 2, (<a href="https://www.ncbi.nlm.nih.gov/gene/7113" style="color:blue; text-decoration:underline">https://www.ncbi.nlm.nih.gov/gene/7113</a>) is a cell-surface protease (Hartenian et al., 2020) that facilitates entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. Viruses found to use this protein for cell entry include Influenza virus and the human coronaviruses HCoV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 (COVID-19 virus).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 is a membrane bound serine protease also known as epitheliasin. TMPRSS2 belongs to the S1A class of serine proteases alongside proteins such as factor Xa and trypsin. Whilst there is evidence that TMPRSS2 autoclaves to generate a secreted protease, its physiological function has not been clearly identified. However, it is known to play a crucial role in facilitating entry of coronavirus particles into cells by cleaving the spike protein (Huggins, 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">After ACE2 receptor binding, SARS-CoV-2 S proteins can be subsequently cleaved and activated by host cell-surface protease at the S1/S2 and S2’ sites, generating the subunits S1 and S2 that remain non-covalently linked. Cleavage leads to activation of the S2 domain that drives fusion of the viral and host membranes (Hartenian et al., 2020, Walls et al., 2016). For other coronaviruses, processing of spike was proposed to be sequential with S1/S2 cleavage preceding that of S2. Cleavage at S1/S2 may be crucial for inducing conformational changes required for receptor binding or exposure of the S2 site to host proteases. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The S1/S2 site of SARS-CoV-2 S protein contains an insertion of four amino acids providing a minimal furin cleavage site (RRAR685↓) (that is absent in SARS-CoV). Interestingly, the furin cleavage site has been implicated in increased viral pathogensis (Bestle et al., 2020b, Huggins, 2020). <span style="color:black">Processing of the spike protein by furin at the S1/S2 cleavage site is thought to occur following viral replication in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) </span><span style="color:black">(Hasan et al., 2020)</span><span style="color:black">. T</span>he spike S2’ cleavage site of SARS-CoV-2 possesses a paired dibasic motif with a single KR segment (KR815↓) (as SARS-CoV) that is recognized by trypsin-like serine proteases such as TMPRSS2. <strong><span style="color:black">The current data support a model for SARS-CoV-2 entry in which furin-mediated cleavage at the S1/S2 site pre-primes spike during biogenesis, facilitating the activation for membrane fusion by a second cleavage event at S2’ by TMPRSS2 following ACE2 binding</span></strong> <span style="color:black">(Bestle et al., 2020b, Johnson et al., 2020)</span><span style="color:black">.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like <span style="color:black">Calu-3 cells but not Huh7.5 and Vero E6 cells</span>. Cell entry was assessed using a viral isolate and viral pseudotypes (artificial viruses) expressing the COVID-19 spike (S) protein. The ability of the viral pseudotypes (expressing S protein from SARS-CoV and SARS-CoV-2) to enter human and animal cell lines was demonstrated, showing that SARS-CoV-2 can enter similar cell lines as SARS-CoV. Amino acid analysis and cell culture experiments showed that, like SARS-CoV, SARS-CoV-2 spike protein binds to human and bat angiotensin-converting enzyme 2 (ACE2) and uses a cellular protease TMPRSS2 for priming. Priming activates the spike protein to facilitate viral fusion and entry into cells. Cell culture experiments were performed using immortalized cell lines and primary human lung cells (Hoffmann et al., 2020b, Rahman et al., 2020).</span></span></p>
<p> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike binding to neuropilin-1:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Neuropilin-1 (NRP1) is a transmembrane glycoprotein that serves as a cell surface receptor for semaphorins and various ligands involved in angiogenesis in vertebrates. NRP1 is expressed in neurons, blood vessels (endothelial cells), immune cells and many other cell types in the mammalian body (maternal fetal interface) and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function (Raimondi et al., 2016). NRP1 is well described as a co-receptor for members of the class 3 semaphorins (SEMA3) or vascular endothelial growth factors (VEGFs) (Gelfand et al., 2014). Structurally, NRP1 comprises seven sub-domains, of which the first five are extracellular; two CUB domains (a1 and a2), two coagulation factor V/VIII domains (FV/VIII; b1 and b2) and a meprin, A5 μ-phosphatase domain (MAM; c). NRP1 contains only a short cytosolic tail with a PDZ-binding domain lacking internal signaling activity. The different ligand families bind to different sites of NRP1; SEMA3A binding requires the first three sub-domains of NRP1 (a1, a2, and b1), whereas binding of VEGF-A requires the b1 and b2 domains (Muhl et al., 2017). Additional studies conducted by means of in silico computational technology to identify and validate inhibitors of the interaction between NRP1 and SARS-CoV-2 Spike protein are reported in (Perez-Miller et al., 2020). Represents a schematic picture of VEGF-A triggered phosphorylation of VEGF-R2. Screening of NRP-1/VEGF-A165 inhibitors by in-cell Western (Perez-Miller et al., 2020).v NRP1 acts as a co-receptor for SARS-CoV-2. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1 is a receptor for <span style="color:black">furin-cleaved SARS-CoV-2 spike peptide </span><span style="color:black">(Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020)</span><span style="color:black">. Blockade of NRP1 reduces infectivity and entry, and alteration of the furin site leads to loss of NRP1 dependence, reduced replication in Calu3, augmented replication in Vero E6, and attenuated disease in a hamster pathogenesis disease model </span><span style="color:black">(Johnson et al., 2020)</span><span style="color:black">.</span> In fact, a small sequence of amino acids was found that appeared to mimic a protein sequence found in human proteins that interact with NRP1. The spike protein of SARS-CoV-2 binding with NRP1 aids viral infection of human cells. This was confirmed by applying a range of structural and biochemical approaches to establish that the spike protein of SARS-CoV-2 does indeed bind to NRP1. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic RRAR C-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. It was reported that the S1 CendR motif directly bound NRP1 by X-ray crystallography and biochemical approaches. Blocking this interaction using RNAi or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture (Daly et al., 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1, known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was revealed by a monoclonal blocking antibody against NRP1. It was found that a SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells faced the nasal cavity (Cantuti-Castelvetri et al., 2020). Furthermore, it has been found that NRP1 is a new potential SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID<span style="font-family:"Cambria Math",serif">‑</span>19. Preclinical studies have suggested that NRP1, a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID<span style="font-family:"Cambria Math",serif">‑</span>19 by enhancing the entry of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory<span style="font-family:"Cambria Math",serif">‑</span>related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic manifestations of COVID<span style="font-family:"Cambria Math",serif">‑</span>19. Accordingly, the neurotropism of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 via NRP1<span style="font-family:"Cambria Math",serif">‑</span>expressing cells in the CNS merits further investigation (Davies et al., 2020).</span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Up-regulation of NRP1 protein in diabetic kidney cells hints at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction (Mayi et al., 2021).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The main feature of NRP1 co-receptor is to form complexes with multiple other receptors. Hence, there is a competition between receptors to complex with NRP-1, which may determine their abilities both quantitatively and qualitatively to transduce signals. It is tempting to hypothesize that the occupancy of NRP-1 with one receptor may thus decrease its availability for virus entry. Recent proteomics work has shown that NRP-1 can form a complex with the α7 nicotinic receptor in mice. Both receptors are expressed in the human nasal and pulmonary epithelium (Mayi et al., 2021).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1, is highly expressed in the respiratory and olfactory epithelium; it is also expressed in the CNS, including olfactory related regions such as the olfactory tubercles and paraolfactory gyri (Davies et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike entry via <span style="color:black">lysosomal cathepsins and endocytosis</span>:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Evidence shows the role of TMPRSS2 and other serine proteases in activating the coronavirus spike protein for plasma membrane fusion. However, studies using various cell culture systems showed that SARS-CoV2 could enter cells via an alternative endosomal–lysosomal pathway.</span> Evidence came from studies<span style="color:black"> demonstrating that lysosomotropic agents reduced SARS-CoV replication in cells lacking TMPRSS2 and other studies, using highly potent and specific small-molecule cathepsin inhibitors, to understand the role of cathepsins in processing and activating the spike for membrane fusion, mainly of cathepsin L (one of the 11 cathepsins) </span><span style="color:black">(Rossi et al., 2004, Simmons et al., 2005)</span><span style="color:black">. SARS-CoV-2 and other coronaviruses can establish infection through endosomal entry in commonly used in vitro cell culture systems. Of relevance, inhibitors of the endosomal pathway, as the cathepsin inhibitor Z-FA-FMK and PIKfyve inhibitor apilimod, blocked viral entry in Huh7.5 and Vero E6 cells but not Calu-3 cells.</span></span></span></p>
<p style="text-align:justify"><strong>Viral entry leads to delivery of virion proteins and translation of viral proteins immediately: </strong></p>
<p style="text-align:justify"><span style="font-size:14px">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D,<em> et al., 2020</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>Huston N. C.<em> et al., 2021</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., <em>et al., 1985</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., <em>et al.</em>, 2018].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting </span>[Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [Sacco M. D. <em>et al., 2020</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>Klein<em> </em>S., <em>et al., 2021</em>, Snijder E. J.<em>, et al., 2020, </em>V'Kovski P. , <em>et al., 2021</em>]<span style="color:black">. This association leads to replication factories or organelles, that are originate new membranous structures that are observed by electron mciroscopy . They are a feature of all coronaviridae and the site of viral replication and transcription hidden from innate immune molecules.</span></span></p>
<h4>How it is Measured or Detected</h4>
<p>SARS-CoV2 entry can be determined by many different ways:</p>
<p>1) quantitative RT-PCR specific to the subgenomic mRNA of the N transcript, in cells manipulated with host factors that express of not TMPRSS2, cathepsinL, neuropilin-1, hACE2 [Glowacka I, et al. (2011)], or exogenous addition of HAT or furin.</p>
<p>2) using spike-pseudotyped viral particles expressing GFP/luciferase/bgalactosidase and comparing with vesicular stomatitis virus G seudotyped particles expressing the same reporter analysed in manipulated cultured with cell lines, followed by determining fluorescence, biolumincescence, luciferase activity in cell lysates [Hoffmann M, et al. (2020)].</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:</span></span></p>
<ol>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">X-ray crystallography and biochemical approaches help to show that the S1 CendR motif directly bound NRP1 (1). Binding of the S1 fragment to NRP1 was assessed and ability of SARS-CoV-2 to use NRP1 to infect cells was measured in angiotensin-converting enzyme-2 (ACE-2)-expressing cell lines by knocking out NRP1 expression, blocking NRP1 with 3 different anti-NRP1 monoclonal antibodies, or using NRP1 small molecule antagonists </span><span style="color:black">(Centers for Disease Control and Prevention, 2020, Daly et al., 2020)</span><span style="color:black">.</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• SARS-CoV-2 utilizes NRP1 for cell entry as evidenced by decreased infectivity of cells in the presence of: NRP1 deletion (p <0.01). Three different anti-NRP1 monoclonal antibodies (p <0.001). Selective NRP1 antagonist, EG00229 (p <0.01).</span></span></p>
<ol start="2">
<li><span style="font-size:11pt"><span style="color:black"><span style="font-family:"Calibri",sans-serif">Cell lines were modified to express ACE2 and TMPRSS2, the two known SARS-CoV-2 host factors, and NRP1 to assess the contribution of NRP1 to infection. Autopsy specimens from multiple airway sites were stained with antibodies against SARS-CoV-2 proteins, ACE2, and NRP1, to visualize co-localization of proteins (6, 15).</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• Infectivity of cells expressing angiotensin converting enzyme-2 (ACE2, receptor for SARS-CoV-2), transmembrane protease serine-2 (TSS2, primes the Spike [S] protein), and neuropilin-1 (NRP1) with pseudovirus expressing the SARS-CoV-2 S1 protein was approximately 3-fold higher than in cells expressing either ACE2 or TSS2 alone (p<0.05).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• Analysis of autopsy tissue from COVID-19 patients showed co-localization of the SARS-CoV-2 spike (S) protein and NRP1 in olfactory and respiratory epithelium.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Virtual screen of nearly 0.5 million compounds against the NRP-1 CendR site, resulting in nearly 1,000 hits. A pharmacophore model was derived from the identified ligands, considering both steric and electronic requirements. Preparation of receptor protein and grid for virtual screening, docking of known NRP-1 targeting compounds, ELISA based NRP1-VEGF-A165 protein binding assay; more details on methodology in the referenced paper </span></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Perez-Miller et al., 2020)</span></span></span></p>
<h4>References</h4>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">BAUGHN, L. B., SHARMA, N., ELHAIK, E., SEKULIC, A., BRYCE, A. H. & FONSECA, R. 2020. Targeting TMPRSS2 in SARS-CoV-2 Infection. <em>Mayo Clin Proc,</em> 95<strong>,</strong> 1989-1999.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">BESTLE, D., HEINDL, M. R., LIMBURG, H., VAN LAM VAN, T., PILGRAM, O., MOULTON, H., STEIN, D. A., HARDES, K., EICKMANN, M., DOLNIK, O., ROHDE, C., KLENK, H.-D., GARTEN, W., STEINMETZER, T. & BÖTTCHER-FRIEBERTSHÄUSER, E. 2020a. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. <em>Life Science Alliance,</em> 3.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">BESTLE, D., HEINDL, M. R., LIMBURG, H., VAN LAM VAN, T., PILGRAM, O., MOULTON, H., STEIN, D. A., HARDES, K., EICKMANN, M., DOLNIK, O., ROHDE, C., KLENK, H. D., GARTEN, W., STEINMETZER, T. & BOTTCHER-FRIEBERTSHAUSER, E. 2020b. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. <em>Life Sci Alliance,</em> 3.</span></span></p>
<p style="margin-left:40px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">BUDZILOWICZ, C.J., WILCZYNSKI, S.P., AND WEISS, S.R. (1985). Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3' end of the viral mRNA leader sequence. <em>J Virol</em><em> 53</em>, 834-840.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">CANTUTI-CASTELVETRI, L., OJHA, R., PEDRO, L. D., DJANNATIAN, M., FRANZ, J., KUIVANEN, S., VAN DER MEER, F., KALLIO, K., KAYA, T., ANASTASINA, M., SMURA, T., LEVANOV, L., SZIROVICZA, L., TOBI, A., KALLIO-KOKKO, H., OSTERLUND, P., JOENSUU, M., MEUNIER, F. A., BUTCHER, S. J., WINKLER, M. S., MOLLENHAUER, B., HELENIUS, A., GOKCE, O., TEESALU, T., HEPOJOKI, J., VAPALAHTI, O., STADELMANN, C., BALISTRERI, G. & SIMONS, M. 2020. Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. <em>Science,</em> 370<strong>,</strong> 856-860.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">CAO, H., LI, Y., HUANG, L., BAI, B. & XU, Z. 2020. Clinicopathological Significance of Neuropilin 1 Expression in Gastric Cancer: A Meta-Analysis. <em>Dis Markers,</em> 2020<strong>,</strong> 4763492.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">CENTERS FOR DISEASE CONTROL AND PREVENTION, U. S. D. O. H. A. H. S. 2020. Covid-19 Science Update 2020.</span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">CHEN, L., LI, X., CHEN, M., FENG, Y. & XIONG, C. 2020. The ACE2 expression in human heart indicates new potential mechanism of heart injury among patients infected with SARS-CoV-2. <em>Cardiovasc Res,</em> 116<strong>,</strong> 1097-1100.</span></span></p>
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<h4><a href="/events/1901">Event: 1901: Interferon-I antiviral response, antagonized by SARS-CoV-2</a></h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Broad mammalian host range based on spike protein tropism for and binding to ACE2 (Conceicao et al. 2020; Wu et al. 2020) and cross-species ACE2 structural analysis (Damas et al. 2020). Some literature found on non-human hosts indicates that NSPs and accessory proteins can interact in a similar manner with bird (chicken) and other mammal proteins in the IFN-I pathway (Moustaqil et al. 2021; Rui et al. 2021).</span></span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). The SAR</span>S-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), <a href="https://www.ncbi.nlm.nih.gov/gene/?term=ORF1a+SARS-CoV-2">ORF1ab</a> (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins (Kim et al. 2020; O’Leary et al. 2020). Viral transcription and replication is explained in depth in <a href="https://aopwiki.org/events/1847" style="color:blue; text-decoration:underline">KE1847</a>. Briefly, the first event upon cell entry is the primary translation of the ORF1a and ORF1b genomic RNA to produce non-structural proteins (NSPs). The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into NSP-1 through NSP-11. A -1-ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 NSPs (duplications of NSP1-11 and five additional proteins, NSP12-16). Viral proteases NSP3 and NSP5 cleave the polypeptides through domains functioning as a papain-like protease and a 3C-like protease, respectively <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). </span> The NSPs, structural proteins, and accessory proteins are encoded by 10 ORFs in the SARS-CoV-2 RNA genome. They may have multiple functions during viral replication as well as in evasion of the host innate immune response, thus augmenting viral replication and spread<strong> (Amor et al. 2020). </strong>Extensive protein-protein interaction <strong>(Gordon et al. 2020) </strong>and viral protein-host RNA interaction networks have been demonstrated between the viral NSPs and accessory proteins and host molecules. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This key event is focused on the specific viral:host protein interactions within the infected cell that are involved in the <a href="https://www.wikipathways.org/index.php/Pathway:WP4868">IFN-I antiviral response pathways</a>. IFN-I is the main component of the innate immune system that is suppressed by the SARS-CoV-2 coronavirus early in infection. </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The primary form of host intracellular virus surveillance detects viral components to induce an immediate systemic type I interferon (IFN) response. Cellular RNA sensors called pattern recognition receptors (PRRs) such as RIG-I, MDA5 and LGP2 detect the presence of viral RNAs and promote nuclear translocation of the transcription factor IRF3, leading to transcription, translation, and secretion of IFN-α and IFN-β. This in turn leads to interaction with the IFN receptor (IFNAR), phosphorylation of STAT1 and 2, and transcription and translation of hundreds of antiviral genes <strong>(Quarleri and Delpino, 2021).</strong></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: NSP1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; NSP8 and NSP9 displace signal recognition particle proteins (SRP54, 27 and 19) to bind to the 7SL RNA and block protein trafficking to the cell membrane (Banerjee et al. 2020; Gordon et al. 2020). Xia et al. (2020) found that NSP6 and NSP13 antagonize IFN-I production via distinct mechanisms: NSP6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and NSP13 binds and blocks TBK1 phosphorylation. NSP14 induces lysosomal degradation of type 1 IFNAR to prevent STAT activation (Hayn et al. 2021). ORF6 hijacks KPNA2 to block IRF3, and Nup98/RAE1 to block STAT nuclear import, to silence IFN-I gene expression (Xia and Shi, 2020). ORF7a suppresses STAT2 phosphorylation and ORF7b suppresses STAT1 and STAT2 phosphorylation to block ISGF3 complex formation with IRF9 (Xia and Shi, 2020). ORF8 interacts and downregulates MHC-I (Zhang et al 2020), and has been reported to block INF</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> expression, but the mechanism has not been identified (Rashid et al. 2021; Li et al. 2020). ORF9b antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO and cGAS-STING signalling (Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Following is a table of the current state of knowledge of SARS-CoV-2 protein putative functions in relation to IFN-I antiviral response antagonism.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">DNA Polymerase Alpha Complex: Regulates the activation of IFN-I through cytosolic<br />
RNA-DNA synthesis (POLA1/2-PRIM1/2) and primes DNA replication in the nucleus (Gordon et al. 2020; Chaudhuri et al. 2020). Can also inhibit host gene expression by binding to ribosomes and modifying host mRNAs (Shi et al. 2020; Schubert et al. 2020; Thoms et al. 2020). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">While not essential for viral replication, deletion of NSP2 diminishes viral growth and RNA synthesis</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses IFN-I: Cleaves IRF3 (Moustaqil et al. 2021); binds/cleaves ISG15 (Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Klemm et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">In complex with NSP8 forms primase as part of multimeric RNA-dependent RNA replicase (RdRp)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP72/54/19 (Gordon et al. 2020) and 7SL RNA to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP and 7SL RNA with NSP8 to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Helicase and triphosphatase that initiates the first step in viral mRNA capping.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with M, S, E and 7a; form viroporins; immune evasion</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds STING (Rui et al 2021)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with RIG-I and MAVS sensors of viral RNA (Fu et al 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Hijacks the nuclear importin Karyopherin a 2 (KPNA2) to block IRF3 (Xia and Shi, 2020) and Nup98/RAE1 to block STAT nuclear import (Miorin et al. 2020; Kato et al. 2020), leading to the silence of downstream ISGs</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts and downregulates MHC-I (Zhang et al. 2020). May inhibit type I interferon (IFN-β) and interferon-stimulated response element (ISRE) (Rashid et al. 2020; Li et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Attenuates stress granule formation: G3BP1/2 (Chen et al. 2020; Cascarina et al. 2020); G3BP1 also interacts with RIG-I (Kim et al. 2019) and STAT1/2 (Mu et al. 2020)</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Membrane protein antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO, and cGAS-STING signaling pathways (Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020)</span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Detection of IFN-I suppression involves measuring gene promoter/transcription activation (luciferase assays), gene up/down regulation (quantitative PCR), protein-protein interaction (immunoprecipitation, immunoblotting) or in-situ co-location of viral and host proteins (immunofluorescent or confocal microscopy) in cell culture. Examples of methods used include the following:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Interferon I decrease (Xia et al. 2020):</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">IFN-I production and signaling luciferase reporter assays</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Co-immunoprecipitation and western blot</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">DNA assembly and RNA transcription of a luciferase replicon for SARS-CoV-2</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Replicon RNA electroporation and luciferase reporter assay</span></span></li>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2-Human Protein-Protein Interaction Map (Gordon et al. 2020)</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cloning and expression of viral proteins via plasmid transfection into HEK293T cell line</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Protein affinity purification using MagStrep beads with detection by anti-strep western blot of cell lysate</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Global analysis of SARS-CoV-2 host interacting proteins using affinity purification-mass spectrometry</span></span></li>
</ul>
<p> </p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amor et al. 2020. Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage. Clinical and Experimental Immunology, 202: 193–209. doi: 10.1111/cei.13523 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Andres et al. 2020. SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells. bioRxiv preprint doi: <a href="https://doi.org/10.1101/2020.08.18.256776" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.08.18.256776</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Banerjee et al. 2020. SARS-CoV-2 disrupts splicing, translation, and protein trafficking to supress host defenses. Cell 183, 1325–1339. <a href="https://doi.org/10.1016/j.cell.2020.10.004" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.10.004</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cascarina and Ross, 2020. A proposed role for the SARS-CoV-2 nucleocapsid protein in the </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">formation and regulation of biomolecular condensates. The FASEB Journal, 34:9832–9842. DOI: 10.1096/fj.202001351 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chaudhuri, A. 2021. Comparative analysis of non-structural protein 1 of SARS-CoV2 with SARS-CoV1 and MERS-CoV: An in-silico study. Journal of Molecular Structure, Volume 1243, 130854, https://doi.org/10.1016/j.molstruc.2021.130854.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chen et al. 2021. SARS-CoV-2 Nucleocapsid Protein Interacts with RIG-I and Represses RIG-Mediated IFN-β Production. Viruses. 13(1):47. <a href="https://doi.org/10.3390/v13010047" style="color:blue; text-decoration:underline">https://doi.org/10.3390/v13010047</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Conceicao et al. 2020. The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016. <a href="https://doi.org/10.1371/journal.pbio.3001016" style="color:blue; text-decoration:underline">https://doi.org/10.1371/journal.pbio.3001016</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Damas et al. 2020. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. PNAS vol. 117 no. 36:22311–22322 <a href="http://www.pnas.org/cgi/doi/10.1073/pnas.2010146117" style="color:blue; text-decoration:underline">www.pnas.org/cgi/doi/10.1073/pnas.2010146117</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fu et al. 2021. SARS-CoV-2 membrane glycoprotein M antagonizes the MAVS-mediated innate antiviral response. Cell Mol Immunol 18: 613–620. https://doi.org/10.1038/s41423-020-00571-x</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. 2020. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 483:459-473. <a href="https://doi.org/10.1038/s41586-020-2286-9" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41586-020-2286-9</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gupta et al. 2021. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. bioRxiv 2021.05.10.443524; doi: https://doi.org/10.1101/2021.05.10.443524</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Han et al. 2020. SARS-CoV-2 ORF9b Antagonizes Type I and III Interferons by Targeting Multiple Components of RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING Signaling Pathways. bioRX <a href="https://doi.org/10.1101/2020.08.16.252973" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.08.16.252973</a> </span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zhang et al. 2020. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv preprint doi: <a href="https://doi.org/10.1101/2020.05.24.111823" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.05.24.111823</a></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020; Wu </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020] and cross-species ACE2 structural analysis [Damas </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]. No literature has been found on primary translation and molecular interactions of nsps in non-human host cells, but it is assumed to occur if the virus replicates in other species.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">This KE1847 "Increase coronavirus production" deals with how the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated, replicated, and transcribed in detail, and how the genomic RNA (gRNA) is packaged, and the virions are assembled and released from the cell. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim<em> et al.</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">[</span>E. J. Snijder<em> et al.</em><span style="color:#131413">] that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>N. C. Huston<em> et al.</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx <em>et al.</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder <em>et al.</em>]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di <em>et al.</em>].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting [E. J. Snider <em>et al.</em>]</span><span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [M. D. Sacco <em>et al.</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>S. Klein<em> et al.</em>, E. J. Snijder<em> et al., </em>P. V'Kovski, <em>et al.</em>]<span style="color:black">. Besides replication, which yields the positive-sense gRNA, the replicase also</span> <span style="color:black">mediates transcription leading to the synthesis of a nested set of subgenomic (sg) mRNAs to express all ORFs downstream of ORF1b that encode structural and accessory viral proteins. </span>These localize to the 3′ one-third of the genome, as stated above, and result in a 3′ coterminal nested set of 7–9 mRNAs that share ~70–90 nucleotide (nt) in the 5′ leader and that is identical to the 5′ end of the genome [P. Liu, and J. Leibowitz]. s<span style="color:black">gRNAs encode conserved structural proteins (spike protein [S], envelope protein [E], membrane protein [M], and nucleocapsid protein [N]), and several accessory proteins. SARS-CoV-2 is known to have at least six accessory proteins (3a, 6, 7a, 7b, 8, and 10). Overall the virus is predicted to express 29 proteins [</span>D. Kim<em> et al.</em>]<span style="color:black">. The gRNA is packaged by the structural proteins to assemble progeny virions.</span></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA [D. Kim<em> et al.</em>]. The SARS-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), ORF1ab (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins [D. Kim<em> et al.</em>]. Other elements of the genome include are shown below [V. B. O'Leary <em>et al.</em>]. <strong>The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).</strong></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">This is completely dependent on the translation machinery of the host cell. Due to fewer rare “slow-codons”, SARS-CoV-2 may have a higher protein translational rate, and therefore higher infectivity compared to other coronavirus groups [V. B. O'Leary <em>et al.</em>]. The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into nsp-1 through nsp-11. A -1 ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 nsps [D. Kim<em> et al.</em>]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel <em>et al.</em>]. Functionally, there are five proteins from pp1ab (nsp-12 through nsp-16) as nsp-1-11 are duplications of the proteins in pp1a due to the ORF overlap. The <span style="color:black">pp1a is approximately 1.4–2.2 times more expressed than pp1ab. </span>After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 <span style="color:black">protease can be referred to as 3C-like protease (3CL<sup>pro</sup>) or as main protease (M<sup>pro</sup>), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein </span><em>et al.</em><span style="color:black">] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, </span><span style="color:#231f20">suppressing host gene expression </span><span style="color:#000000">[</span>M. Thoms<em> et al.]</em><span style="color:black">. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors.</span> N<span style="color:black">sp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, w</span>hilst <span style="color:black">nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [</span>P. V'Kovski <em>et al.</em>]<span style="color:black">. </span>nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. <span style="color:black">nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function.</span> Nsp-10 composes the RNA <span style="color:black">capping machinery</span> nsp-9. <span style="color:black">nsp13 provides the RNA 5′-triphosphatase activity</span>. Nsp-14 is a <em><span style="color:black">N</span></em><span style="color:black">7-methyltransferase and nsp-16 the 2′-<em>O</em>-methyltransferase. </span>Many of the nsps have multiple functions and many viral proteins are involved in innate immunity inhibition. Nsp-3 is involved in vesicle formation along with nsp-4 and nsp-6 where viral replication occurs. Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: nsp-16 binds to U1 and U2 splicing RNAs to suppress global mRNA splicing; nsp-1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; nsp-8 and nsp-9 bind to the 7SL RNA to block protein trafficking to the cell membrane [A. K. Banerjee<em> et al.</em>]. Xia et al. [H. Xia<em> et al.</em>] found that nsp-6 and nsp-13 antagonize IFN-I production via distinct mechanisms: nsp-6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and nsp-13 binds and blocks TBK1 phosphorylation.</span></span></p>
<p> </p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Viral transcription and replication:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder<em> et al.</em>]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to <span style="color:black">small spherular invaginations, and large vesiculotubular clusters, consisting of single- and/or double-membrane vesicles (DMV), convoluted membranes (CM) and double-membrane spherules invaginating from the ER [</span>S. Klein<em> et al., </em>E. J. Snijder<em> et al.</em>]<span style="color:black">. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-<sup>3</sup>[H]uridine) and detection by EM autoradiography</span> <span style="color:#000000">[</span>E. J. Snijder<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Viral replicative proteins and specific host factors are recruited</span> to ROs [E. J. Snijder<em> et al.</em>]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist <em>et al.</em>]. Viral RTC act in combination with other viral and host factors involved in selecting template RNAs, elongating RNA synthesis, differentiating genomic RNA replication from mRNA transcription, modifying product RNAs with 5’ caps or 3’ polyadenylate [P. Ahlquist <em>et al.</em>]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz<em> et al.</em>]. The intermediates comprise <span style="color:black">full-length negative-sense complementary copies of the genome, which functions as templates for the generation of new positive-sense gRNA, and a nested set of sg mRNAs that lead to the expression of proteins encoded in all ORFs downstream of ORF1b. </span>The transcription of coronaviruses <span style="color:black">is a discontinuous process that produces nested 3′ and 5′ co-terminal sgRNAs. Of note, the synthesis of sg mRNAs is not exclusive to the order <em>Nidovirales</em> but a discontinuous minus-strand synthesis strategy to produce a nested set of 3′ co-terminal sg mRNAs with a common 5′ leader in infected cells</span> <span style="color:black">are unique features of the <em>coronaviruses</em> and <em>arteriviruses</em> [</span>W. A. Miller and G. Koev.]<span style="color:black">. Of note, the produced genomic RNA represents a small fraction of the total vRNA [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The discontinuous minus-strand synthesis of a set of nested sg mRNAs happens during the synthesis of the negative-strand RNA, by an interruption mechanism of the RTC as it reads the TRS-B preceding each gene in the 3′ one-third of the viral genome [</span>I. Sola, F. Almazan <em>et al., </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">. The synthesis of the negative-strand RNA stops and is re-initiated at the TRS-L of the genome sequence close from the 5′ end of the genome [</span>H. Di <em>et al.</em>]<span style="color:black">. Therefore, t</span><span style="color:black">he mechanism by which the leader sequence is added to the 5' end requires that the RTC switches template by a jumping mechanism. This interruption process involves the interaction between complementary TRSs of the nascent negative-strand RNA TRS-B and the positive-strand gRNA at the positive-sense TRS-L. The TRS-B site has a 7-8 bp conserved core sequence (CS) that facilitates RTC template switching as it hybridizes with a near complementary CS in the TRS-L [</span>I. Sola, F. Almazan <em>et al. </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">.</span> <span style="color:black">Upon re-initiation of RNA synthesis at the TRS-L region, a negative-strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. This means that all sg mRNAs as well as the genomic RNA share a common 5' sequence, named leader sequence [</span>X. Zhang et al.]<span style="color:black">. This programmed template switching leads to the generation of sg mRNAs with identical 5' and 3' sequences, but alternative central regions corresponding to the beginning of each structural ORF [</span>I. Sola <em>et al.</em> 2015, S. G. Sawicki <em>et al.</em>, Y. Yang <em>et al.</em>]<span style="color:black">. Of note, the existence of TRSs also raises the possibility that these sites are at the highest risk of recombining through TRS-B mediated template switching [</span>Y. Yang]<span style="color:black">.</span> <span style="color:black">The set of sg mRNAs is then translated to yield </span>29 identified different proteins [F. Wu<em> et al.</em>], although many papers have identified additional ORFs [D. Kim<em> et al.. </em>Y. Finkel<em> et al., </em>A. Vandelli<em> et al.</em>]. The translation of the linear single-stranded RNA conducts to the generation of the following proteome: 4 are structural proteins, S, N, M, and E; 16 proteins nsp: the first 11 are encoded in ORF1a whereas the last 5 are encoded in ORF1ab. In addition, 9 accessory proteins named ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10 have been identified [F. Wu<em> et al.</em>]. At the beginning of infection, there is the predominant expression of the nsp that result from ORF1a and ORF1ab, however, at 5 hpi, the proteins encoded by the <span style="color:black">5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [</span>Y. Finkel<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The final step of viral production requires virion assembly and this process is not well explored for SARS-CoV-2. For example, the role of the structural proteins of SARS-CoV-2 in virus assembly and budding in not known. In general, all beta-coronavirus structural proteins assemble at the endoplasmic reticulum (ER)-to-Golgi compartment [</span>J. R. Cohen <em>et al.</em><em>, </em>A. Perrier<em> et al.</em>]<span style="color:black"> and v</span>iral assembly requires two steps: Genome packaging which is a process in which the SARS-CoV-2 gRNA must be coated by the viral protein nucleoprotein (N) protein, <span style="color:black">forming viral ribonucleoprotein (vRNPs) complexes, </span>before being selectively packaged into progeny virions [P. V'Kovski <em>et al.</em>], a step in which vRNPs<span style="color:black"> bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">. This results in viral particles enveloped with host membranes containing viral M, E, and S transmembrane structural proteins that need to be released from the cell.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 gRNA packaging involves the N protein. The N protein of human coronaviruses is highly expressed in infected cells. It is considered a multifunctional protein, promoting efficient sub-genomic viral RNA transcription, viral replication, virion assembly, and interacting with multiple host proteins [P. V'Kovski <em>et al.</em>, D. E. Gordon<em> et al.</em>, R. McBride, and M. van Zyl, B. C.]. In relation to transcription and replication, the N protein could provide a cooperative mechanism to increase protein and RNA concentrations at specific localizations S. Alberti, and S. Carra, S. F. Banani <em>et al.</em>], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) <em>in vitro</em> [A. Savastano <em>et al.</em>, H. Chen<em> et al.</em>, C. Iserman<em> et al.</em>, T. M. Perdikari<em> et al.</em>, J. Cubuk<em> et al.</em>], dependent on its C-terminal domain (CTD) [H. Chen<em> et al.]</em>. It has been hypothesised that N could be involved in replication close to the ER and in packaging of gRNA into vRNPs near the ERGIC where genome assembly is thought to take place [A. Savastano<em> et al.</em>], but so far this is still speculative. Phosphorylation of N could adjust the physical properties of condensates differentially in ways that could accommodate the two different functions of N: transcription and progeny genome assembly [A. Savastano <em>et al.</em>, C. Iserman<em> et al., </em>C. R. Carlson<em> et al.</em>]. </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The ERGIC constitutes the main assembly site of coronaviruses [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black"> and budding events have been seen by EM studies.</span> For SARS-CoV-2, v<span style="color:black">irus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black">. The ectodomain of S trimers were found facing the ERGIC lumen and not induce membrane curvature on its own, therefore proposing that vRNPs and spike trimers</span> <span style="color:black">[</span>S. Klein<em> et al.</em>]<span style="color:black">.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Finally, it has been shown that SARS-CoV-2 virions de novo formed traffic to lysosomes for unconventional egress by Arl8b-dependent lysosomal exocytosis [S. Ghosh<em> et al.</em>]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh<em> et al.</em>].</span></span></p>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">In vivo translation assay</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay</span></span></li>
</ul>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]</span></span></p>
<ul>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 viral protein binding to RNA</span></span></li>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">The mRNA transcripts are detected by the real-time reverse transcription-PCR (RT-PCR) assay. Several methods targeting the mRNA transcripts have been developed, which includes the RT-PCR assays targeting RdRp/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 [Chan <em>et al.</em>]. RT-PCR assays detecting SARS-CoV-2 RNA in saliva include quantitative RT-PCR (RT-qPCR), direct RT-qPCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) [Nagura-Ikeda M, <em>et al.</em>]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">viral replication is measured by RT-qPCR in infected cells, formation of liquid organelles is assessed in vitro reconstitution systems and in infected cells. Labelling by radioactive nucleosides.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.</span></span></p>
<h4>References</h4>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:Arial,Helvetica,sans-serif">1. D. Kim<em> et al.</em>, The Architecture of SARS-CoV-2 Transcriptome. <em>Cell</em> <strong>181</strong>, 914-921 e910 (2020).</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">2. E. J. Snijder<em> et al.</em>, Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage. <em>Journal of Molecular Biology</em> <strong>331</strong>, 991-1004 (2003).</span></span></p>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">19. W. A. Miller, G. Koev, Synthesis of subgenomic RNAs by positive-strand RNA viruses. <em>Virology</em> <strong>273</strong>, 1-8 (2000).</span></span></p>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">23. X. Zhang, C. L. Liao, M. M. Lai, Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. <em>J Virol</em> <strong>68</strong>, 4738-4746 (1994).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">24. S. G. Sawicki, D. L. Sawicki, S. G. Siddell, A contemporary view of coronavirus transcription. <em>J Virol</em> <strong>81</strong>, 20-29 (2007).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">25. Y. Yang, W. Yan, B. Hall, X. Jiang, Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">26. F. Wu<em> et al.</em>, A new coronavirus associated with human respiratory disease in China. <em>Nature</em> <strong>579</strong>, 265-269 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">27. A. Vandelli<em> et al.</em>, Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. <em>Nucleic Acids Res</em> <strong>48</strong>, 11270-11283 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">28. J. R. Cohen, L. D. Lin, C. E. Machamer, Identification of a Golgi complex-targeting signal in the cytoplasmic tail of the severe acute respiratory syndrome coronavirus envelope protein. <em>J Virol</em> <strong>85</strong>, 5794-5803 (2011).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">29. A. Perrier<em> et al.</em>, The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal. <em>J Biol Chem</em> <strong>294</strong>, 14406-14421 (2019).</span></span></p>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">32. S. Alberti, S. Carra, Quality Control of Membraneless Organelles. <em>Journal of Molecular Biology</em> <strong>430</strong>, 4711-4729 (2018).</span></span></p>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">34. A. Savastano, A. I. de Opakua, M. Rankovic, M. Zweckstetter, Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates. (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">35. H. Chen<em> et al.</em>, Liquid-liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA. <em>Cell Res</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">36. C. Iserman<em> et al.</em>, Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">37. T. M. Perdikari<em> et al.</em>, SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">38. J. Cubuk<em> et al.</em>, The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">39. C. Iserman<em> et al.</em> (Cold Spring Harbor Laboratory, 2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">40. C. R. Carlson<em> et al.</em>, Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests abiophysical basis for its dual functions. <em>Molecular Cell</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">41. L. Mendonca<em> et al.</em>, SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">42. S. Ghosh<em> et al.</em>, beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. <em>Cell</em> <strong>183</strong>, 1520-1535 e1514 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">43. Schubert, K., Karousis, E.D., Jomaa, A. <em>et al.</em> SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. <em>Nat Struct Mol Biol</em> <strong>27, </strong>959–966 (2020). </span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">44. Chan, Jasper Fuk-Woo et al. Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated <em>In Vitro</em> and with Clinical Specimens. J Clin Microbiol. 2020:58(5)e00310-20. doi:10.1128/JCM.00310-20</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">45. Nagura-Ikeda M, Imai K, Tabata S, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020;58(9):e01438-20. doi:10.1128/JCM.01438-20</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">46. Conceicao C, Thakur N, Human S, Kelly JT, Logan L, Bialy D, et al. (2020) The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016. https://doi.org/10.1371/journal.pbio.3001016</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">47. Damas J, Hughes GM, Keough KC, Painter CA, Persky NS, Corbo M, Hiller M, Koepfli KP, Pfenning AR, Zhao H, Genereux DP, Swofford R, Pollard KS, Ryder OA, Nweeia MT, Lindblad-Toh K, Teeling EC, Karlsson EK, Lewin HA. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. Proc Natl Acad Sci U S A. 2020 Sep 8;117(36):22311-22322. doi: 10.1073/pnas.2010146117. Epub 2020 Aug 21. PMID: 32826334; PMCID: PMC7486773.</span></span></p>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/406">Aop:406 - SARS-CoV-2 infection leading to hyperinflammation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/407">Aop:407 - SARS-CoV-2 infection leading to pyroptosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/488">Aop:488 - Increased reactive oxygen species production leading to decreased cognitive function</a></td>
<p>Response to ROS occurs in many cell types and tissues in all life stages and the broad range of mammals.</p>
<h4>Key Event Description</h4>
<p>Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids, and DNA. Severe oxidative stress can trigger apoptosis and necrosis. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)</p>
<p>The "Diminished Protective Oxidative Stress Response" is a critical key event in the Adverse Outcome Pathway (AOP) framework that plays a central role in understanding how exposure to various stressors can lead to adverse outcomes.</p>
<p>Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids, and DNA. Severe oxidative stress can trigger apoptosis and necrosis. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)</p>
<p>One essential component of this key event is the activity of Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that plays a pivotal role in the regulation of the oxidative stress response. Detecting Nrf2 activity is crucial for assessing the status of the oxidative stress response.</p>
<p>The cellular defence/defense response to oxidative stress includes induction of detoxifying enzymes and antioxidant enzymes. Nuclear factor-erythroid 2-related factor 2 (Nrf2) binds to the antioxidant response elements (ARE) within the promoter of these enzymes and activates their transcription. Inactive Nrf2 is retained in the cytoplasm by association with an actin-binding protein Keap1. Upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs, and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, cytochrome P450, NAD(P)H quinone oxidoreductase, heme oxygenase, and superoxide dismutase. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)</p>
<p>Nrf2, a master regulator of oxidative stress through enhanced expression of anti-oxidant genes of glutathione and thioredoxin-antioxidant systems, has anti-inflammatory, anti-apoptotic, and antioxidant effects. Dimethyl fumarate (DMF), an activator of Nrf2, can decrease inflammation and reactive oxygen species (ROS) through the inhibition of NF-kappaB by inducing anti-oxidant enzymes (Jackson et al, 2014; Hassan et al, 2020; Timpani et al, 2021).</p>
<p>Inactivation of Nrf2 causes diminished protective responses to ROS.</p>
<h4>How it is Measured or Detected</h4>
<p>Oxidative stress can be measured as follows:</p>
<p>1. Direct detection of reactive oxygen species (ROS)</p>
<p>ROS can be detected by intracellular ROS assay, in vitro ROS/RNS assay. Nitric oxide can be detected in intracellular nitric oxide assay (Ashoka et al, 2020).</p>
<p>Hydroxyl, peroxyl, or other ROS can be measured using a fluorescence probe, 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA), at fluorescence detection at 480 nm/530 nm.</p>
<p>Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range.</p>
<p>ROS can be detected by PEGylated bilirubin-coated iron oxide nanoparticles in whole blood (Lee et al, 2020).</p>
<p>2. Measurement of anti-oxidants</p>
<p>The level of catalase, glutathione, or superoxide dismutase can be measured as anti-oxidants. Catalase is an anti-oxidative enzyme that catalyses the resolution of hydrogen peroxide (H2O2) into H2O and O2. The chemiluminescence or fluorescence of HRP catalytic reaction can be detected with residual H2O2 and probes (DHBS+AAP, or ADHP (10-Acetyl-3, 7-dihydroxyphenoxazine)).</p>
<p>Anti-oxidant capacity is also one of the oxidative stress markers. Oxygen radical antioxidant capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), total antioxidant capacity (TAC), the cell-based exogenous antioxidant assay can be used for measuring the antioxidant capacity.</p>
<p>3. Detection of damages in protein, lipid, DNA or RNA</p>
<p>Oxidation of protein can be measured by the detection of protein carbonyl content (PCC), 3-nitrotyrosine, advanced oxidation protein products, or BPDE protein adduct. </p>
<p>DNA oxidation can be detected with 8-oxo-dG / 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA or HPLC (Chepelev et al, 2015; Valavanidis et al, 2009).</p>
<p>Lipid peroxides decompose to form malondialdehyde (MDA) and 4, hydroxynonenal (4-HNE), natural bi-products of lipid peroxidation. Lipid peroxidation can be monitored by thiobarbituric acid (TBA) reactive substances in biological samples. MDA and TBA form MDA-TBA adduct in a 1:2 stoichiometry and detected by colorimetric or fluorometric measurement.</p>
<p> </p>
<p>4. Detection of Nrf2 activity</p>
<p>Measuring Nrf2 activity involves assessing its transcriptional activity or protein abundance.</p>
<p>Several methods can be employed to detect Nrf2 activity, and these include:</p>
<p>a. Luciferase Reporter Assay:</p>
<p> - This widely used method involves creating a reporter plasmid containing Nrf2-responsive antioxidant response element (ARE) sequences and a luciferase gene.</p>
<p> - Cells of interest are transfected with the Nrf2-ARE luciferase reporter construct.</p>
<p> - After exposure to the stressor of interest, cells are lysed, and luciferase activity is measured.</p>
<p> - Assessing Nrf2 activity at the transcriptional level can be achieved through qPCR.</p>
<p> - Specific Nrf2 target genes (e.g., NQO1, HO-1) are selected and their mRNA levels are quantified.</p>
<p> - Increased expression of these genes is indicative of Nrf2 activation, while reduced expression suggests diminished Nrf2 activity.</p>
<p>c. Western Blotting:</p>
<p> - This method allows the detection of Nrf2 protein levels in cell or tissue samples.</p>
<p> - After exposure to a stressor, proteins are extracted, separated by electrophoresis, and transferred to a membrane.</p>
<p> - Specific antibodies against Nrf2 are used to detect its abundance.</p>
<p> - Increased Nrf2 protein levels suggest Nrf2 activation, while reduced levels indicate diminished activity.</p>
<p>d. Immunofluorescence:</p>
<p> - Immunofluorescence can be used to assess the cellular localization of Nrf2.</p>
<p> - Cells are fixed and probed with antibodies specific to Nrf2, followed by fluorescently labeled secondary antibodies.</p>
<p> - Nrf2 localization within the cell (e.g., cytoplasm or nucleus) can indicate its activation status.</p>
<p> - EMSA is a technique that measures the binding of Nrf2 to ARE sequences.</p>
<p> - Radioactively labeled ARE sequences are incubated with nuclear extracts, and the formation of DNA-protein complexes is visualized on a gel.</p>
<p> - The intensity of the complex can indicate Nrf2 binding activity.<br />
</p>
<h4>References</h4>
<p>Ashoka, A.H. et al. (2020), “Recent Advances in Fluorescent Probes for Detection of HOCl and HNO”, ACS omega, 5(4), 1730-1742. https://doi.org/10.1021/acsomega.9b03420.</p>
<p>Chepelev, N.L. et al. (2015), “HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues”, J Vis Exp, e52697-e52697, https://doi.org/10.3791/52697.</p>
<p>Hassan, S.M. et al. (2020), “The Nrf2 Activator (DMF) and Covid-19: Is there a Possible Role?”, Med Arch, 74(2), 134-138. https://doi.org/10.5455/medarh.2020.74.134-138.</p>
<p>Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan”, Toxicol Appl Pharmacol, 274, 63-77, https://doi.org/10.1016/j.taap.2013.10.019.</p>
<p>Lee, D.Y. et al. (2020), “PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood”, Theranostics, 10(5), 1997-2007. https://doi.org/10.7150/thno.39662.</p>
<p>Timpani, C.A, E. Rybalka. (2021), “Calming the (Cytokine) Storm: Dimethyl Fumarate as a Therapeutic Candidate for COVID-19.”, Pharmaceuticals, 14(1), 15. https://doi.org/10.3390/ph14010015.</p>
<p>Valavanidis, A. et al. (2009), “8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis”, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 27, 120-39. https://doi.org/10.1080/10590500902885684</p>
<p>The KE is applicable to broad species/life stage/sex. </p>
<h4>Key Event Description</h4>
<p>Coagulation is a process that responds to injury by the rapid formation of a clot. Activation of coagulation factor proteins are involved in coagulation. In the extrinsic pathway, platelets, upon the contact with collagen in the injured blood vessel wall, release thromboxane A2 (TXA2) and adenosine 2 phosphates (ADP), leading to the clot formation. Extravascular tissue factor (TF) binds to plasma factor VIIa (FVIIa) and promotes the activation of FXa. Activated FXa assembles with cofactors FVa and FVIIIa on the surface of aggregated platelets, which lead to generation of thrombin (FIIa). Thrombin catalyzes the production of fibrin (FG) which creates a clot.<br />
The binding of prekallikrein and high-molecular weight kininogen activate FXIIa in the intrinsic pathway.<br />
Many regulators are involved in coagulation system. Plasmin is one of the modulators required for dissolution of the fibrin clot. Plasmin is activated by tissue plasminogen activator (tPA) and urokinase plasminogen activation (uPA). SERPINs inhibit thrombin, plasmin and tPA. For example, SERPINE1 or plasminogen activator inhibitor-1 (PAI-1) inhibits tPA/uPA and results in hypofibrinolysis [Bernard I,et al. Viruses. 2021; 13(1):29.]. In addition, SERPING1 inhibits FXII, and thus down-regulation of SERPING1 lifts suppression of FXII of the intrinsic coagulation cascade [Garvin et al. eLife 2020;9:e59177]. Protein C, protein S and thrombomodulin degrade FVa and FVIIIa. [Ref. IPA, Coagulation System, version60467501, release date: 2020-11-19]</p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Coagulation and inflammatory parameters are measured in COVID-19 patients [Di Nisio et al. 2021]. Coagulation parameters include platelet count, prothrombin time, activated partial thromboplastin time, D-dimer, fibrinogen, antithrombin III [Di Nisio et al. 2021]. These parameters are measured in the blood.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Whole human blood model for testing the activation of coagulation and complement system, as well as clot formation [<span style="color:#222222">Ekstrand-Hammarström, B.</span> et al. <span style="color:#222222">Biomaterials 2015, 51, 58-68</span>, <span style="color:#222222">Ekdahl, K.N., et al.</span> <span style="color:#222222">Nanomedicine: Nanotechnology, Biology and Medicine 2018, 14, 735-744, Ekdahl, K.N., et al.</span> <span style="color:#222222">Science and Technology of Advanced Materials, 20:1, 688-698,]</span> </span></span></p>
<h4>References</h4>
<ol>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Bernard I, Limonta D, Mahal LK, Hobman TC. Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19. <em>Viruses</em>. 2021; 13(1):29. DOI: </span></span></span><a href="https://doi.org/10.3390/v13010029"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.3390/v13010029</span></span></span></a></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Garvin et al. A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm. <em>eLife</em> 2020;9:e59177. DOI: </span></span></span><a href="https://doi.org/10.7554/eLife.59177"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.7554/eLife.59177</span></span></span></a></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Di Nisio, Marcello et al. Interleukin-6 receptor blockade with subcutaneous tocilizumab improves coagulation activity in patients with COVID-19 European Journal of Internal Medicine, Volume 83, 34 - 38 DOI: </span></span></span><a href="https://doi.org/10.1016/j.ejim.2020.10.020"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.1016/j.ejim.2020.10.020</span></span></span></a></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Ekstrand-Hammarström, B.; Hong, J.; Davoodpour, P.; Sandholm, K.; Ekdahl, K.N.; Bucht, A., Nilsson, B. TiO2 nanoparticles tested in a novel screening whole human blood model of toxicity trigger adverse activation of the kallikrein system at low concentrations. </span></span></span><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Biomaterials 2015, 51, 58-68 DOI:</span></span></span><a href="https://doi.org/10.1016/j.biomaterials.2015.01.031"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.1016/j.biomaterials.2015.01.031</span></span></span></a></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Ekdahl, K.N.; Davoodpour, P.; Ekstrand-Hammarström, B.; Fromell, K.; Hamad, O.A.; Hong, J.; Bucht, A.; Mohlin, C.; Seisenbaeva, G.A.; Kessler, V.G., Nilsson, B. Contact (kallikrein/kinin) system activation in whole human blood induced by low concentrations of α-Fe2O3 nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine 2018, 14, 735-744 [DOI: </span></span></span><a href="https://doi.org/10.1016/j.nano.2017.12.008"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.1016/j.nano.2017.12.008</span></span></span></a><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">]</span></span></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9.0pt"><span style="background-color:white"><span style="color:#222222">Kristina N Ekdahl, Karin Fromell, Camilla Mohlin, Yuji Teramura & Bo Nilsson (2019) A human whole-blood model to study the activation of innate immunity system triggered by nanoparticles as a demonstrator for toxicity, Science and Technology of Advanced Materials, 20:1, 688-698, DOI: 10.1080/14686996.2019.1625721</span></span></span></span></span></li>
</ol>
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/1846">Event: 1846: Thrombosis and Disseminated Intravascular Coagulation</a></h4>
<td><a href="/aops/379">Aop:379 - Binding to ACE2 leading to thrombosis and disseminated intravascular coagulation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/395">Aop:395 - Binding of Sars-CoV-2 spike protein to ACE 2 receptors expressed on pericytes leads to disseminated intravascular coagulation resulting in cerebrovascular disease (stroke)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/377">Aop:377 - Dysregulated prolonged Toll Like Receptor 9 (TLR9) activation leading to Multi Organ Failure involving Acute Respiratory Distress Syndrome (ARDS)</a></td>
<p><span style="font-family:Times New Roman,Times,serif">Homo sapiens</span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Thrombosis is defined as the formation or presence of a thrombus. Clotting within a blood vessel may cause infarction of tissues supplied by the vessel. Extreme aggravation of blood coagulation induces multiple thrombi in the microvasculature, which leads to consumption coagulopathy followed by disseminated intravascular coagulation (DIC).</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">DIC is a pathological syndrome resulting from the formation of thrombin, subsequent activation and consumption of coagulation proteins, and the production of fibrin thrombi. The initial pathologic events are thrombotic in nature resulting in thrombotic vascular occlusions. The initial clinical events are usually hemorrhagic resulting in oozing from mucosa and massive gastrointestinal blood loss. The occlusive events occur as a result of fibrin microthrombi or platelet microthrombi that obstruct the microcirculation of organs. This obstruction can result in organ hypoperfusion and ischemia, infarction, and necrosis. All organs are potentially vulnerable to the effects of thrombotic occlusions.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">The renal effects of DIC are multifactorial and may be associated with hypovolemia or hypotension. If the hypotension is not corrected it may lead to renal failure due to acute tubular necrosis. Fibrin thrombi may also block glomerular capillaries causing ischemic, renal cortical necrosis (Colman, 1984).</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">The cerebral effects of DIC often result in nonspecific changes such as altered state of consciousness, convulsions, and coma. Major vascular occlusions, subarachnoid hemorrhage, multiple cortical and brain stem hemorrhages may occur following microvascular occlusions (Schwartzman RJ, 1982).</span></span></span></span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">The pulmonary effects of DIC may be caused by interstitial hemorrhage resulting in a clinical effect resembling acute respiratory distress syndrome (Schwartzman RJ,1973; Shahl RL, 1984).</span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Clinical laboratory tests are used to diagnose DIC.</span></span></span></span></span></p>
<p><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#202124">Prothrombin time</span></span></span></strong><span style="background-color:white; font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:#202124"> (<strong>PT</strong>) is a blood test that measures how long it takes blood to clot.</span></span></span><span style="background-color:white; font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:#2e2e2e"> PT measures the time required for fibrin clot formation after the addition of tissue thromboplastin and calcium.</span></span></span><span style="background-color:white; font-family:Calibri,sans-serif; font-size:11pt"> </span><span style="background-color:white; font-family:Calibri,sans-serif; font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:#111111">The average time range for blood to clot is about 10 to 13 seconds.</span></span></span></p>
<p><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Activated partial prothrombin time (APTT). </span></span></span></strong><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:#333333">Platelet poor plasma [PPP] is incubated at 37°C then phospholipid (cephalin) and a contact activator (e.g. Kaolin, micronized silica, or ellagic acid) are added. This leads to the conversion of Factor XI [FXI] to FXIa. The remainder of the pathway is not activated as no calcium is present. The addition of calcium (pre-warmed to 37°C) initiates clotting. The APTT is the time taken from the addition of calcium to the formation of a fibrin clot.</span></span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#333333"> The clotting time for the APTT lies between 27-35 seconds.</span></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Decreased fibrinogen concentrations</span></span></span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Diluted plasma is clotted with a high concentration of Thrombin. The tested plasma is diluted (usually 1:10 but this may vary if the Fibrinogen concentration is very low or very high) to minimize the effect of 'inhibitory substances' within the plasma e.g. heparin, elevated levels of FDPs. The use of a high concentration of Thrombin (typically 100 U/ml) ensures that the clotting times are independent of Thrombin concentration over a wide range of Fibrinogen levels.<br />
<br />
The test requires a reference plasma with a known Fibrinogen concentration and that has been calibrated against a known international reference standard. A calibration curve is constructed using this reference plasma by preparing a series of dilutions (1:5 –1:40) in the buffer to give a range of Fibrinogen concentrations. The clotting time of each of these dilutions is established (using duplicate samples) and the results (clotting time(s)/Fibrinogen concentration (g/L) are plotted on Log-Log graph paper. The 1:10 concentration is considered to be 100% i.e. normal. There should be a linear correlation between clotting times in the region of 10-50 sec.<br />
<br />
The test platelet-poor diluted plasma (diluted 1:10 in buffer) is incubated at 37°C, Thrombin [~100 U/mL] added (all pre-warmed to 37°C). The time taken for the clot to form is compared to the calibration curve and the Fibrinogen concentration deduced. Test samples whose clotting times fall out with the linear part of the calibration curve should be re-tested using different dilutions.<br />
<br />
Most laboratories use an automated method in which clot formation is deemed to have occurred when the optical density of the mixture has exceeded a certain threshold.</span></span></span></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Platelet Measurements-</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">A platelet count is the number of platelets a person has per microliter. The ideal platelet range is 150,000 – 400,000 per microliter in most healthy people.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Fibrinolysis measurements-</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">D-Dimer can be measured by a fluorescence immunoassay. To determine cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood and plasma specimens. The test is used as an aid in the assessment and evaluation of patients suspected of having disseminated intravascular coagulation or thromboembolic events including pulmonary embolism</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Procedure: </span></span></strong></span></span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Commercially available kits are available to measure d-dimer in whole blood or plasma. The kits contain all the reagents necessary for the quantification of cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood or plasma specimens.</span></span></p>
</td>
</tr>
</tbody>
</table>
<h4>Regulatory Significance of the AO</h4>
<p><span style="font-size:16px"><span style="font-family:"Times New Roman",serif">Thrombosis is one of the world’s main concerns in terms of severe symptoms or adverse responses of the vaccine for COVID-19 which is caused by SARS-CoV-2. Excess thrombosis leads to DIC, which might be mortal. For safely developing the therapeutics and vaccines of COVID-19, it is regulatory significant to understand the cellular and molecular mechanisms in the pathogenesis of coronaviral infection, which may include thrombosis and DIC, AO1846.</span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Hemostasis and Thrombosis Basic Principles and Clinical Practices Robert W Colman, Jack Hirsh, Victor J. Marder, Edwin W. Salzman (ed) Philadelphia, 1994.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Schwartzman RJ, Hill JB: Neurologic complications of DIC. Neurology 32:791, 1982</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Robboy SJ, Minna JD, Colman RW et.al. Pulmonary hemorrhage syndrome as a manifestation of DIC: Analysis of 10 cases. Chest 63:718, 1973.</span></span></span></span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#444444">Stahl RL, Javid JP, Lackner H: Unrecognized pulmonary embolism presenting as DIC. SM J Med 76:772, 1984.</span></span></span></p>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2056">Relationship: 2056: Binding to ACE2 leads to SARS-CoV-2 cell entry </a></h4>
<p>This KER applies to humans in all life stages and all sexes.</p>
<p> </p>
<p> </p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This KER deals with the evidence supporting the individual weight that the surface protein of SARS-CoV-2 spike needs to bind:ACE2, and of being cleaved in two different sites, for viral entry to occur. Viral entry is essential for initiating a cascade of events leading to COVID19.</span></span></p>
<p> </p>
<p> </p>
<p> </p>
<h4>Evidence Supporting this KER</h4>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:#0070c0">Binding of SARS-CoV-2 S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells) :</span></em></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers, and this may in part explain the susceptibility of many COVID-19 patients to severe respiratory distress (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2, thus highlighting the vulnerability of the nervous system to SARS-CoV-2 infection. Additionally, they also express transmembrane serine protease 2 (TMPRSS2) and furin, which facilitate virus entry into the host (Jakhmola et al. 2020).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">Once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. These cells express ACE-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (Jakhmola et al. 2020). Specifically in the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>Binding of S protein to ACE2 receptors present in the intestines</em></strong></span></span></span></p>
<strong>Biological Plausibility</strong>
<p>Upon binding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to angiotensin-converting enzyme 2 (ACE2) on the surface of the host cells, SARS-CoV-2 enters inside the cells with an internalization mechanism.</p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptor (Nature 579, 270–273, 2020 ; J. Virol. 94, e00127-20; Nature 588, 327–330). The <span style="color:#0e101a">SARS-CoV-2</span><strong><span style="color:#0e101a"> </span></strong>surface spike (S) protein mediates the binding to the receptor and requires 2 cleavage steps for viral entry to occur, as follows. The spike protein contains 1273 aminoacids divided into two subunits, S1 and S2. The subunits are cleaved by furin-like enzymes, as spike of sars-cov-2 contains an insertion <sup>680</sup>S<u>PRRA</u>R↓SV<sup>687</sup> forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. In addition, there is a second cleavage site <sup>808</sup>PSKPS<strong>KR</strong>|SFIEDL<sup>822</sup> just before the fusion peptide that needs to occur for viral entry. The S1 subunit contains a receptor-binding domain (RBD) encompassing the receptor-binding motif (RBM) that binds ACE2. The S2 contains a fusion peptide (FP), that penetrates into cell membranes and mediates fusion between the viral and host membranes to release viral proteins and genome. </span></span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-family:Arial, Helvetica, sans-serif"><span style="font-size:12px">When TMPRSS2 is not available, it is hypothesized that the virus may use alternative proteases to get in the cells either by fusion with the plasma membrane or entry via endosomes and fusion with endocytic membranes at low pH when proteases for priming become active, but the evidence is less robust (Jackson et al, 2022).</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">PFAS (PFOS, PFOA, PFNA, PFHxS, and GenX)</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Short-term (10 days), high dose (20 mg/kg/day) exposure to PFOA leads to about 1.6 fold upregulation of the pulmonary mRNA level of <em>Ace2</em> and to about 1.5 upregulation of the pulmonary mRNA level of <em>Tmprss2</em> in CD1 mice. [1]</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Long-term (12 weeks) of an environmentally relevant PFAS mixture (PFOS, PFOA, PFNA, PFHxS, and GenX; each in 2 mg/l concentration) exposure leads to downregulation of pulmonary mRNA expression of Ace2 2.5-fold in C57BL/6 J male mice. A similar decreasing trend was observed in PFAS-exposed male mice for <em>Tmprss2. </em>[2]</span></span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">female sex (XX chromosomes)</span></span></td>
<td>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">ACE2 localizes to the X sex chromosome and displays a sex-dependent expression profile with higher expression in female than in male tissues [1,2]. Estradiol inhibits TMPRSS2, needed to facilitate SARS-CoV-2 entry into the cell [3]</span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. Estrogen therapy has been shown to mitigate endoplasmic reticulum stress induced by SARS-CoV-2 invasion through activation of cellular unfold protein response and regulation of inositol triphosphate (IP3) and phospholipase C [4].</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Different studies have also illustrated that estradiol increases the expression of ADAM17, leading to high-circulating soluble ACE2 potentially neutralizing SARS-CoV-2 and preventing its binding to mACE2. [5] Thus, Estradiol might reduce SARS-CoV-2 infectivity through modulation of cellular ACE2/TMPRSS2/ADAM17 axis expression.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Male sex (XY chromosomes)</span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Androgen receptors (ARs) play a key role in increasing transcription of TMPRSS2. This may explain the predominance of males to COVID-19 fatality and severity. [6]</span></span></p>
<td><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">ACE2 protein expression is increased with aging in several tissues [1], including lungs and particularly in patients requiring mechanical ventilation [2]. During aging, telomere dysfunction activates a DNA damage response leading to higher ACE2 expression. Thus, telomere shortening could contribute to make elderly more susceptible to SARS-CoV-2 infection [3].</span></span></td>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222">Lipids, as important structural components of cellular and sub-cellular membranes, are crucial in the infection process [1]. Changes in intracellular cholesterol alter cell membrane composition, impacting structures such as lipid rafts, which accommodate many cell-surface receptors [2], including ACE2 and TMPRSS2 [3, 4]. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222"><strong>In COVID-19.</strong> In an<em> in vitro</em> study, the depletion of membrane-bound cholesterol in ACE2-expressing cells led to a reduced infectivity of SARS-CoV [3]. In vitro, higher cellular cholesterol increased uptake of SARS-CoV-2 S protein; this effect was decreased with Methyl-beta-cyclodextrin, a compound which extracts cholesterol from cell membranes [5]. HDL scavenger receptor B type 1 (SR-B1), a receptor found in pulmonary and many other cells, could facilitate ACE2-dependent entry of SARS-CoV-2 [6]. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="background-color:white"><span style="color:#222222"><strong>In COVID-19.</strong> ACE2 is highly expressed in adipose tissue, thus excess adiposity may drive more infection (8). Obese patients have more adipose tissue and therefore more ACE2-expressing cells [9]. SARS-CoV-2 dysregulates lipid metabolism in the host and the effect of such dysregulated lipogenesis on the regulation of ACE2, specifically in obesity [10]. Lung epithelial cells infected with SARS-CoV-2 showed upregulation of genes associated with lipid metabolism [11], including the SOC3 gene. A mouse model of diet-induced obesity showed higher Ace2 expression in the lungs, which negatively correlated with the expression sterol response element binding proteins 1 and 2 (SREBP) genes. Suppression of Srebp1 showed a significant increase in Ace2 expression in the lung. Lipids, including fatty acids, could interact directly with SARS-CoV-2 influencing spike configuration and modifying the affinity for ACE2 and thus its infectivity [12]. The dysregulated lipogenesis and the subsequently high ACE2 expression in obese patients might be one mechanism underlying the increased risk for severe complications [10]. </span></span></span></span></p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Vitamin D </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black">Vitamin D deficiency</span></span></span></p>
<p> </p>
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<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black">Vitamin D administration enhanced mRNA expression of VDR and ACE2 in a rat model of acute lung injury [</span>1<span style="color:black">]. In particular, vitamin D upregulates the soluble ACE2 form [</span>2<span style="color:black">]. Thus, low vitamin D status may impair the trapping protective mechanism of soluble ACE2 [</span>3<span style="color:black">]. Furthermore, vitamin D deficiency has been shown to reduce the expression of antimicrobial peptides (-defensin, cathelicidin), which act against enveloped viruses [</span>4,5<span style="color:black">]. </span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:black"><strong>In COVID-19. </strong>Decreased sACE2 and cellular viral defense might be some mechanisms explaining how low vitamin D modulate SARS-CoV-2 infectibility.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Gut dysbiosis (alteration of gut microbiota)</span></span></p>
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<div>
<div>
<div>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Some evidence shows that gut microbiota influences Ace2 expression in the gut. Colonic Ace2 expression decreased significantly upon microbial colonization in mice and rats [1,2]. <em>Coprobacillus</em> enrichment was associated with severe COVID-19 in patients [3] and was shown to upregulate colonic ACE2 in mice [4]. The abundance of<em> Bacteroides </em>species was associated with reduced ACE2 expression in the murine gut [4] and negatively correlated with fecal SARS-CoV-2 load [3,5]. Thus, gut dysbiosis might lead to higher levels of ACE2 in the gut, potentially increasing the ability of SARS-CoV-2 to enter enterocytes. </span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Polymorphisms inducing amino acid residue changes of ACE2 in the binding interface would influence affinity for the viral S protein. Evidence exists that K353 and K31 in hACE2, the main hotspots that form hydrogen bonds with the main chain of N501 and Q493 in receptor-binding motif respectively, play a role in tightly binding to the S protein of SARS-CoV-2 [</span>1<span style="color:black">]. Around the twenty natural ACE2 variants, three alleles of 17 variants were found to affect the attachment stability [</span>2<span style="color:black">]. Thus, the ACE2 variants modulating the interaction between the virus and the host have been reported to be rare, consistently with the overall low appearance of ACE2 polymorphisms. In this context, it is key to approach both the ACE2 genotypes and the clinical descriptions of the phenotypes in a population-wide manner, in order to better understand how ACE2 variations are relevant in the susceptibility for SARS-CoV-2 infection [</span>3<span style="color:black">]. In addition, since ACE2 is X-linked, the rare variants that enhance SARS-CoV-2 binding are expected to increase susceptibility to COVID-19 in males [</span>4<span style="color:black">]. On the other hand, the X-chromosome inactivation of the female causes a “mosaic pattern”, which might be an advantage for females in terms of reduced viral binding [</span>5<span style="color:black">]. TMPRSS2 single-nucleotide polymorphisms (SNPs) were associated with a frequent “European haplotype” [</span>6<span style="color:black">], which not observed in Asians, is suggested to upregulate TMPRSS2 gene expression in an androgen-specific way. Thus, there is a need for in vitro validation studies to assess the involvements of population-specific SNPs of both ACE2 and TMPRSS2 in susceptibility toward SARS-CoV-2 infection. The occurrence of a pandemic is related to the genetics of the infecting agent. In the case of SARS-CoV-2, through genomic surveillance it is possible to track the spread of SARS-CoV-2 lineages and variants, and to monitor changes to its genetic code that can influence viral entry and</span> <span style="color:black">production. Consequently, genomic surveillance is crucial to understand how mutations occurring on SARS-CoV-2 genome influence and drive the pandemic [</span>7<span style="color:black">]. For example, a recent study [</span>8<span style="color:black">] highlights that through genomic surveillance it is possible to trace co-infections by distinct SARS-CoV-2 genotypes, which are expected to have a different impact on factors modulating COVID-19. Genomic surveillance of SARS-CoV-2 is able to reveal tremendous genomic diversity [</span>9<span style="color:black">], and coupled with language models and machine learning approaches, contributes to predicting the impact of mutations (such as those occurring in the spike protein), and thus can better address challenging aspects, like an estimation of the efficacy of therapeutic treatments [</span>10<span style="color:black">].</span></span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:10.0pt"><span style="font-family:URWPalladioL-Roma"><span style="color:black"> </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Are monoclonal antibodies designed to recognize and attach to two</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">different sites of the Receptor-Binding Domain (RBD) of the S protein of SARS-CoV-2,</span></span></p>
<span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">blocking the virus to enter cells [1,2,3].</span></span></td>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">2) EMA Starts Rolling Review of REGN-COV2 Antibody Combination (Casirivimab / Imdevimab). EMA 2021. Available online:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(accessed on 12 May 2022)</span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">3) EMA Starts Rolling Review of Sotrovimab (VIR-7831) for COVID-19. EMA 2021. Available online:</span></span></p>
<span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">https://www.ema.europa.eu/en/news/ema-starts-rolling-review-sotrovimab-vir-7831-covid-19 (accessed on 12 May 2022)</span></span></td>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Interacts directly with viral</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">particles and has been shown to bind to the SARS-CoV-2 S1 Spike RBD, causing significant</span></span></p>
<p>Air pollution induces Increased expression of ACE2 which may result in increased viral entry and coronavirus production. </p>
<p>Increased ACE2 expression has been reported in the respiratory system in response to air pollution exposure (1-4). Increased expression may affect susceptibility to SARS-CoV-2 infection. Similarly, some constituents of air pollution (PM, ozone) have been reported to increase the expression of TMPRSS2 (3, 5-6). </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">ACE2 mRNA and protein levels, as well as enzymatic activity, were </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">shown to be upregulated in explanted hearts from patients with end-stage HF, as well </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">as in the HF rat model [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">1-3</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Myocytes, fibroblasts, vascular smooth muscle cells, pericytes [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">4</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and endothelial cells of the coronaries [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">5</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] express ACE2, while myocytes in patients suffering from heart disease exhibit higher ACE2 expression [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">6</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pericytes - the mural cells lining microvasculature, interacting with endothelial cells notably to maintain </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">microvascular stability - exhibited the strongest ACE2 expression in HF patients [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">7</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">], rendering these cells involved in the coronary vasculature of the myocardium, more susceptible to infection. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Furthermore, SARS-CoV-2 infects and replicates in pericytes, and a decrease in their numbers follows [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">8</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Patients with pre-existing HF showed increased ACE2 levels in myocytes and pericytes, having thereby higher risk of heart injury [</span><span style="color:#0875b8">7, 9</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">In addition, sACE2 levels are higher in HF patients [</span><span style="color:#0875b8">10, 11</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and sACE2 activity is increased in HF [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">12</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">In contrast to a protective role of sACE2, it has been proposed that viral binding to </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">circulating sACE2 forms SARS-CoV-2/sACE2 complexes, which might mediate infection of </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">cells in distal tissues [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">13</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">]; hence, pre-existing HF might disseminate SARS-CoV-2 infection.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Interestingly, the increase in sACE2 activity is associated with HF with reduced ejection fraction (HFrEF) but not with HF with preserved ejection fraction (HFpEF), suggesting (i) a rather complex role of HF in regulating ACE2-mediated infection by SARS-CoV-2 [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">10</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] and (ii) the potential of sACE2 activity to be used as a biomarker to distinguish between </span></span></span></span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">the two HF types. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Lastly, it is noteworthy that Khoury et al. provided evidence in a different direction, by showing that ADAM17 and TMPRSS2 [</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#0875b8">14</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">] expression levels are downregulated in a HF rat model, thus potentially conferring a protective role against infection by SARS-CoV-2 in HF [</span><span style="color:#0875b8">3</span></span></span><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">].</span></span></span></span></span></p>
<td>Chemicals in foods affect ACE3 expression</td>
<td>Chemicals in foods affect ACE2 expression</td>
<td>
<ul>
<li>Geranium and lemon oils were found to reduce in vitro ACE2 activity and expression, as well as ACE2 and TMPRSS2 mRNA levels [207].</li>
<li>Several molecular modelling and docking studies indicate the potential for compounds found in garlic [208], turmeric (curcumin) [209], thyme and oregano (carvacrol) [210], green tea [211] and other plant foods (quercetin) [212] to inhibit binding of SARS-CoV-2.</li>
<li>Pelargonidin, found in red and black berries, was shown to dose-dependently block SARS-CoV-2 binding to ACE2, reduce SARS-CoV-2 replication in vitro and reduce ACE2 expression [213].</li>
<li>Quercetin and related compounds inhibit recombinant human ACE2 activity [214] at physiologically relevant concentrations in vitro.</li>
<li>In a human crossover study, 30-day supplementation with resveratrol decreased ACE2 in adipose tissue [216], potentially attenuating an increased risk for infection and viral replication in humans with obesity. In vitro, resveratrol inhibited the replication of SARS-CoV-2 [217].</li>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">COVID19 References related to CNS:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
<p> </p>
<p><strong>References in general:</strong></p>
<p>Benton DJ, Wrobel AG, Xu P, Roustan C, Martin SR, Rosenthal PB, Skehel JJ, Gamblin SJ. Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature. 2020 Dec;588(7837):327-330. doi: 10.1038/s41586-020-2772-0.</p>
<p>Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol. 2022 Jan;23(1):3-20. doi: 10.1038/s41580-021-00418-x.</p>
<p>Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol. 2020 Mar 17;94(7):e00127-20. doi: 10.1128/JVI.00127-20.</p>
<p>Zhou, P., Yang, XL., Wang, XG. <em>et al.</em> A pneumonia outbreak associated with a new coronavirus of probable bat origin. <em>Nature</em> <strong>579</strong>, 270–273 (2020). doi: 10.1038/s41586-020-2012-7</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It has been shown that in human populations males are more likely to suffer severe infections and deaths due to COVID-19 than females. However, in the viral entry and infection phase, one study found that women of working age had higher infection rates than men, but the suggested cause was higher contact rates among women (Doerre and Doblhammer, 2022). Contact rate increase is an important transmission factor but would not constitute a gender-based biological difference in viral entry or IFN-I pathway antagonism. A biological basis for females having higher levels of Type I IFN has been proposed concerning Toll-like receptor (TLR) 7. TLR7 is expressed in plasmacytoid dendritic cells (pDCs), an immune cell type that on infection with SARS-CoV-2 migrates from peripheral blood into the respiratory tract epithelium. TLR7 stimulates higher IFN-I production in pDCs in women than in men (Van der Sluis et al. 2022). It is proposed that this is due to the TLR7 gene being on the X chromosome, and that X inactivation in males is incomplete regarding the TLR7 gene, creating a double gene-dose effect in females (Spiering and de Vries, 2021). In a mouse SARS-CoV model, XY males had more adverse outcomes than XX females and XXY males (Gadi et al. 2020). Additionally, loss-of-function TLR7 mutations have been identified that are associated with increased COVID-19 severity (Szeto et al. 2021). However, these results focus on disease outcome as the endpoint, where factors beyond the initial antiviral response could be involved. Also note that the nasal and upper respiratory tract (URT) epithelial cells express ACE2 receptors for SARS-CoV-2 entry while the pDCs do not, relying on viral endocytosis (Van der Sluis et al. 2022). There is not a clear picture in the literature of the timing of pDC arrival in the epithelium after exposure, and the role of TLR7 in sex differences is currently hypothetical (Spiering and de Vries, 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In a large study modelling URT viral load dynamics drawn from measurements in 605 human subjects, variations over 5 orders of magnitude in URT viral load from the time of symptom onset was not explained by age, sex, or severity of illness. Additionally, these variables did not explain modelling results concerning control of viral load by immune responses in the early (innate) or late (adaptive) phases (Challenger et al. 2022). Other sources also support that rate of infection and measured viral load does not differ by gender (e.g., Arnold et al. 2022; Qi et al. 2021; Cheemarla et al. 2021). Therefore, evidence exists that the components of cell entry and the early antiviral response are not influenced by gender specific differences such as sex hormone levels or sex chromosomes to the extent of affecting viral load. Elderly people are more susceptible to severe disease than children and young adults, but Challenger et al. (2022) found no evidence indicating that life stage increases infectability or early viral load generation. However, Sharif-Askari et al. (2022) reported that children had higher expression of IFN-I and associated ISGs than adults.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Generally, most mammals are likely susceptible to the SARS-CoV-2 virus based on reports of naturally and experimentally infected animals (See AO 1939). No infections have been reported in other classes of vertebrates. Other than bioinformatic studies on the ACE2 sequence across vertebrates however, there have been few studies on the mechanisms of susceptibility to infection of non-human hosts. Three studies were found on protein targets in the IFN-I innate immune response pathway that included other vertebrates. Rui et al. (2021) showed that SARS-CoV-2 3CLpro and ORF3a inhibit vertebrate (human, mouse, and chicken) STING ability to induce IFNβ promoter activity in a dose-dependent manner in HEK293T cells transfected with IFNβ-luciferase reporter plasmid vectors, together with tagged STING and cGAS vectors and increasing amounts of the SARS-CoV-2 3CLpro or ORF3a expression vectors. This study shows that the vulnerability of the host IFN-I pathway protein components to inhibition by SARS-CoV-2 protein stressors is not limited to humans, however Rui et al. (2021) did not determine the specific amino acids involved in the STING-ORF3a or STING-3CLpro interactions. Mostaquil et al. (2020) studied the cleavage site of IRF3 by PLpro (SARS-CoV-2 NSP3) and compared sequences across mammals. They determined that the IRF3 cleavage site in mammalian species in the taxonomic orders of primates, carnivora, artiodactyla, chiroptera (bats) and a few other mammals was conserved and would generally be susceptible to cleavage, and therefore IFN-I antagonism, but rodentia IRF3 would likely not be susceptible. Hameedi et al. (2022) compared molecular dynamic simulations of 3CLpro cleavage of NEMO in humans and mice showing a decrease in the average number of contacts between mNEMO and 3CLpro compared to hNEMO. Also, hNEMO may be more strongly bound to the catalytic site, and the mNEMO/3CLpro interaction appears more prone to destabilization (Hameedi et al., 2022).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px">Upon entry of a virus into the host cell (KE1738), the virus is unpackaged from the structural nucleocapsid (N), envelope (E), and membrane (M) proteins. <span style="font-family:Calibri"><span style="color:black">The viral RNA is detected by Pattern Recognition Receptor (PRR) proteins including RIG-I and MDA5</span></span> but the M proteins can interact with these PRRs directly, and block this initial host reaction (Fu et al., 2021). The viral genomic RNA can then be translated directly at the host ribosomes. The viral proteins are processed through cleavage by viral protease enzymes. This releases a repertoire of non-structural proteins (NSPs) and accessory open reading frame (ORF) proteins that has evolved, for example in the SARS-CoV-2 virus, to bind and block the proteins in the interferon I (IFN-I) antiviral cascade (KE1901). <span style="font-family:"Calibri",sans-serif">The normal function of the host’s IFN-I response to other viruses is the expression of IFN-I which in turn stimulates the expression of many interferon-stimulated gene (ISG) proteins with antiviral functions. The SARS-CoV-2 antagonism of the IFN-I pathway delays or curtails the expression of IFN-I and ISG proteins. </span></span></p>
<h4>Evidence Supporting this KER</h4>
<p>Empirical evidence supporting this relationship is described below.</p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This relationship is concerned with how entry of the virus into the host cell and subsequent release and transcription of viral proteins affects the downstream innate immune response. In particular, literature suggests the main pathway antagonized is the expression of type I interferons (IFN-I), consisting primarily of IFNα and IFNβ, and IFN-I stimulated genes (ISGs) (Banerjee et al., 2020; Blanco-Melo et al., 2020; Cheemarla et al., 2021; Xia et al., 2020; Sharif-Askari et al., 2022). Although there are few studies with evidence for cell entry leading directly to reduced IFN expression (Xia et al., 2020; Hatton et al. 2021), several studies demonstrate individual viral protein interactions with and blocking of host proteins in the IFN-I pathway or ISG proteins (Schubert et al. 2020; Thoms et al. 2020; Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Mostaqil et al., 2021; Xia et al. 2020; Quarleri and Delpino, 2021; Xia and Shi, 2020; Miorin et al. 2020; Kato et al. 2020; Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020; see below and also key event 1901). These studies provide the biological rationale that SARS-CoV-2 entry into the host cell causes interactions between viral proteins and known protein components of the host IFN-I antiviral response, resulting in inhibition of IFN-I and ISG expression.</span></span></p>
<p> </p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Empirical evidence in support of temporal concordance comes from patient reports, showing that interferon expression is delayed by SARS-CoV-2 compared to other viruses like influenza, which is also described as an untuned or imbalanced response between interferons being initially low in moderate to severe cases (Banco-Melo et al. 2020; Galani et al., 2021; Hadjadj et al., 2020; Hatton et al., 2021; Rouchka et al., 2021). This indicates that SARS-CoV-2 stressors are suppressing the interferon response and highlights an important point regarding the difference between SARS-CoV-2 and other viruses in the stressors produced upon viral entry. Other viruses, as well as non-viral compounds used in research (e.g., polyinosinic:polycytidylic acid or poly[I:C]) enter the cell and stimulate the normal functional operation of the immune response, while SARS-CoV-2 blocks the response at multiple points, acting as a true prototypical stressor. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hatton et al. (2021) used human nasal epithelium differentiated at the air-liquid interface (ALI) cultures (organoids) with several cell types. Secretory cells were the cell type with the highest expression of viral transcripts, with ciliated and deuterosomal cells also showing expression. The SARS-CoV-2-infected secretory and ciliated cells also had many downregulated ISGs. Compared to SARS-CoV-2, influenza A virus induced significantly higher levels of IFN-I (IFNβ) and IFN-III (IFNλ1) at 6 and 24 hours post infection, as well as ISGs Ubiquitin specific peptidase 18 (USP18), radical s-adenosyl methionine domain containing 2 (RSAD2), and ubiquitin-like protein ISG15 at 24 hours post infection (Hatton et al., 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Individual stressors from the virus were investigated by Xia et al. (2020) using an IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> promoter luciferase assay. HEK293T cells were co-transfected with luciferase reporter plasmids, the specific viral protein expressing plasmid, and stimulator plasmid RIG-I (2CARD). Of the viral proteins tested (NSPs 1, 2, 4-16, S, N, E, M, and ORFs 3a, 3b, 6, 7a, 7b, 8, and 10), four proteins (NSPs 1, 6, and 13 and ORF6) significantly reduced INF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β induction compared to the control (empty vector). A similarly conducted ISRE-promoter luciferase assay showed significant inhibition of the IFN-I signaling pathway (normally resulting in induction of ISGs) by NSPs 1, 6, 7, 13 and 14, ORFs 3a, 6, </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">7a and 7b, and M protein (Xia et al., 2020). See Xia et al. (2020) and Xia and Shi (2020) for schematics depicting the actions of the SARS-CoV-2 proteins on the protein components of the IFN-I antiviral response pathway.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 stressor proteins and the IFN-I pathway responses were investigated individually in the following studies:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reductions in IFNβ mRNA induction were seen when SARS-CoV-2 N protein was co-transfected into A549 cells with RIG-I, MAVS, or TBK1, and similar transfections resulted in IFNβ promoter activity reduction in poly(I:C)-stimulated HEK293T cells (Chen et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sun et al. (2022) determined that SARS-CoV-2 and avian coronavirus infectious bronchitis virus (IBV) NSP3 PLpro N-terminal domain directly interacts with MDA5 to inhibit IFNβ expression when co-transfected in HEK293T cells.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fu et al. (2020) found M interaction with MAVS (as determined by coimmunoprecipitation and in vitro pull-down assay) interferes with recruitment of downstream pathway proteins TRAF, TBK1, and IRF3, inhibiting IFNβ1 promoter, IFN-stimulated response element (ISRE), and NFκB promoter activity in a dose-dependent manner. The M protein inhibited the transcription of ISGs (ISG56, CXCL10, and TNF) based on mRNA levels, and inhibited IFNβ and TNFα secretion based on measures of these proteins in HEK293 cell culture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shin et al. (2020) generated a crystal structure and found that SARS-CoV-2 Plpro preferentially cleaves ISG15. ISG15 functions in antiviral immunity to directly inhibit viral replication (Perng and Lenschow, 2018).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between TOMM70 and ORF9b via affinity purification-mass spectrometry (AP-MS). TOMM70-ORF9b interaction is supported by several studies (Gao et al., 2021; Brandherm et al., 2021; Ayinde et al., 2022). Jiang et al. (2020) used a dual luciferase reporter assay to show human IFN-β promoter activity was significantly reduced in the presence SARS-CoV-2 Orf9b compared to controls.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between ORF6 and the host Nup98-RAE1 protein pair via AP-MS. The interaction was confirmed by Miorin et al., 2020 and Li et al., 2021 (see crystal structures). Miorin et al. (2020) also demonstrate that upon treatment with recombinant IFN-I in HEK293T cells, Nup98 binding to SARS-CoV-2 Orf6 blocks translocation of STAT1 into the nucleus, resulting in suppression of ISRE-dependent gene expression.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Using co-immunoprecipitation, Xia et al. (2020) showed that ORF6 selectively bound with KPNA2. Expression of ORF6 blocked nuclear translocation of IRF3, suggesting that ORF6 inhibited IFN-β production by binding to KPNA2 to block IRF3 nuclear translocation.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Biswal et al. (2022) solved the X-ray crystal structure of the G3BP1 N-terminal nuclear transport factor 2-like domain bound to the first intrinsically disordered region of SARS-CoV-2 N protein.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The interaction of the N-terminus of ORF9b with NEMO upon viral infection interrupts its K63-linked polyubiquitination, thereby inhibiting viral-RNA-induced IFNβ1 activation in HEK293T cells in an ORF9b-dose-dependent manner (Wu et al., 2021)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hameedi et al. (2022) solved the X-ray crystal structure of 3CLpro bound to NEMO and characterized 3CLpro cleavage of NEMO. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kilkenny et al., 2021 demonstrate that components of the host DNA polymerase α (Pol α)–primase complex or primosome directly bind with SARS-CoV-2 NSP1. They also provide a cryo-electron microscopy structure of NSP1 bound to the primosome. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Schubert et al. (2020) provide cryo-EM structures of NSP1 bound to the 40S ribosome subunit, inhibiting translation of host proteins.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sui et al. (2022) show that NSP13 recruits TBK1 to an aggregation of ubiquitinated proteins (p62) for autophagic degradation, resulting in inhibition of IFNβ production, and that NSP13 impaired IRF3 luciferase reporter activity induced by TBK1 in a dose-dependent manner. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia et al. (2020) co-transfected HEK293T cells with plasmids containing TBK1 and either nsp6 or nsp13. Only NSP13 inhibited TBK1 phosphorylation, and did so in a dose-dependent manner, but both NSP6 and NSP13 suppressed IRF3 phosphorylation. Both NSP6 and NSP13 bind TBK1, as shown by co-immunoprecipitation. NSP6 binds to TBK1 without affecting TBK1 phosphorylation but this decreases IRF3 phosphorylation, while NSP13/TBK1 binding inhibits TBK1 phosphorylation. In both cases, IFN-β production is reduced (Xia et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rui et al. (2021) SARS-CoV-2 ORF3a and 3CLpro inhibited IFNβ promoter activity through cyclic GMP-AMP synthase (cGAS)-STING pathways, specifically through interaction with STING, as indicated by co-immunoprecipitation. 3CLpro also bound to STING and specifically inhibited K63-ubiquitin-mediated modification of STING, which is required for signaling and downstream expression of IFN-I. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mostaquil et al. (2020) showed with a fluorescent-based cleavage assay that NSP3 (Plpro) cleaves IRF-3, and thereby reduces IRF-3 available for induction of IFN-I expression.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>STAT1/STAT2</strong>: Signal transducer and activator of transcription</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mu et al. (2020) used Sendai virus (SeV)-induced ISRE-promoter activation via the luciferase reporter assay to determine that SARS-CoV-2 N protein can inhibit the phosphorylation of STAT1 and STAT2 resulting in decrease in ISG production. They also showed through co-immunoprecipitation that N interacts with both STAT1 and STAT2, and that N inhibits STAT1/2 phosphorylation by blocking interactions with kinases including JAK1.</span></span></p>
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</tbody>
</table>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">There are uncertainties based on differing disease outcomes, especially associated with timing of IFN increase or suppression under different cell culture circumstances and in different people infected with SARS-CoV-2. Effectiveness of IFN treatment is still uncertain due to some studies evaluating IFN along with other drugs (Sodeifian et al., 2021).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are ISGs that have been implicated in SARS-CoV-2 entry as well as antiviral activity (Prelli Bozzo et al., 2021), in addition to the fact that the SARS-CoV-2 entry receptor ACE2 is an IFN-I stimulated gene (Ziegler et al., 2020). These are some of the paradoxes that confound transcriptomic studies that determine up- or downregulation of IFNs and ISGs in response to infection, and responses are highly dependent on the time points sampled. Efforts to address uncertainties around when and under what circumstances IFNs and ISGs either promote or supress the virus are ongoing. </span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>The current quantitative understanding of this relationship is described below.</p>
<strong>Response-response relationship</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">A specific titer of virus can be used for infection, but as shown by Hatton et al. (2021), different cell types may express different levels of the actual stressors (viral protein transcripts). Because there are many stressors from each viral particle, which might be differentially expressed and also differentially inhibit each of their targets, a consistent whole viral entry dose leading to IFN-I or ISG response is difficult to measure. However, Chen et al. (2020), Xia et al. (2020), Fu et al. (2021), Wu et al. (2021) and Sui et al., (2022) all showed that individual protein stressor components of SARS-CoV-2 reduced IFN-I expression in a dose-dependent manner. </span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (infector) and secondary case (infectee). V</span></span><span style="font-size:14px">iral entry leading to antagonism of the IFN response occurs d</span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">uring t</span></span><span style="font-size:14px">he latent period of the disease prior to symptom onset. </span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Latent period calculation is based on serial interval and median pre-symptomatic infectious period: Serial interval 5.2 days (Rai et al. 2021) – 2.5 days pre-symptom infectious period (Byrne et al. 2020) equals approximately 2.7 days. The latent period was longer in asymptomatic cases (4-9 days); pre-symptomatic transmission occurs from about 3 days after exposure to symptom onset at about day 5-7, viral load peaks from about day 5-7 to day 9-11, and the host can remain infectious to symptom clearance or death (Byrne et al. 2020).</span></span><span style="font-size:14px"> As noted, IFN administered prior to exposure or within the latent period window can stop replication. However, IFN administered too late, in the inflammatory stage (post-symptom onset), led to long-lasting harm and worsened disease outcome (Sodeifian et al., 2022). </span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In a study using a primary nasal cell model (differentiated at air-liquid interface), the virus did not proliferate beyond the limit of assay detection if treated with IFN beta or lambda 16 hours prior to infection, and virus was significantly reduced in cultures treated 6h post-infection compared to untreated cultures. Treatments 24h post infection were not significantly different from untreated controls for either type of IFN (Hatton et al., 2021)</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In humans, the viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (infector) and secondary case (infectee). <span style="font-size:14px">V</span></span></span><span style="font-size:14px">iral entry leading to antagonism of the IFN response occurs d</span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">uring t</span></span><span style="font-size:14px">he latent period of the disease prior to symptom onset. </span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Latent <span style="font-size:14px">period</span> calculation is based on serial interval and median pre-symptomatic infectious period: Serial interval 5.2 days (Rai et al. 2021) – 2.5 days pre-symptom infectious period (Byrne et al. 2020) equals approximately 2.7 days. The latent period was longer in asymptomatic cases (4-9 days).</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<strong>Known modulating factors</strong>
<div><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Genetic mutations.</strong> Autoimmunity to IFNs has been found in some COVID-19 patients. These individuals produce autoantibodies that attack IFN (Bastard et al., 2021 and 2022), which may be associated with human leukocyte antigen (HLA) gene mutations (Ku et al., 2016; Chi et al., 2013). Zhang et al., 2020 note inborn errors (genetic mutations) in IFN-I immunity that result in severe COVID-19, but some are also genes for proteins involved in the initial response (TBK1, IRF3, NEMO, IFNAR1, IFNAR2, STAT1, and STAT2). Zhang et al. (2022) also found similar mutations (STAT2 and IFNAR1) in children with COVID-19 pneumonia.</span></span></div>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Pollutant exposures.</strong> Most studies have been conducted with the endpoints to determine whether prior or concurrent exposure to chemical or air particulate pollutants exacerbates COVID-19 symptoms resulting in <em>more severe disease or higher mortality rates.</em> This would point to effects downstream of viral replication usually relating to antibody suppression, inflammation and organ/tissue damage. Fewer studies can be found that study pollutant effects on <em>susceptibility to infection</em>, which are relevant to this KER, specifically cell entry or interferon response antagonism. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Marques et al., (2022) reviews associations between COVID-19 and outdoor air pollutants including PM<sub>2.5</sub>, PM<sub>10</sub>, O<sub>3</sub>, NO<sub>2</sub>, SO<sub>2</sub> and CO, reporting that environmental air pollution increases both disease incidence and severity. Physiological mechanism is not investigated for most studies. One relevant study estimated significant odds ratios for increased risk of severe COVID-19 and gene transcriptional analysis showing downregulation of genes associated with the IFN-I pathway in patients with high short-term NO<sub>2</sub> exposure (Feng et al., 2023).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Per- and polyfluoroalkyl substances (PFAS) are a large group of contaminants of current concern, due to their potential for toxicity, ubiquitous presence in the environment and consumer products, as well as their resistance to degradation. Although most community exposure to PFAS is through diet and drinking water, airborne and dermal exposures may also occur, especially in the workplace (CDC/NIOSH 2022). Statistical links between high measured serum or urine concentrations of specific PFAS compounds or mixtures and higher rates of COVID-19-positive cases have been found. One study in Sweden calculated a sex- and age-Standardized Incidence Ratio (SIR) for the town of Ronneby that had highly PFAS-contaminated drinking water compared to a demographically matched town with background PFAS levels (Nielsen et al. 2021). Serum PFAS concentrations were previously measured in 2014-15 for 3507 participants (Xu et al. 2021), after the Ronneby drinking water contamination issue was identified in 2013. Ronneby residents had higher infection risk, with a SIR of 1.19 [95% CI: 1.12-1.27]. Ji et al. (2021) measured urine and serum in a smaller study in China with 160 subjects. They reported statistically significant odds ratios for infection of 1.94 [95% CI: 1.39–2.96] for perfluorooctane sulfonate (PFOS), 2.73 [1.71–4.55] for perfluorooctanoic acid (PFOA), and 2.82 [1.97–3.51] for Σ (12) PFASs, after controlling for age, sex, body mass index (BMI), comorbidities, and urine albumin-to-creatinine ratio (UACR). These odds of infection were clearly higher even though the PFAS-exposed subjects in China had serum concentrations lower than in the Ronneby study participants. Additionally, the risk of infection was similar for residents in a significantly more contaminated section of Ronneby compared with a less contaminated section, so there was no dose-response relationship (Nielsen et al. 2021). However, these associations warrant more study to determine causality. Ji et al. (2021) also found elevated PFAS to be associated with altered mitochondrial metabolism. A potential consideration is that inhibition of mitochondrial oxidative phosphorylation impairs MAVS-mediated induction of IFNs, indicating the coordination between antiviral response and mitochondrial metabolism (Yoshizumi et al., 2017). Another study proposes </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">modulation of ACE2 and TMPRSS2 expression in the lungs of </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">PFAS-exposed mice may play a role in PFAS-associated immune suppression </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yang et al. 2022). Houck et al., (2022)</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">report testing 147 PFAS substances in screening platforms including the BioMAP® Diversity PLUS panel, which is used to model complex tissue adverse effects of pharmaceuticals and environmental chemicals.</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Toxicity Signatures within the BioMAP profile indicated the Skin Rash (MEK-Associated) Signature for PFOA, with IFN</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">α/</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> as one of the target mechanisms.</span></span> <span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">While not specific to COVID-19, one study found that exposure to aryl hydrocarbons and dioxins may block IFN production (Franchini and Lawrence, 2018).</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 uses the host ACE2-receptor for entry, upon which the host IFN response could upregulate ACE2 to enhance infection (Ziegler et al., 2020), a positive feedback loop for viral entry, while the IFN response also induces antiviral protein expression to help restore homeostasis as a positive feedback loop to KE 1901.</span></span></p>
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<h4><a href="/relationships/2497">Relationship: 2497: IFN-I response, antagonized leads to SARS-CoV-2 production</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Sex.</strong> In a large study modelling URT viral load dynamics drawn from measurements in 605 human subjects, variations over 5 orders of magnitude in URT viral load from the time of symptom onset was not explained by age, sex, or severity of illness. Additionally, these variables did not explain modelling results concerning control of viral load by immune responses in the early (innate) or late (adaptive) phases (Challenger et al. 2022). Other sources also support that rate of infection and measured viral load does not differ by gender (e.g., Arnold et al. 2022; Qi et al. 2021; Cheemarla et al. 2021). This evidence suggests that the components of the early antiviral response are not influenced by gender specific differences such as sex hormone levels or sex chromosomes to the extent of affecting viral load.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Life Stage. </strong>To apply to this KER, studies would need to show differences in IFN or ISGs correlated with viral load and differing by age. Saheb Sharif-Askari et al. (2022) reported that children had higher expression of IFN-I and associated ISGs than adults, but did not measure viral loads. Euser et al. (2021) found that SARS-CoV-2 viral loads increase with age, but did not measure IFN or ISGs. Literature that connects the two factors for age in humans was not found.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Taxonomic.</strong> No non-mammalian vertebrates have been found to become infected with SARS-CoV-2. Many mammals have tested positive and several are known to shed and transmit the virus, however the prevalent aspects of non-human mammalian infection and transmission found in the literature are ACE2 binding capacity and measures of viral load. For the few species for which IFN is mentioned in the literature (Mostaquil et al., 2020; Rui et al., 2021; Hameedi et al., 2022), the potential IFN antagonism is not linked to resulting increase in viral replication, except in the golden hamster, <em>Mesocricetus auratus</em> (Hoagland et al., 2021). The hamsters were Infected with SARS-CoV-2 resulting in high levels of virus in the upper and lower respiratory tracts and an </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">IFN-I response that was not sufficient to control COVID-19 progression. Direct contact resulted in inoculated hamsters transmitting the virus to naïve hamsters. When intranasal IFN-I was administered to the hamsters, viral replication was reduced and transmission was prevented (Hoagland et al., 2021). For bats, IFN and ISGs are constitutively expressed and therefore may contribute to immune tolerance and lack of replication of SARS-CoV-2 in many bat species (Irving et al., 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">). Differential susceptibility and viral shedding has been found across mammalian species (EFSA/Nielson et al., 2023), and it is likely that differences in IFN-I response may be involved. Therefore, more studies are needed in diverse taxa to assess the tDOA for IFN-I antagonism leading to increase in SARS-CoV-2 replication across the potentially susceptible species.</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The normal function of the host’s innate immune response to viruses is the expression of interferons (IFN) which in turn stimulates the expression of many interferon-stimulated gene (ISG) proteins with antiviral functions (Amor et al., 2020; Harrison et al., 2020). ISGs generally function to inhibit viral replication (Yang and Li, 2020). The SARS-CoV-2 antagonism of the IFN-I pathway delays or curtails the expression of IFN-I and ISG proteins. This results in the downstream event, SARS-CoV-2 production, increased. The increase in SARS-CoV-2 viral production can be measured as viral load, which can contribute to both transmission to new hosts and more severe disease. This KER details the specific ISGs that inhibit viral replication, and demonstrates the difference in how SARS-CoV-2 negates the function of these proteins or delays their expression compared to other viruses to successfully increase its numbers.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<p>See below.</p>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The functional relationships between the upstream IFN-I antagonism and downstream increase in SARS-CoV-2 viral replication is biologically plausible via the suppression of IFN through interaction inhibition of the host pathway proteins by viral proteins. This in turn would lead to suppressing the expression of ISGs that have been demonstrated to inhibit replication. The effects of ISGs on viral replication has been demonstrated for several viruses (Schoggins et al., 2011). SARS-CoV-2 replication may be impacted by different ISGs than other families of viruses. A gain-of-function analysis evaluating the impacts of ISGs on SARS-CoV-2 viral replication (Martin-Sancho et al., 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">) showed that a specific subset of ISGs when stably overexpressed in cultured human cells infected with SARS-CoV-2 controlled viral infection, including RNA binding proteins that suppress viral RNA synthesis and ISGs inhibiting viral assembly and egress. Therefore, the lack of these ISGs due to antagonism of the IFN-I pathway leads to increased viral replication.</span></span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Evidence from patients who contracted COVID-19 supports the relationship between IFN antagonism and viral production:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Busnadiego et al. (2020) found that different IFNs upregulate ACE2 to differing degrees, but all IFNs elevated ISGs and inhibited SARS-CoV-2 replication in a dose-dependent manner. Some people have developed autoimmunity toward their own IFN proteins (Bastard et al., 2021; Lopez et al., 2021). They produce autoantibodies that block even exogenously administered IFN, and this has resulted in more severe COVID-19 disease in these patients. Also, Zhang et al., (2020) note inborn errors (genetic mutations) in IFN-I immunity that result in severe COVID-19.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cheemarla et al. (2021) used patient nasopharyngeal samples and airway epithelial organoids. COVID-19 patient samples had upregulated ISG RNAs in the upper respiratory tract. SARS-CoV-2 replicated exponentially when unchecked, doubling in 6 h. ISGs rose with viral replication and peaked as viral load declined. Rhinovirus infection before SARS-CoV-2 exposure caused ISG induction to accelerate and stopped SARS-CoV-2 replication, while blocking ISG induction increased viral replication.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hadjadj et al. (2020) report a phenotype in severe COVID-19 patients with no IFNβ, low IFNα, persistent blood viral load and exacerbated inflammatory response.</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Hatton et al. (2021) use human nasal epithelium differentiated at the air-liquid interface (ALI) cultures (organoids) to show delayed induction of IFN-I and -III in SARS-CoV-2 compared to influenza A virus. They found that exogenous IFNs administered pre-exposure or early in infection controlled SARS-CoV-2 replication.</span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schuhenn et al. (2022) found that differential immune signatures of IFNα subtypes suppress SARS-CoV-2 infection by treating primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection. The most effective antiviral subtype was IFNα5, against both in vitro and in vivo infected mice, and additive effects with the antiviral drug remdesivir in cell culture.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rouchka et al., (2021) found that there is not only wide variation in nasopharyngeal viral loads in COVID-19 patients early in infection, but also that viral loads were strongly correlated with host gene expression associated with IFNα-inducible cellular antiviral response genes (ISGs). Also, patients with mild symptoms were often found to have a higher viral load than those with severe disease, indicating lack of correlation between susceptibility to severe disease, and susceptibility to viral replication.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In review articles, Yang and Li (2020) and Samuel (2023) discuss the relationship between the IFN antiviral response and viral replication. Yang et al. focus on ISGs with multiple mechanisms that inhibit viral replication by sensing, degrading, or repressing expression of viral RNA. These ISGs may use a variety of co-factors, which indicates the highly complex nature of the type I IFN response. Samuel et al. report that overall genetic variability of both SARS-CoV-2 and the human host affect the IFN response, and viral replication is in turn sensitive to variation in IFN antiviral action.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">These studies point out inconsistencies in quantity and type of IFN expression or administration in patients and COVID-19 disease outcome, but confirming the link between IFN-I response and viral replication. There is uncertainty in the fact that several IFN-I pathway components have been variously implicated. Because many different IFN subtypes and subsequently many different ISGs and cofactors may be involved, not only the specific repertoire of ISGs expressed may differ among individuals, but also the quantity of each ISG may influence viral production.</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11.0pt"><span style="background-color:white"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">The current quantitative understanding of this relationship is described below.</span></span></span></span></p>
<strong>Response-response relationship</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Busnadiego et al., (2020) found an inverse, linear relationship between IFNβ or IFNλ1 concentration and viral titer, measured as plaque forming units (PFU) in primary human bronchial epithelial cells (BEpCs) differentiated and grown at an air-liquid interface (ALI). However, the upstream event of IFN antagonism is not represented by administered IFN but by antagonism of the IFN response, and does not answer the question of what dose of antagonist results in increased viral replication in a host system, where viral replication is not normal biology. Comparatively, difference in IFN expression between cells infected with influenza A virus vs. SARS-CoV-2 showed significantly higher IFNβ and IFNλ1 for influenza at both 6 and 24 hours post-infection, but this was not tied to relative viral production (Hatton et al., 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The key event of IFN-I response antagonism encompasses a broad range of stressors and targets: 1) viral proteins interacting with pathway proteins leading to IFN expression, 2) the IFN subtypes that induce the expression of ISGs, and 3) the variation in type and amount of ISGs expressed, which also varies with cell/tissue type. Viral replication related to these factors is also dependent on the dose of virus to which the individual host is exposed and the genetic make-up and overall condition of that individual. These factors may explain the variable results in IFN dose-viral production response determination, and why the actual response-response relationship for this KER, between the viral dose resulting in antagonism and viral replication increase, have not been determined. Saheb Sharif-Askari et al., 2022 concluded that more mechanistic studies are needed to quantify the amount of early IFN required to overcome SARS-CoV-2 antagonism and prevent replication. Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analog of double-strand RNA (dsRNA) that can stimulate IFN production. The use of poly(I:C) administered before and during SARS-CoV-2 infection in mice increased ISGs and lowered viral loads (Tamir et al., 2022) but was administered at different time points rather than at different dose concentrations. Poly(I:C) dosing may be a potential method to quantify the IFN stimulation needed to overcome SARS-CoV-2 antagonism.</span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The viral entry MIE and early KEs coincide with the time from exposure to symptoms, within which are the latent period, or time from exposure to infectiousness, and the serial interval, or the time interval between the onset of symptoms in the primary (index) and secondary (contact) case. Pre-symptomatic transmission occurs from about 3 days after exposure to symptom onset at about day 5-7, viral load peaks from about day 5-7 to day 9-11, and the host can remain infectious to symptom clearance or death (Byrne et al. 2020). IFN administered prior to exposure or within the latent period window can stop replication (Sodeifian et al., 2021). In a study using a primary nasal cell model (differentiated at air-liquid interface), the virus did not proliferate beyond the limit of assay detection if treated with IFN beta or lambda 16 hours prior to infection, and virus was significantly reduced in cultures treated 6h post-infection compared to untreated cultures. Treatments 24h post infection were not significantly different from untreated controls for either type of IFN (Hatton et al., 2021). This would suggest that viral antagonism of IFN occurring during the first 24h post viral entry allows viral loads to be generated likely concurrently, reaching transmissible levels within 72h post viral entry.</span></span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">IFN has been the subject of studies for potential therapeutic value to enhance the antiviral response. However, IFN administered too late, in the inflammatory stage (post-symptom onset), led to long-lasting harm and worsened disease outcome (Sodeifian et al., 2021). </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Therapeutics used in COVID-19 patients tend to target either the ACE2 binding, downstream inflammatory response, or viral replication via inhibition of the viral RNA-dependent, RNA polymerase to block viral genome replication (i.e., Remdesivir) (Narayanan and Parimon, 2022). No other therapeutics were found to be relevant to this KER, i.e., specifically targeted to IFN components or ISGs leading to supressed viral replication (see WHO 2021 and Terracciano et al., 2021).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It is known that per- and poly-fluorinated alkyl substances (PFAS), air pollutants, and other environmental chemicals are implicated in SARS-CoV-2 susceptibility and COVID-19 disease severity (Marques et al., 2022; Nielsen et al., 2021; Xu et al., 2021). However, it is currently unknown whether or how the mechanisms of action are related to blocking IFN components or ISGs, leading to viral replication.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Genetic factors are of importance to this KER: Autoantibodies against IFN, as noted, block even exogenously administered IFN, resulting in more severe disease (Quarleri and Delpino, 2021</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">;</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Bastard et al., 2021; Busnadiego et al., 2020; Lopez et al., 2021). There are 15 known clinically recessive and inborn errors of type I IFN immunity (Zhang et al., 2022). Four of these including X-linked recessive TLR7 deficiency, and autosomal recessive IFNAR1, STAT2, or TYK2 deficiencies were found in children with moderate to critical pneumonia due to COVID-19. Zhang et al. (2022</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> also reported enhanced SARS-CoV-2 replication measured as expression of viral nucleocapsid (N-protein) in STAT2- and TYK2-deficient patients’ cells.</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">RIG-Like Receptors (RLRs) including MDA5 are Pattern Recognition Receptors (PRRs) that recognize Pathogen-Associated Molecular Patterns (PAMPs) like viral RNA and start signalling cascades to express IFNs. These PRRs and other proteins in the pathway, including STAT1 and STAT2 involved in transcription of the ISGs, are also regulated by IFN, and therefore are themselves ISGs (Yang and Li, 2020). As RNA from most viruses is detected, signalling to express more ISGs increases, and more IFN is expressed (Michalska et al., 2018). However, SARS-CoV-2 inhibits these and other components of the IFN pathway to delay expression of ISGs, and viral production goes unchecked, actually disrupting the normal antiviral positive feedback loop. In fact, SARS-CoV-2 can co-opt another ISG, interferon-induced transmembrane protein 2 (IFITM2), for efficient replication in human lung, heart, and gut cells (Nchioua et al., 2022), which might also be considered a positive feedback loop (i.e., the more IFITM2 is expressed, the more the virus replicates). However, IFITM2 and 3 have also shown antiviral activity toward SARS-CoV-2 (Shi et al., 2021), therefore the conflicting results require more research. </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xu, Y., Nielsen, C., Li, Y., Hammarstrand, S., Andersson, E. M., Li, H., Olsson, D. S., Engström, K., Pineda, D., Lindh, C. H., Fletcher, T., & Jakobsson, K. (2021). Serum perfluoroalkyl substances in residents following long-term drinking water contamination from firefighting foam in Ronneby, Sweden. <em>Environment International</em>, <em>147</em>, 106333. <a href="https://doi.org/10.1016/j.envint.2020.106333" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.envint.2020.106333</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yang, E., & Li, M. M. H. (2020). All About the RNA: Interferon-Stimulated Genes That Interfere With Viral RNA Processes. <em>Frontiers in Immunology</em>, <em>11</em>, 605024. <a href="https://doi.org/10.3389/fimmu.2020.605024" style="color:blue; text-decoration:underline">https://doi.org/10.3389/fimmu.2020.605024</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., Bastard, P., Liu, Z., Le Pen, J., Moncada-Velez, M., Chen, J., Ogishi, M., Sabli, I. K. D., Hodeib, S., Korol, C., Rosain, J., Bilguvar, K., Ye, J., Bolze, A., Bigio, B., Yang, R., Arias, A. A., Zhou, Q., Zhang, Y., … Zhang, X. (2020). Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. <em>Science</em>, <em>370</em>(6515), eabd4570. <a href="https://doi.org/10.1126/science.abd4570" style="color:blue; text-decoration:underline">https://doi.org/10.1126/science.abd4570</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., Matuozzo, D., Le Pen, J., Lee, D., Moens, L., Asano, T., Bohlen, J., Liu, Z., Moncada-Velez, M., Kendir-Demirkol, Y., Jing, H., Bizien, L., Marchal, A., Abolhassani, H., Delafontaine, S., Bucciol, G., COVID Human Genetic Effort, Abel, L., Abolhassani, H., … Casanova, J.-L. (2022). Recessive inborn errors of type I IFN immunity in children with COVID-19 pneumonia. <em>Journal of Experimental Medicine</em>, <em>219</em>(8), e20220131. <a href="https://doi.org/10.1084/jem.20220131" style="color:blue; text-decoration:underline">https://doi.org/10.1084/jem.20220131</a></span></span></p>
</div>
<div>
<h4><a href="/relationships/2358">Relationship: 2358: SARS-CoV-2 production leads to Diminished Protective Response to ROS</a></h4>
<p>The KER applies to species including <em>Homo sapiens</em> which have ACE2 receptors to bind to SARS-CoV-2 and protective responsive system to ROS, as ROS scavenging system.<br />
The KER has relatively broad applicability among <em>Homo sapiens</em>.<br />
</p>
<h4>Key Event Relationship Description</h4>
<p>ROS is generated upon the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19), and induces oxidative stress (Janardhan VV and Kalousek V, 2020). SARS-CoV-2 infection induces cytokine storm (Frisoni P et al., 2021, Kaidashev I et al., 2021). Cytokine storm includes ROS-induced oxidative stress and immune cell dysregulation. Glutathione S-transferase genes, which have functions in the elimination of ROS, involves the morbidity and mortality from COVID-19 (Kaidashev I et al., 2021). The heme oxygenase-1 (HO-1) induction may be involved in the inflammation-induced coagulation in COVID-19 (Kaidashev I et al., 2021). ROS quenching by vitamin C, E, beta-carotene and polyphenols has been suggested in COVID-19 in the point of view of the nutrient, since oxidative stress causes inflammation (Iddir M et al., 2020). Potential roles of omega-3 fatty acids accompanied by antioxidants have been suggested in the cytokine storm due to SARS-CoV-2 infection (Rogero MM, 2020).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Increased coronavirus production increase in angiotensin II (Ang II). Ang II increases ROS production via NADPH oxidase activation (Nishida et al., 2005). Coronavirus pathogenesis pathway is involved in molecules in production of reactive oxygen species (ROS), oxidative stress responses (Tanabe, 2021). The fixation of SARS-CoV-2 in angiotensin-converting enzyme 2 (ACE2) receptor results in reduction of AC2 activity, leading to dysfunction of the renin-angiotensin system (RAS) and excessive production of pro-inflammatory and pro-oxidant agents (Ramdani and Bachari, 2022). </p>
<strong>Empirical Evidence</strong>
<p>Nrf2 activator, dimethyl fumarate(DMF), which was recently approved for the treatment of multiple sclerosis, and is a potent anti-oxidant and anti-inflammatory agent inhibiting the NF-kappaB pathway through binding and activation of the IKKbeta at Cys-179, inhibited SARS-CoV-2 entry (Hassan et al., 2020). As SARS-CoV-2 is attached to ACE2, ACE2 is not available within the renin-angiotensin system to convert Ang II to angiotensin-(1,7) resulting in Ang II to accumulate. Ang II stimulates membrane-bound NADPH oxidase, which in turn generate ROS and oxidative stress (Janardhan et al., 2020). Resveratrol, a natural compound possessing anti-oxidant properties by trapping ROS, has a potential to inhibit SARS-CoV-2 infection (Ramdani and Bachari, 2020, van Brummelen and van Brummelen, 2022).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The transient up-regulation of ROS may serve as an inhibitory factor of pathogen (Zhu et al., 2021).</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Quercetin inhibits one of the main proteases highly conserved among coronaviruses, SARS-CoV-2 3CLpro (Ki = ~7 uM) (Abian, Ortega-Alarcon et al. 2020).</p>
<strong>Response-response relationship</strong>
<p>Angiotensin II dose-dependently increases ROS.</p>
<p>Angiotensin II dose-dependently increases ROS (Nishida, Tanabe et al. 2005).</p>
<strong>Time-scale</strong>
<p>Catalytic activity can be measured in 10-100 sec, or 10-40 min.</p>
<p>Catalytic activity of SARS-CoV-2 3CLpro or inhibitory effect of the SARS-CoV-2 3CLpro can be measured in 10-100 sec or 10-40 min <em>in vitro</em> (Abian, Ortega-Alarcon et al. 2020).</p>
<td>Lower level of selenium and higher levels of reactive oxygen species are observed in COVID-19 patients.</td>
<td>Šķesters et al. 2023</td>
</tr>
<tr>
<td>Vitamin D</td>
<td> </td>
<td> </td>
<td> </td>
<td>Nutrient</td>
<td>Vitamin D supplementation reduce the number of virus particles that could attach to ACE2 and enter the cell.</td>
<td>Iddir M et al. 2020</td>
</tr>
</tbody>
</table>
</div>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>Fibrin may induce oxidative stress.</p>
<h4>References</h4>
<p>Abian, O., D. Ortega-Alarcon, A. Jimenez-Alesanco, L. Ceballos-Laita, S. Vega, H. T. Reyburn, B. Rizzuti and A. Velazquez-Campoy (2020). "Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening." International Journal of Biological Macromolecules 164: 1693-1703.</p>
<p>Frisoni P, Neri M, D’Errico S, Alfieri L, Bonuccelli D, et al. (2021) Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1β, IL-6, IL-15 and TNF-α. Forensic Sci Med Pathol. 31:1-15.</p>
<p>Hassan, S. M., M. J. Jawad, S. W. Ahjel, R. B. Singh, J. Singh, S. M. Awad and N. R. Hadi (2020). "The Nrf2 Activator (DMF) and Covid-19: Is there a Possible Role?" Med Arch 74(2): 134-138.</p>
<p>Iddir M, Brito A, Dingeo G, Fernandez Del Campo SS, Samouda H, et al. (2020) Strengthening the Immune System and Reducing Inflammation and Oxidative Stress through Diet and Nutrition: Considerations during the COVID-19 Crisis. Nutrients. 12(6):1562.</p>
<p>Janardhan, V., V. Janardhan and V. Kalousek (2020). "COVID-19 as a Blood Clotting Disorder Masquerading as a Respiratory Illness: A Cerebrovascular Perspective and Therapeutic Implications for Stroke Thrombectomy." Journal of Neuroimaging 30(5): 555-561.</p>
<p>Kaidashev I, Shlykova O, Izmailova O, Torubara O, Yushchenko Ya, et al. (2021) Host gene variability and SARS-CoV-2 infection: A review article. Heliyon. 7(8): e07863.</p>
<p>Nishida, M., S. Tanabe, Y. Maruyama, S. Mangmool, K. Urayama, Y. Nagamatsu, S. Takagahara, J. H. Turner, T. Kozasa, H. Kobayashi, Y. Sato, T. Kawanishi, R. Inoue, T. Nagao and H. Kurose (2005). "G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes." J Biol Chem 280(18): 18434-18441.</p>
<p>Ramdani, L.H., K. Bachari. (2020). "Potential therapeutic effects of Resveratrol against SARS-CoV-2 " Acta virologica 64.</p>
<p>Rogero MM. (2020) Potential benefits and risks of omega-3 fatty acids supplementation to patients with COVID-19. Free Radic Biol Med. 156:190-99.</p>
<p>Šķesters A, Lece A, Kustovs D, Zolovs M. Selenium Status and Oxidative Stress in SARS-CoV-2 Patients. Medicina (Kaunas). 2023 Mar 8;59(3):527. doi: 10.3390/medicina59030527.</p>
<p>Tanabe S. (2021) Involvement of Reactive Oxygen Species (ROS) and Coagulation in Coronaviral Infection. Adv Clin Med Res. 2(2):21.</p>
<p>van Brummelen, R. and A. C. van Brummelen (2022). "The potential role of resveratrol as supportive antiviral in treating conditions such as COVID-19 – A formulator’s perspective." Biomedicine & Pharmacotherapy 148: 112767.</p>
<p>Zhu, Z., Z. Zheng and J. Liu (2021). "Comparison of COVID-19 and Lung Cancer via Reactive Oxygen Species Signaling." Front Oncol 11: 708263.</p>
<p> </p>
<p> </p>
</div>
<div>
<h4><a href="/relationships/2359">Relationship: 2359: Diminished Protective Response to ROS leads to Coagulation</a></h4>
<p>ROS are oxygen-derived molecules that oxidize molecules or are converted into oxygen radicals (André-Lévigne D, et al., 2017). ROS have dual-effects which are cell damaging or beneficial roles (André-Lévigne D, et al., 2017, Beckman KB and Ames BN, 1998, Bedard K and KH Krause, 2007). ROS generated by NOX2, a NADPH oxidase, in macrophage play an important role in killing of phagocytosed microorganisms (Bedard K and KH Krause, 2007). The ROS accumulation cause mitochondrial dysfunction which leads to coagulopathy associated with inflammatory signaling pathways (Saleh J et al., 2020). Polymorphonuclear leucocytes, commonly referred to as neutrophils, generate large amounts of ROS via the NADPH oxidase complex (Barrett CD et al., 2018).</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify">The ROS released from the polymorphonuclear leucocytes following trauma and haemorrhagic shock led to lung injury and coagulopathy (Barrett CD et al., 2018). Serpin family A member 1 (SERPINA1/alpha-1-antitrypsin), a serine protease inhibitor, inhibits coagulation factor 2a (thrombin) (Cohen AB., 1973). SERPINA1 is a member of low-density lipoprotein (LDL) and involved in ROS network (Lubrano V, and Balzan S. 2020). ROS are required for release of granzyme B (GzmB), a cytotoxic lymphocyte protease, into the cytosol (Mangan MS et al., 2016). SERPINA1 is converted into a ROS-sensitive granzyme B (GzmB) inhibitor by replacing the P4-P3’ reactive center loop residues (Mangan MS et al., 2016). Thrombin activates NADPH oxidase and produces ROS, which leads to fibroblast proliferation (Zhou SY et al., 2010). Endothelial exposure of thrombin induces NOX-dependent superoxide superoxide anion and hydrogen peroxide (Pai WY et al., 2017, Holland JA et al., 1998).</p>
<strong>Empirical Evidence</strong>
<p>The presence of ROS assists in the transformation of a circulating, non-oxidized, circular-shaped beta2-glycoprotein 1 into an oxidized J-shape, which binds to antiphospholipid antibodies such as anticardiolipin, lupus anticoagulant, and anti-beta2-GP1 antibodies (Janardhan et al., 2020). Domain V of beta2gP1 binds with the phospholipid layer of platelets or endothelial cells via Annexin (Janardhan et al., 2020).</p>
<p>Nrf2, a basic leucine zipper transcription factor, plays a crucial role in cellular defense against oxidative stress by inducing the expression of cytoprotective and detoxifying genes. <a href="https://biosignaling.biomedcentral.com/articles/10.1186/s12964-022-00906-3" target="_blank">Activation of Nrf2 has protective roles against cancer onset and progression, while persistent activation can lead to malignant progression, chemo/radio resistance, and poor prognosis</a> Pouremamali, F., et al. (2022).</p>
<p>Nrf2’s involvement in inflammatory responses and erythrocyte homeostasis suggests a potential impact on blood coagulation. <a href="https://seikagaku.jbsoc.or.jp/10.14952/SEIKAGAKU.2021.930674/data/index.html" target="_blank">Additionally, the interaction between Nrf2 and NF-κB, and the increase in inflammatory factors due to Nrf2 deficiency, highlight the complex role of Nrf2 in cellular processes</a> (Motohashi et al., 2021).</p>
<p>Keap1, a major regulator of Nrf2, ubiquitinates Nrf2 under basal conditions, targeting it for proteasomal degradation in the cytoplasm. <a href="https://cancerci.biomedcentral.com/articles/10.1186/s12935-022-02660-5" target="_blank">This regulation is crucial for maintaining cellular homeostasis and responding to oxidative stress</a><span style="font-size:small"> </span><span style="font-size:16px">(Khodakarami et al., 2022)</span>.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Considering the effect of Nrf2 inactivation on blood coagulation, it is hypothesized that inactivation could lead to increased inflammatory responses, potentially promoting blood coagulation. However, this relationship requires further research for confirmation.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>Fibrin may induce oxidative stress.</p>
<h4>References</h4>
<p>André-Lévigne D, Modarressi A, Pepper MS, Cuenod BP. (2017) Reactive Oxygen Species and NOX Enzymes Are Emerging as Key Players in Cutaneous Wound Repair. Int J Mol Sci. 18(10):2149.</p>
<p>Barrett CD et al., (2018) Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy. Clin Exp Immunol. 194(1):103-117.</p>
<p>Beckman KB, BN Ames. (1998) The free radical theory of aging matures. Physiol Rev. 78(2):547-81.</p>
<p>Bedard K, KH Krause. (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 87(1):245-313.</p>
<p>Cohen AB. (1973) Mechanism of action of alpha-1-antitrypsin. J Biol Chem. 248(20):7055-9.</p>
<p>Holland JA, Meyer JW, Donnell RW, Johnson DK, Ziegler LM. (1998) Thrombin Stimulated Reactive Oxygen Species Production in Cultured Human Endothelial Cells. Endothelium. 6(2):113-121.</p>
<p>Janardhan, V., V. Janardhan and V. Kalousek (2020). "COVID-19 as a Blood Clotting Disorder Masquerading as a Respiratory Illness: A Cerebrovascular Perspective and Therapeutic Implications for Stroke Thrombectomy." Journal of Neuroimaging 30(5): 555-561.</p>
<p>Janardhan, V., V. Janardhan and V. Kalousek. (2020) COVID-19 as a Blood Clotting Disorder Masquerading as a Respiratory Illness: A Cerebrovascular Perspective and Therapeutic Implications for Stroke Thrombectomy. Journal of Neuroimaging 30(5): 555-561.</p>
<p>Khodakarami A. (2022) The molecular biology and therapeutic potential of Nrf2 in leukemia. Cancer Cell International. 22:241.</p>
<p>Lubrano V, S Balzan. (2020) Role of oxidative stress-related biomarkers in heart failure: galectin 3, α1-antitrypsin and LOX-1: new therapeutic perspective? Mol Cell Biochem. 464(1-2):143-152.</p>
<p>Mangan MS, Bird HS, Kaiserman D, Matthews AY, Hitchen C, et al. (2016) A Novel Serpin Regulatory Mechanism: SerpinB9 is reversibly inhibited by vicinal disulfide bond formation in the reactive center loop. J Biol Chem. 291(7):3626-38.</p>
<p>Motohashi H. (2021) <a href="https://cancerci.biomedcentral.com/articles/10.1186/s12935-022-02660-5" target="_blank">NRF2によるストレス応答と硫黄代謝制御.</a> Journal of Japanese Biochemical Society 93(5): 674-683 . </p>
<p>Pai WY, Lo WY, Hsu T, Peng CT, Wang Hj. (2017) Angiotensin-(1-7) Inhibits Thrombin-Induced Endothelial Phenotypic Changes and Reactive Oxygen Species Production via NADPH Oxidase 5 Downregulation. Front Physiol. 8:994.</p>
<p>Pouremamali F, et al. (2022) An update of Nrf2 activators and inhibitors in cancer prevention/promotion. Cell Communication and Signaling, 20, Article number: 100.</p>
<p>Saleh J, Peyssonaux, Singh KK, Edeas M. (2020) Mitochondria and microbiota dysfunction in COVID-19 pathogenesis. Mitochondrion. 54:1-7.</p>
<p>Zhou SY, Xiao W, Pan XJ, Zhu MX, Yang ZH, et al. (2010) Thrombin promotes human lung fibroblasts to proliferate via NADPH oxidase/reactive oxygen species/extracellular regulated kinase signaling pathway. Chin Med J (Engl). 123(17):2432-9.<br />
</p>
<p>Zhou SY, Xiao W, Pan XJ, Zhu MX, Yang ZH, et al. (2010) Thrombin promotes human lung fibroblasts to proliferate via NADPH oxidase/reactive oxygen species/extracellular regulated kinase signaling pathway. Chin Med J (Engl). 123(17):2432-9.</p>
</div>
<div>
<h4><a href="/relationships/2360">Relationship: 2360: Coagulation leads to Diminished Protective Response to ROS</a></h4>
<p>The KER applies to Homo sapiens (Robea, Balmus et al. 2023), Mus musculus (Alabanza, Esmon et al. 2013).</p>
<h4>Key Event Relationship Description</h4>
<p>Blood coagulation through activation and recruitment of platelets following vessel-wall injury induces formation of thrombin through coagulation cascade, which induces reactive oxygen species generation by NOX enzymes in vascular cells (André-Lévigne D et al., 2017). Coagulation is a complex process that involves the formation of blood clots to prevent excessive bleeding. During injury or infection, tissue damage activates the coagulation cascade. Coagulation factors, such as tissue factor (TF), initiate clot formation by converting fibrinogen to fibrin. Simultaneously, inflammation is triggered, involving immune cells, cytokines, and chemokines. Reactive oxygen species (ROS) are produced during cell metabolism. ROS include radicals (e.g., superoxide anion, hydroxyl radical, nitric oxide) and non-radicals (e.g., hydrogen peroxide) and play dual roles: harmful (e.g., cell death pathways) and beneficial (e.g., microbial killing). Neutrophils and macrophages use ROS to kill engulfed pathogens. Chronic granulomatous disease (CGD), caused by NADPH oxidase deficiency, impairs ROS-dependent pathogen killing. ROS contribute to inflammation, signal transduction, cell migration, and gene expression. Hydrogen peroxide (H₂O₂) modulates gene expression via redox-based epigenetic modifications and transcriptional regulation. ROS are essential for immune system function. Coagulation and ROS intersect in immune responses, impacting both protective immunity and potential harm.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Coagulation induces extracellular ROS production in a C5a-dependent manner that contributes to organ injury (Barrett, Hsu et al. 2018). Coagulation balance that refers to the interaction between the procoagulant pathways specific for clot formation and hose mechanisms included in the fibrinolysis system influences dysregulation of homeostasis maintenance, which lead sot oxidative stress (Robea, Balmus et al. 2023).<br />
</p>
<strong>Empirical Evidence</strong>
<p>Inhibition of activated protein C, an anti-coagulant involved in the interactions between the coagulation and immune systems, induces decreases in peripheral CD4+ T cells, which induces immune suppression (Alabanza, Esmon et al. 2013).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Relationship between coagulation and diminished protective oxidative stress response is both directions and the amplification of the magnitude may be involved in feedback loop. There is uncertainty in terms of the relations in amount of the coagulation factors and reactive oxygen species.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p>Thrombin (50 mU/ml, 2 min) induced ROS production (Balykina, Naida et al. 2024). The flavonoid aglycones (100 uM, 30 min) such as luteolin, myricetin, quecetin, eriodictyol, kaempferol, and apigenin inhibited thrombin-induced ROS formation (Panth, Paudel et al. 2016, Jomova, Raptova et al. 2023).</p>
<strong>Time-scale</strong>
<p>Thrombin treatment for 2 min induced ROS production in human platelets (Balykina, Naida et al. 2024).</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>Increased ROS caused by diminished response to ROS causes oxidative stress and coagulation (Gutmann, Siow et al. 2020).</p>
<p>Increased oxidative stress has been reported in various cardiovascular-related diagnoses (Panth, Paudel et al. 2016, Ranneh, Ali et al. 2017, Jomova, Raptova et al. 2023).</p>
<h4>References</h4>
<p style="text-align:justify">Alabanza, L. M., N. L. Esmon, C. T. Esmon and M. S. Bynoe (2013). "Inhibition of endogenous activated protein C attenuates experimental autoimmune encephalomyelitis by inducing myeloid-derived suppressor cells." J Immunol 191(7): 3764-3777.</p>
<p style="text-align:justify">André-Lévigne D, Modarressi A, Pepper MS, Cuenod BP. (2017) Reactive Oxygen Species and NOX Enzymes Are Emerging as Key Players in Cutaneous Wound Repair. Int J Mol Sci. 18(10):2149.</p>
<p style="text-align:justify">Balykina, A., L. Naida, K. Kirkgöz, V. O. Nikolaev, E. Fock, M. Belyakov, A. Whaley, A. Whaley, V. Shpakova, N. Rukoyatkina and S. Gambaryan (2024). "Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases." Int J Mol Sci 25(9).</p>
<p style="text-align:justify">Barrett, C. D., A. T. Hsu, C. D. Ellson, Y. M. B, Y. W. Kong, J. D. Greenwood, S. Dhara, M. D. Neal, J. L. Sperry, M. S. Park, M. J. Cohen, B. S. Zuckerbraun and M. B. Yaffe (2018). "Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy." Clin Exp Immunol 194(1): 103-117.</p>
<p style="text-align:justify">Bassoy, E.Y.; Walch, M.; Martinvalet, D. Reactive Oxygen Species: Do They Play a Role in Adaptive Immunity? Frontiers in Immunology 2021, 12, doi:10.3389/fimmu.2021.755856.</p>
<p style="text-align:justify">Gutmann, C.; Siow, R.; Gwozdz, A.M.; Saha, P.; Smith, A. Reactive Oxygen Species in Venous Thrombosis. International Journal of Molecular Sciences 2020, 21, 1918.</p>
<p style="text-align:justify">Jomova, K.; Raptova, R.; Alomar, S.Y.; Alwasel, S.H.; Nepovimova, E.; Kuca, K.; Valko, M. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Archives of Toxicology 2023, 97, 2499-2574, doi:10.1007/s00204-023-03562-9.</p>
<p style="text-align:justify">Martinvalet, D.; Walch, M. Editorial: The Role of Reactive Oxygen Species in Protective Immunity. Frontiers in Immunology 2022, 12, doi:10.3389/fimmu.2021.832946.</p>
<p style="text-align:justify">Panth, N.; Paudel, K.R.; Parajuli, K. Reactive Oxygen Species: A Key Hallmark of Cardiovascular Disease. Advances in Medicine 2016, 2016, 9152732, doi:https://doi.org/10.1155/2016/9152732.</p>
<p style="text-align:justify">Ranneh, Y.; Ali, F.; Akim, A.M.; Hamid, H.A.; Khazaai, H.; Fadel, A. Crosstalk between reactive oxygen species and pro-inflammatory markers in developing various chronic diseases: a review. Applied Biological Chemistry 2017, 60, 327-338, doi:10.1007/s13765-017-0285-9.</p>
<p style="text-align:justify">Robea, M. A., I.-M. Balmus, I. Girleanu, L. Huiban, C. Muzica, A. Ciobica, C. Stanciu, C. D. Cimpoesu and A. Trifan (2023). "Coagulation Dysfunctions in Non-Alcoholic Fatty Liver Disease&mdash;Oxidative Stress and Inflammation Relevance." Medicina 59(9): 1614.</p>
</div>
<div>
<h4><a href="/relationships/2290">Relationship: 2290: Coagulation leads to Thrombosis and DIC</a></h4>
<p>This KER applies to Homo sapiens (Tsantes, Petrou et al. 2024).</p>
<h4>Key Event Relationship Description</h4>
<p>Many regulators are involved in coagulation system. Plasmin is one of the modulators required for dissolution of the fibrin clot. Plasmin is activated by tissue plasminogen activator (tPA) and urokinase plasminogen activation (uPA). SERPINs inhibit thrombin, plasmin and tPA. For example, SERPINE1 or plasminogen activator inhibitor-1 (PAI-1) inhibits tPA/uPA and results in hypofibrinolysis (Bernard I et al., 2021). In addition, SERPING1 inhibits FXII, and thus down-regulation of SERPING1 lifts suppression of FXII of the intrinsic coagulation cascade (Garvin et al., 2020). Protein C, protein S and thrombomodulin degrade FVa and FVIIIa. [Ref. IPA, Coagulation System, version60467501, release date: 2020-11-19]</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Overexpression of procoagulant molecules, among which tissue factor is the most well studied, induces thrombosis. Thrombotic complications include disseminated intravascular coagulation (DIC) (Tsantes, Petrou et al. 2024).</p>
<strong>Empirical Evidence</strong>
<p>Coagulation induced by activation of polymorphonuclear leucocytes leads to thrombosis and coagulopathy (Barrett, Hsu et al. 2018).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>It is unclear how the balance between coagulation and hemolysis is involved in the induction of thrombosis and DIC.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Not well known</p>
<strong>Response-response relationship</strong>
<p>Not well known</p>
<strong>Time-scale</strong>
<p>Not well known</p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p>Decreased fibrinolysis is involved in coagulation system. Coagulopathy induced by excess coagulation may be key for thrombosis and disseminated intravascular coagulation. (Mast AE et al., 2021, Garvin MR et al.,2020).</p>
<h4>References</h4>
<p><span style="font-size:14px"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#222222">Bernard I, Limonta D, Mahal LK, Hobman TC. Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19. <em>Viruses</em>. 2021; 13(1):29. DOI: </span></span><a href="https://doi.org/10.3390/v13010029"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.3390/v13010029</span></span></a></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#222222">Barrett, C. D., A. T. Hsu, C. D. Ellson, Y. M. B, Y. W. Kong, J. D. Greenwood, S. Dhara, M. D. Neal, J. L. Sperry, M. S. Park, M. J. Cohen, B. S. Zuckerbraun and M. B. Yaffe (2018). "Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy." Clin Exp Immunol 194(1): 103-117.</span></span></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#222222">Bernard I, Limonta D, Mahal LK, Hobman TC. Endothelium Infection and Dysregulation by SARS-CoV-2: Evidence and Caveats in COVID-19. <em>Viruses</em>. 2021; 13(1):29. DOI: </span></span><a href="https://doi.org/10.3390/v13010029"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.3390/v13010029</span></span></a></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#222222">Garvin et al. A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm. <em>eLife</em> 2020;9:e59177. DOI: </span></span><a href="https://doi.org/10.7554/eLife.59177"><span style="background-color:white"><span style="color:#1155cc">https://doi.org/10.7554/eLife.59177</span></span></a></span></span></p>
<p><span style="font-size:14px">Mast AE, Wolberg AS, Gailani D, Garvin MR, Alvarez C, Miller JI, Aronow B, Jacobson D (2021) SARS-CoV-2 suppresses anticoagulant and fibrinolytic gene expression in the lung. eLife 10:e64330. doi:10.7554/eLife.64330</span></p>
<p><span style="font-size:14px">Tsantes, A. G., E. Petrou, K. A. Tsante, R. Sokou, F. Frantzeskaki, A. Domouchtsidou, A. E. Chaldoupis, S. P. Fortis, D. Piovani, G. K. Nikolopoulos, N. Iacovidou, S. Bonovas, G. Samonis and A. E. Tsantes (2024). "Cancer-Associated Thrombosis: Pathophysiology, Laboratory Assessment, and Current Guidelines." Cancers (Basel) 16(11).</span></p>