AOP ID and Title:

Short Title: SARS-CoV-2 leads to acute respiratory distress

Authors

 

Young Jun Kim¹, Yongoh Lee¹, Jihun Yang², Chung Seok^2,3 Brigitte Landesmann⁴, Laure-Alix Clerbaux⁴ , Penny Nymark⁵, Jukka Sund⁶, Filipovska, Julija^7, Shihori Tanabe⁸, Gillian Bezemer⁹, Maria Joao Amorim¹⁰ and HyunJoon Kong¹¹ 

¹Korea Institute of Science and Technology (KIST) Europe, Saarbrücken 66123, Germany, ² NEXT&BIO, South Korea,  ³KU-KIST Graduate School of Converging Science and Technology, South Korea ⁴ F3 Chemical Safety and Alternative Methods Unit incorporating EURL ECVAM, Directorate F – Health, Consumers and Reference Materials Joint Research Centre, European Commission  ⁵ Institute of Environmental Medicine, Karolinska Institutet, Solna, Sweden ⁶ F.3 unit, EURL-ECVAM Joint Research Centre, European Commission ⁷ Independent,  North Macedonia  ⁸ Division of Risk Assessment, Center for Biological Safety and Research National Institute of Health Sciences, Japan ⁹ Impact Station, Universiteit Utrecht, Nederland ¹⁰ Instituto Gulbenkian de Ciência, Portugal ¹¹ University of Illinois at Urbana-Champaign, USA

Status

Abstract

Inhalation of substances, including viral particles,  the RNA virus capsid (S) glycoprotein binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. The S protein is cleaved into S1 and S2 units by a human cell-derived protease (proteolytic enzyme) that is assumed to be Furin.S1 units then bind to its receptor, ACE2. The other fragment, S2, is cleaved by TMPRSS2, a human cell surface serine protease, resulting in cell membrane fusion. The S protein binds the catalytic domain of ACE2 with high affinities likewise, COVID-19 shares 79.6% homology of SARS-CoV and 96% identical at the whole-genome level to a bat coronavirus. The binding of the coronavirus S protein to ACE2 triggers a conformational change in the S protein of the coronavirus, allowing for proteolytic digestion by host cell proteases called TMPRSS2. The AOP reports the S glycoprotein of viral capsid in complex with its host cell receptor ACE2 resulted in acute respiratory distress associated with mortality by cytokine storms and enhanced inflammation in pulmonary tissue. S-glycoprotein of the virus uses ACE2 to get into cells that are found on the surface of epithelial cells in Kidney, Heart, Liver and Lung. However, there is an unexplored relationship for ACE2 levels between fibrotic hypersensitivity and Renin-Angiotensin Pathway which caused acute respiratory distress associated with mortality.

Background

The ACE2 gene encodes the angiotensin-converting enzyme-2, which has been proved to be the receptor for both the SARS-coronavirus (SARS-CoV) and the human respiratory coronavirus. ACE2 is a key component of blood pressure regulation in the renin-angiotensin system. Angiotensin (Ang) converting enzyme 2 (ACE2) is a homolog of ACE.  ACE2 negatively regulates the renin-angiotensin system (RAS) by converting Ang II to Ang-(1-7) and AngI to Ang(1-9). The higher levels of receptor expression achieved by the expression of recombinant ACE2 could be relevant for cell-cell fusion. The underlying mechanisms remain to be elucidated and could play a role in the entry of the cell-free virus into cells and finally increase the acute respiratory distress associated with mortality.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

SARS-CoV

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2,https://doi.org/10.1371/journal.ppat.1007236

 Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P, Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu Y, Zheng D, Leibbrandt A, Wada T, Slutsky AS, Liu D, Qin C, Jiang C, Penninger JM (Aug 2005). "A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury". Nature Medicine. 11 (8): 875–9. doi:10.1038/nm1267. PMID 16007097.

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Considerations for Potential Applications of the AOP (optional)

This AOP not only contributes new tools to study entry of the viral particles or Inhalation of stressors into cells and localize its receptor-binding domain of ACE2 but also could serve in the development of novel vaccine immunogens against TMPRSS2 proteases which may inhibit cell entry of COVID-19.

References

1.  Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P, Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu Y, Zheng D, Leibbrandt A, Wada T, Slutsky AS, Liu D, Qin C, Jiang C, Penninger JM (Aug 2005). "A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury". Nature Medicine. 11 (8): 875–9. doi:10.1038/nm1267. PMID 16007097.
2. "What are the official names of the disease and the virus that causes it?". Q&A on coronaviruses. World Health Organization. Retrieved 22 February 2020.
3. Zhou P, Yang X (2020-02-03). "A Pneumonia Outbreak Associated With a New Coronavirus of Probable Bat Origin". Nature. doi:10.1038/s41586-020-2012-7. PMID 32015507.
4. Xintian, Xu; Chen, Ping (2020). "Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission". Science China Life Sciences. doi:10.1007/s11427-020-1637-5. PMID 32009228.
5. Understanding COVID-19 through adverse outcome pathways – 2nd CIAO AOP Design Workshop doi.org/10.14573/altex.2102221
6.  Lewis, Ricki. "COVID-19 Vaccine Will Close in on the Spikes". DNA Science Blog. Public Library of Science. Retrieved 22 February 2020.
7.  Walls, Alexandra; et al. (2020). "Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein". bioRxiv. bioRxiv. doi:10.1101/2020.02.19.956581. Retrieved 22 February 2020.
8. He L,et al (2006).Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS.J Pathol. 2006 Nov;210(3):288-97.​
9. Daniel Wrapp, Nianshuang Wang, Kizzmekia S. Corbett , Jory A. Goldsmith, Ching-Lin Hsieh , Olubukola Abiona , Barney S. Graham , Jason S. McLellan Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Wrapp et al., Science 367, 1260–1263 (2020)
10. Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H 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
11. Xie Xudong et al.Age- and gender-related difference of ACE2 expression in rat lung Life Sciences Volume 78, Issue 19, 4 April 2006, Pages 2166-2171
12. Kim Y et al. Advanced Adverse Outcome Pathways Potentially Bridging the Pathogenesis of COVID-19, 2021,  doi: 10.20944/preprints202101.0065.v1 
13. Penny Nymark et al. Systematic Organization of COVID-19 Data Supported by the Adverse Outcome Pathway Framework, 2021,doi: 10.20944/preprints202101.0573.v1
14. Mathieu Vinke,  A putative AOP for pneumonia related to COVID-19, 2020, Archives of Toxicology 94(9)

Appendix 1

List of MIEs in this AOP

Event: 1739: Binding to ACE2

Short Name: Binding to ACE2

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The KE is applicable to broad species/life stage/sex. The binding of ACE2 occure in the cells which express ACE2. 

Key Event Description

Angiotensin-converting enzyme 2 (ACE2) 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).

A table on ACE2 expression levels according to tissues (Kim et al.)

	Sample size	ACE2 mean expression	Standard deviation of expression
Intestine	51	9.50	1.183
Kidney	129	9.20	2.410
Stomach	35	8.25	3.715
Bile duct	9	7.23	1.163
Liver	50	6.86	1.351
Oral cavity	32	6.23	1.271
Lung	110	5.83	0.710
Thyroid	59	5.65	0.646
Esophagus	11	5.31	1.552
Bladder	19	5.10	1.809
Breast	113	4.61	0.961
Uterus	25	4.37	1.125
Protaste	52	4.35	1.905

ACE2 receptors in the brain (endothelial, neuronal and glial cells):

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.

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).

ACE2 receptors in the intestines

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).

 

 

How it is Measured or Detected

In vitro methods supporting interaction between ACE2 and SARS-CoV-2 spike protein

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,

Reference	ACE2 protein	SARS-CoV S	SARS-CoV2 S	Method	Measured Kd
doi:10.1126/science.abb2507	1–615 aa	306–577 aa		SPR	325.8 nM
			1–1208 aa		14.7 nM
doi:10.1001/jama.2020.3786	19–615 aa	306–527 aa		SPR	408.7 nM
			319–541 aa		133.3 nM
Lan et al., 2020	19–615 aa	306–527 aa		SPR	31.6 nM
			319–541 aa		4.7 nM
doi:10.1016/j.cell.2020.02.058	1–614 aa	306–575 aa		BLI	1.2 nM
			328–533 aa		5 nM
doi:10.1126/science.abb2507	1–615 aa	306–577 aa		BLI	13.7 nM
			319–591 aa		34.6 nM

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.

References

de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.

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.

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.

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.

Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.

Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.

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.

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

Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.

Xia, H. and Lazartigues, E.  Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010  Rep. 12 (3), 170– 175

List of Key Events in the AOP

Event: 1738: SARS-CoV-2 cell entry

Short Name: SARS-CoV-2 cell entry

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

TMPRSS2 vertebrates (Lam et al., 2020)

NRP1 in human & rodents (but also present in monkey and other vertebrates (Lu and Meng, 2015)

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 (Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018). 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 (Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020). However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors (Kawase et al., 2012, Hoffmann et al., 2020a). 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 (Lamers et al., 2020), as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors (Hoffmann et al., 2020b). Therefore it is critical to map which cells contain the different types of proteases.

In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:

• AT2 and clara cells of lungs
• sinusoidal endothelium, and hepatocyte of the liver,
• endocrine cells of the prostate,
• goblet cells , and enterocytes of the small intestine,
• intercalated cells, and the proximal tubular of the kidney.
• Ciliated, secretory and suprabasal of nasal
• spermatogonial stem cells of testes
• cyto tropoblast and peri vascular cells of placenta
• 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.

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).

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).

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).

 

NRP-1:

All life stages

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).

The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).

 

Sex Applicability:

TMPRSS2:

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).

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.

NRP-1:

For more information difference of NRP1 expression between male and female see https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue.

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).

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 (Moutal et al., 2020).

Key Event Description

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) (Hoffmann et al., 2020b). 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 (Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020).

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).

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 (Millet and Whittaker, 2018). 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 virus infection cycle: (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). 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 (Hoffmann et al., 2020b); or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L (Yang and Shen, 2020). 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 (Kawase et al., 2012). When TMPRSS2 (or other serine proteases such as TMPRSS4 (Zang et al., 2020) or human airway trypsin-like protease [HAT] (Bestle et al., 2020a)) is expressed, fusion of the virus with the cell surface membrane is preferred (Shirato et al., 2018), while in their absence, the virus can penetrate the cell by endocytosis (Kawase et al., 2012). 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) (Cantuti-Castelvetri et al., 2020). This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.

TMPRSS2 Spike cleavage:

TMPRSS2 (transmembrane serine protease 2, (https://www.ncbi.nlm.nih.gov/gene/7113) 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).

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).

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.

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). 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) (Hasan et al., 2020). The 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. 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 (Bestle et al., 2020b, Johnson et al., 2020).

Virus	S1/S2 site	S2’ site
SARS-CoV-2	TNSPRRAR|SVA	PSKPSKR|SFIEDL
SARS-CoV	S----LLR|STS	PLKPTKR|SFIEDL

Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like Calu-3 cells but not Huh7.5 and Vero E6 cells. 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).

 

Spike binding to neuropilin-1:

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.

NRP1 is a receptor for furin-cleaved SARS-CoV-2 spike peptide (Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020). 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 (Johnson et al., 2020). 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).

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‑CoV‑2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID‑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‑19 by enhancing the entry of SARS‑CoV‑2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory‑related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS‑CoV‑2 infection mediator implicated in the neurologic manifestations of COVID‑19. Accordingly, the neurotropism of SARS‑CoV‑2 via NRP1‑expressing cells in the CNS merits further investigation (Davies et al., 2020).

 

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).

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).

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).

More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1

Spike entry via lysosomal cathepsins and endocytosis:

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. Evidence came from studies 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) (Rossi et al., 2004, Simmons et al., 2005). 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.

Viral entry leads to delivery of virion proteins and translation of viral proteins immediately:

Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D, et al., 2020]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively that contain cis-acting secondary RNA structures essential for RNA synthesis [Huston N. C. et al., 2021]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., et al., 1985]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., et al., 2003]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., et al., 2018]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [Snijder E. J., et al., 2003], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [Sacco M. D. et al., 2020]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [Klein S., et al., 2021, Snijder E. J., et al., 2020, V'Kovski P. , et al., 2021]. 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.

How it is Measured or Detected

SARS-CoV2 entry can be determined by many different ways:

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.

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)].

TMPRSS2:

TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).

Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)

NRP-1:

Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:

1. 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 (Centers for Disease Control and Prevention, 2020, Daly et al., 2020).

Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020):

• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1).

• 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).

2. 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).

Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020):

• 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).

• 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.

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 (Perez-Miller et al., 2020)

References

BAUGHN, L. B., SHARMA, N., ELHAIK, E., SEKULIC, A., BRYCE, A. H. & FONSECA, R. 2020. Targeting TMPRSS2 in SARS-CoV-2 Infection. Mayo Clin Proc, 95, 1989-1999.

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. Life Science Alliance, 3.

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. Life Sci Alliance, 3.

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. J Virol 53, 834-840.

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. Science, 370, 856-860.

CAO, H., LI, Y., HUANG, L., BAI, B. & XU, Z. 2020. Clinicopathological Significance of Neuropilin 1 Expression in Gastric Cancer: A Meta-Analysis. Dis Markers, 2020, 4763492.

CENTERS FOR DISEASE CONTROL AND PREVENTION, U. S. D. O. H. A. H. S. 2020. Covid-19 Science Update 2020.

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. Cardiovasc Res, 116, 1097-1100.

DALY, J. L., SIMONETTI, B., KLEIN, K., CHEN, K. E., WILLIAMSON, M. K., ANTON-PLAGARO, C., SHOEMARK, D. K., SIMON-GRACIA, L., BAUER, M., HOLLANDI, R., GREBER, U. F., HORVATH, P., SESSIONS, R. B., HELENIUS, A., HISCOX, J. A., TEESALU, T., MATTHEWS, D. A., DAVIDSON, A. D., COLLINS, B. M., CULLEN, P. J. & YAMAUCHI, Y. 2020. Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science, 370, 861-865.

DAVIES, J., RANDEVA, H. S., CHATHA, K., HALL, M., SPANDIDOS, D. A., KARTERIS, E. & KYROU, I. 2020. Neuropilin1 as a new potential SARSCoV2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID19. Mol Med Rep, 22, 4221-4226.

DI, H., MCINTYRE, A.A., AND BRINTON, M.A. (2018). New insights about the regulation of Nidovirus subgenomic mRNA synthesis. Virology 517, 38-43.

GELFAND, M. V., HAGAN, N., TATA, A., OH, W. J., LACOSTE, B., KANG, K. T., KOPYCINSKA, J., BISCHOFF, J., WANG, J. H. & GU, C. 2014. Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding. Elife, 3, e03720.

HARTENIAN, E., NANDAKUMAR, D., LARI, A., LY, M., TUCKER, J. M. & GLAUNSINGER, B. A. 2020. The molecular virology of coronaviruses. J Biol Chem, 295, 12910-12934.

HASAN, A., PARAY, B. A., HUSSAIN, A., QADIR, F. A., ATTAR, F., AZIZ, F. M., SHARIFI, M., DERAKHSHANKHAH, H., RASTI, B., MEHRABI, M., SHAHPASAND, K., SABOURY, A. A. & FALAHATI, M. 2020. A review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin. J Biomol Struct Dyn, 1-9.

HOFFMANN, M., KLEINE-WEBER, H. & POHLMANN, S. 2020a. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell, 78, 779-784 e5.

HOFFMANN, M., KLEINE-WEBER, H., SCHROEDER, S., KRUGER, N., HERRLER, T., ERICHSEN, S., SCHIERGENS, T. S., HERRLER, G., WU, N. H., NITSCHE, A., MULLER, M. A., DROSTEN, C. & POHLMANN, S. 2020b. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 181, 271-280 e8.

HUGGINS, D. J. 2020. Structural analysis of experimental drugs binding to the SARS-CoV-2 target TMPRSS2. J Mol Graph Model, 100, 107710.

HUSTON, N.C., WAN, H., STRINE, M.S., DE CESARIS ARAUJO TAVARES, R., WILEN, C.B., AND PYLE, A.M. (2021). Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms. Mol Cell 81, 584-598 e585.

JING, Y., RUN-QIAN, L., HAO-RAN, W., HAO-RAN, C., YA-BIN, L., YANG, G. & FEI, C. 2020. Potential influence of COVID-19/ACE2 on the female reproductive system. Mol Hum Reprod, 26, 367-373.

JOHNSON, B. A., XIE, X., KALVERAM, B., LOKUGAMAGE, K. G., MURUATO, A., ZOU, J., ZHANG, X., JUELICH, T., SMITH, J. K., ZHANG, L., BOPP, N., SCHINDEWOLF, C., VU, M., VANDERHEIDEN, A., SWETNAM, D., PLANTE, J. A., AGUILAR, P., PLANTE, K. S., LEE, B., WEAVER, S. C., SUTHAR, M. S., ROUTH, A. L., REN, P., KU, Z., AN, Z., DEBBINK, K., SHI, P. Y., FREIBERG, A. N. & MENACHERY, V. D. 2020. Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis. bioRxiv.

JU, B., ZHANG, Q., GE, J., WANG, R., SUN, J., GE, X., YU, J., SHAN, S., ZHOU, B., SONG, S., TANG, X., YU, J., LAN, J., YUAN, J., WANG, H., ZHAO, J., ZHANG, S., WANG, Y., SHI, X., LIU, L., ZHAO, J., WANG, X., ZHANG, Z. & ZHANG, L. 2020. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature, 584, 115-119.

JURASZEK, J., RUTTEN, L., BLOKLAND, S., BOUCHIER, P., VOORZAAT, R., RITSCHEL, T., BAKKERS, M. J. G., RENAULT, L. L. R. & LANGEDIJK, J. P. M. 2021. Stabilizing the closed SARS-CoV-2 spike trimer. Nat Commun, 12, 244.

KANG, Y.-L., CHOU, Y.-Y., ROTHLAUF, P. W., LIU, Z., SOH, T. K., CURETON, D., CASE, J. B., CHEN, R. E., DIAMOND, M. S., WHELAN, S. P. J. & KIRCHHAUSEN, T. 2020. Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2. bioRxiv, 2020.04.21.053058.

KAWASE, M., SHIRATO, K., VAN DER HOEK, L., TAGUCHI, F. & MATSUYAMA, S. 2012. Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry. J Virol, 86, 6537-45.

KIM, D., LEE, J.Y., YANG, J.S., KIM, J.W., KIM, V.N., AND CHANG, H. (2020). The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914-921 e910.

KLEIN, S., CORTESE, M., WINTER, S.L., WACHSMUTH-MELM, M., NEUFELDT, C.J., CERIKAN, B., STANIFER, M.L., BOULANT, S., BARTENSCHLAGER, R., AND CHLANDA, P. (2020). SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nat Commun 11, 5885.

LAM, S. D., BORDIN, N., WAMAN, V. P., SCHOLES, H. M., ASHFORD, P., SEN, N., VAN DORP, L., RAUER, C., DAWSON, N. L., PANG, C. S. M., ABBASIAN, M., SILLITOE, I., EDWARDS, S. J. L., FRATERNALI, F., LEES, J. G., SANTINI, J. M. & ORENGO, C. A. 2020. SARS-CoV-2 spike protein predicted to form complexes with host receptor protein orthologues from a broad range of mammals. Sci Rep, 10, 16471.

LAMERS, M. M., BEUMER, J., VAN DER VAART, J., KNOOPS, K., PUSCHHOF, J., BREUGEM, T. I., RAVELLI, R. B. G., PAUL VAN SCHAYCK, J., MYKYTYN, A. Z., DUIMEL, H. Q., VAN DONSELAAR, E., RIESEBOSCH, S., KUIJPERS, H. J. H., SCHIPPER, D., VAN DE WETERING, W. J., DE GRAAF, M., KOOPMANS, M., CUPPEN, E., PETERS, P. J., HAAGMANS, B. L. & CLEVERS, H. 2020. SARS-CoV-2 productively infects human gut enterocytes. Science, 369, 50-54.

LAN, J., GE, J., YU, J., SHAN, S., ZHOU, H., FAN, S., ZHANG, Q., SHI, X., WANG, Q., ZHANG, L. & WANG, X. 2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature, 581, 215-220.

LETKO, M., MARZI, A. & MUNSTER, V. 2020. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol, 5, 562-569.

LI, F. 2016. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol, 3, 237-261.

LIN, B., FERGUSON, C., WHITE, J. T., WANG, S., VESSELLA, R., TRUE, L. D., HOOD, L. & NELSON, P. S. 1999. Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2. Cancer Res, 59, 4180-4.

LIU, L., WANG, P., NAIR, M. S., YU, J., RAPP, M., WANG, Q., LUO, Y., CHAN, J. F., SAHI, V., FIGUEROA, A., GUO, X. V., CERUTTI, G., BIMELA, J., GORMAN, J., ZHOU, T., CHEN, Z., YUEN, K. Y., KWONG, P. D., SODROSKI, J. G., YIN, M. T., SHENG, Z., HUANG, Y., SHAPIRO, L. & HO, D. D. 2020. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature, 584, 450-456.

LU, Y. & MENG, Y. G. 2015. Quantitation of Circulating Neuropilin-1 in Human, Monkey, Mouse, and Rat Sera by ELISA. Methods Mol Biol, 1332, 39-48.

LUCAS, J. M., TRUE, L., HAWLEY, S., MATSUMURA, M., MORRISSEY, C., VESSELLA, R. & NELSON, P. S. 2008. The androgen-regulated type II serine protease TMPRSS2 is differentially expressed and mislocalized in prostate adenocarcinoma. J Pathol, 215, 118-25.

LUKASSEN, S., CHUA, R. L., TREFZER, T., KAHN, N. C., SCHNEIDER, M. A., MULEY, T., WINTER, H., MEISTER, M., VEITH, C., BOOTS, A. W., HENNIG, B. P., KREUTER, M., CONRAD, C. & EILS, R. 2020. SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J, 39, e105114.

MADJID, M., SAFAVI-NAEINI, P., SOLOMON, S. D. & VARDENY, O. 2020. Potential Effects of Coronaviruses on the Cardiovascular System: A Review. JAMA Cardiol, 5, 831-840.

MAYI, B. S., LEIBOWITZ, J. A., WOODS, A. T., AMMON, K. A., LIU, A. E. & RAJA, A. 2021. The role of Neuropilin-1 in COVID-19. PLoS Pathog, 17, e1009153.

MILLET, J. K. & WHITTAKER, G. R. 2018. Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells. Virology, 517, 3-8.

MIRABELLI, C., WOTRING, J. W., ZHANG, C. J., MCCARTY, S. M., FURSMIDT, R., FRUM, T., KADAMBI, N. S., AMIN, A. T., O’MEARA, T. R., PRETTO, C. D., SPENCE, J. R., HUANG, J., ALYSANDRATOS, K. D., KOTTON, D. N., HANDELMAN, S. K., WOBUS, C. E., WEATHERWAX, K. J., MASHOUR, G. A., O’MEARA, M. J. & SEXTON, J. Z. 2020. Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19. bioRxiv, 2020.05.27.117184.

MOUTAL, A., MARTIN, L. F., BOINON, L., GOMEZ, K., RAN, D., ZHOU, Y., STRATTON, H. J., CAI, S., LUO, S., GONZALEZ, K. B., PEREZ-MILLER, S., PATWARDHAN, A., IBRAHIM, M. M. & KHANNA, R. 2020. SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signaling to induce analgesia. bioRxiv.

MUHL, L., FOLESTAD, E. B., GLADH, H., WANG, Y., MOESSINGER, C., JAKOBSSON, L. & ERIKSSON, U. 2017. Neuropilin 1 binds PDGF-D and is a co-receptor in PDGF-D-PDGFRbeta signaling. J Cell Sci, 130, 1365-1378.

MUKHERJEE, S. & PAHAN, K. 2021. Is COVID-19 Gender-sensitive? J Neuroimmune Pharmacol, 16, 38-47.

MURGOLO, N., THERIEN, A. G., HOWELL, B., KLEIN, D., KOEPLINGER, K., LIEBERMAN, L. A., ADAM, G. C., FLYNN, J., MCKENNA, P., SWAMINATHAN, G., HAZUDA, D. J. & OLSEN, D. B. 2021. SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and development. PLoS Pathog, 17, e1009225.

PAN, X. W., XU, D., ZHANG, H., ZHOU, W., WANG, L. H. & CUI, X. G. 2020. Identification of a potential mechanism of acute kidney injury during the COVID-19 outbreak: a study based on single-cell transcriptome analysis. Intensive Care Med, 46, 1114-1116.

PEREZ-MILLER, S., PATEK, M., MOUTAL, A., CABEL, C. R., THORNE, C. A., CAMPOS, S. K. & KHANNA, R. 2020. In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein. bioRxiv.

PREMKUMAR, L., SEGOVIA-CHUMBEZ, B., JADI, R., MARTINEZ, D. R., RAUT, R., MARKMANN, A., CORNABY, C., BARTELT, L., WEISS, S., PARK, Y., EDWARDS, C. E., WEIMER, E., SCHERER, E. M., ROUPHAEL, N., EDUPUGANTI, S., WEISKOPF, D., TSE, L. V., HOU, Y. J., MARGOLIS, D., SETTE, A., COLLINS, M. H., SCHMITZ, J., BARIC, R. S. & DE SILVA, A. M. 2020. The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol, 5.

QIAO, Y., WANG, X. M., MANNAN, R., PITCHIAYA, S., ZHANG, Y., WOTRING, J. W., XIAO, L., ROBINSON, D. R., WU, Y. M., TIEN, J. C., CAO, X., SIMKO, S. A., APEL, I. J., BAWA, P., KREGEL, S., NARAYANAN, S. P., RASKIND, G., ELLISON, S. J., PAROLIA, A., ZELENKA-WANG, S., MCMURRY, L., SU, F., WANG, R., CHENG, Y., DELEKTA, A. D., MEI, Z., PRETTO, C. D., WANG, S., MEHRA, R., SEXTON, J. Z. & CHINNAIYAN, A. M. 2020. Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Proc Natl Acad Sci U S A.

RAHMAN, N., BASHARAT, Z., YOUSUF, M., CASTALDO, G., RASTRELLI, L. & KHAN, H. 2020. Virtual Screening of Natural Products against Type II Transmembrane Serine Protease (TMPRSS2), the Priming Agent of Coronavirus 2 (SARS-CoV-2). Molecules, 25.

RAIMONDI, C., BRASH, J. T., FANTIN, A. & RUHRBERG, C. 2016. NRP1 function and targeting in neurovascular development and eye disease. Prog Retin Eye Res, 52, 64-83.

RIVA, L., YUAN, S., YIN, X., MARTIN-SANCHO, L., MATSUNAGA, N., BURGSTALLER-MUEHLBACHER, S., PACHE, L., DE JESUS, P. P., HULL, M. V., CHANG, M., CHAN, J. F.-W., CAO, J., POON, V. K.-M., HERBERT, K., NGUYEN, T.-T., PU, Y., NGUYEN, C., RUBANOV, A., MARTINEZ-SOBRIDO, L., LIU, W.-C., MIORIN, L., WHITE, K. M., JOHNSON, J. R., BENNER, C., SUN, R., SCHULTZ, P. G., SU, A., GARCIA-SASTRE, A., CHATTERJEE, A. K., YUEN, K.-Y. & CHANDA, S. K. 2020. A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals. bioRxiv, 2020.04.16.044016.

ROBBIANI, D. F., GAEBLER, C., MUECKSCH, F., LORENZI, J. C. C., WANG, Z., CHO, A., AGUDELO, M., BARNES, C. O., GAZUMYAN, A., FINKIN, S., HAGGLOF, T., OLIVEIRA, T. Y., VIANT, C., HURLEY, A., HOFFMANN, H. H., MILLARD, K. G., KOST, R. G., CIPOLLA, M., GORDON, K., BIANCHINI, F., CHEN, S. T., RAMOS, V., PATEL, R., DIZON, J., SHIMELIOVICH, I., MENDOZA, P., HARTWEGER, H., NOGUEIRA, L., PACK, M., HOROWITZ, J., SCHMIDT, F., WEISBLUM, Y., MICHAILIDIS, E., ASHBROOK, A. W., WALTARI, E., PAK, J. E., HUEY-TUBMAN, K. E., KORANDA, N., HOFFMAN, P. R., WEST, A. P., JR., RICE, C. M., HATZIIOANNOU, T., BJORKMAN, P. J., BIENIASZ, P. D., CASKEY, M. & NUSSENZWEIG, M. C. 2020. Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature, 584, 437-442.

ROSSI, A., DEVERAUX, Q., TURK, B. & SALI, A. 2004. Comprehensive search for cysteine cathepsins in the human genome. Biol Chem, 385, 363-72.

SACCO, M.D., MA, C., LAGARIAS, P., GAO, A., TOWNSEND, J.A., MENG, X., DUBE, P., ZHANG, X., HU, Y., KITAMURA, N., et al. (2020). Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L. Sci Adv 6(50):eabe0751.

SHANG, J., WAN, Y., LUO, C., YE, G., GENG, Q., AUERBACH, A. & LI, F. 2020. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A, 117, 11727-11734.

SHIRATO, K., KAWASE, M. & MATSUYAMA, S. 2018. Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry. Virology, 517, 9-15.

SIMMONS, G., GOSALIA, D. N., RENNEKAMP, A. J., REEVES, J. D., DIAMOND, S. L. & BATES, P. 2005. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci U S A, 102, 11876-81.

SINGH, M., BANSAL, V. & FESCHOTTE, C. 2020. A single-cell RNA expression map of human coronavirus entry factors. bioRxiv.

SNIJDER, E.J., BREDENBEEK, P.J., DOBBE, J.C., THIEL, V., ZIEBUHR, J., POON, L.L.M., GUAN, Y., ROZANOV, M., SPAAN, W.J.M., AND GORBALENYA, A.E. (2003). Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage. Journal of Molecular Biology 331, 991-1004.

SNIJDER, E.J., LIMPENS, R., DE WILDE, A.H., DE JONG, A.W.M., ZEVENHOVEN-DOBBE, J.C., MAIER, H.J., FAAS, F., KOSTER, A.J., AND BARCENA, M. (2020). A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis. PLoS Biol 18, e3000715.

SUBRAMANIAN, A., VERNON, K. A., SLYPER, M., WALDMAN, J., LUECKEN, M. D., GOSIK, K., DUBINSKY, D., CUOCO, M. S., KELLER, K., PURNELL, J., NGUYEN, L., DIONNE, D., ROZENBLATT-ROSEN, O., WEINS, A., REGEV, A. & GREKA, A. 2020. RAAS blockade, kidney disease, and expression of ACE2, the entry receptor for SARS-CoV-2, in kidney epithelial and endothelial cells.

UNIPROTKB - O14786 (NRP1_HUMAN)

V'KOVSKI, P., KRATZEL, A., STEINER, S., STALDER, H., AND THIEL, V. (2021). Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol. 19, 155–170.

WALLS, A. C., TORTORICI, M. A., BOSCH, B. J., FRENZ, B., ROTTIER, P. J. M., DIMAIO, F., REY, F. A. & VEESLER, D. 2016. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. Nature, 531, 114-117.

WANG, Y., LIU, M. & GAO, J. 2020. Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions. Proc Natl Acad Sci U S A, 117, 13967-13974.

YAMAMOTO, M., KISO, M., SAKAI-TAGAWA, Y., IWATSUKI-HORIMOTO, K., IMAI, M., TAKEDA, M., KINOSHITA, N., OHMAGARI, N., GOHDA, J., SEMBA, K., MATSUDA, Z., KAWAGUCHI, Y., KAWAOKA, Y. & INOUE, J. I. 2020. The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner. Viruses, 12.

YANG, N. & SHEN, H.-M. 2020. Targeting the Endocytic Pathway and Autophagy Process as a Novel Therapeutic Strategy in COVID-19. International Journal of Biological Sciences, 16, 1724-1731.

YUAN, M., WU, N. C., ZHU, X., LEE, C. D., SO, R. T. Y., LV, H., MOK, C. K. P. & WILSON, I. A. 2020. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science, 368, 630-633.

ZANG, R., GOMEZ CASTRO, M. F., MCCUNE, B. T., ZENG, Q., ROTHLAUF, P. W., SONNEK, N. M., LIU, Z., BRULOIS, K. F., WANG, X., GREENBERG, H. B., DIAMOND, M. S., CIORBA, M. A., WHELAN, S. P. J. & DING, S. 2020. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol, 5.

ZIEGLER, C. G. K., ALLON, S. J., NYQUIST, S. K., MBANO, I. M., MIAO, V. N., TZOUANAS, C. N., CAO, Y., YOUSIF, A. S., BALS, J., HAUSER, B. M., FELDMAN, J., MUUS, C., WADSWORTH, M. H., 2ND, KAZER, S. W., HUGHES, T. K., DORAN, B., GATTER, G. J., VUKOVIC, M., TALIAFERRO, F., MEAD, B. E., GUO, Z., WANG, J. P., GRAS, D., PLAISANT, M., ANSARI, M., ANGELIDIS, I., ADLER, H., SUCRE, J. M. S., TAYLOR, C. J., LIN, B., WAGHRAY, A., MITSIALIS, V., DWYER, D. F., BUCHHEIT, K. M., BOYCE, J. A., BARRETT, N. A., LAIDLAW, T. M., CARROLL, S. L., COLONNA, L., TKACHEV, V., PETERSON, C. W., YU, A., ZHENG, H. B., GIDEON, H. P., WINCHELL, C. G., LIN, P. L., BINGLE, C. D., SNAPPER, S. B., KROPSKI, J. A., THEIS, F. J., SCHILLER, H. B., ZARAGOSI, L. E., BARBRY, P., LESLIE, A., KIEM, H. P., FLYNN, J. L., FORTUNE, S. M., BERGER, B., FINBERG, R. W., KEAN, L. S., GARBER, M., SCHMIDT, A. G., LINGWOOD, D., SHALEK, A. K., ORDOVAS-MONTANES, J., LUNG-NETWORK@HUMANCELLATLAS.ORG, H. C. A. L. B. N. E. A. & NETWORK, H. C. A. L. B. 2020. SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell, 181, 1016-1035 e19.

Event: 1901: Interferon-I antiviral response, antagonized by SARS-CoV-2

Short Name: IFN-I response, antagonized

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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).

Key Event Description

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 (doi:https://doi.org/10.1016/j.cell.2020.04.01). 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 (Kim et al. 2020; O’Leary et al. 2020). Viral transcription and replication is explained in depth in KE1847. 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 (doi:https://doi.org/10.1016/j.cell.2020.04.01). 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 (Amor et al. 2020). Extensive protein-protein interaction (Gordon et al. 2020) and viral protein-host RNA interaction networks have been demonstrated between the viral NSPs and accessory proteins and host molecules. 

This key event is focused on the specific viral:host protein interactions within the infected cell that are involved in the IFN-I antiviral response pathways. IFN-I is the main component of the innate immune system that is suppressed by the SARS-CoV-2 coronavirus early in infection. 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 (Quarleri and Delpino, 2021).

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 INFβ 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).

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.

 

Gene	Protein	Function	Role in early innate immune evasion
ORF1a	NSP1	NSP1 antagonizes interferon induction to suppress host antiviral response.	DNA Polymerase Alpha Complex: Regulates the activation of IFN-I through cytosolic 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).
	NSP2	While not essential for viral replication, deletion of NSP2 diminishes viral growth and RNA synthesis	Translation repression through binding GIGYF2and EIF4E2 (4EHP) (Gupta et al. 2021)
	NSP3	Papain-like protease (Plpro); Cleaves the ORF1a and 1ab polypeptides	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)
	NSP5	3C-like protease (3CLpro); Cleaves the ORF1a and 1ab polypeptides	Binds STING (Rui et al. 2021)
	NSP6	Limits autophagosome expansion	Suppresses IFN-I expression: Binds TBK-1 to supress IRF3 phosphorylation (Xia et al. 2020; Quarleri and Delpino, 2021)
	NSP7	In complex with NSP8 forms primase as part of multimeric RNA-dependent RNA replicase (RdRp)
	NSP8	Replication complex with NSP7, NSP9 and NSP12	Binds SRP72/54/19 (Gordon et al. 2020) and 7SL RNA to block IFN membrane transport (Banerjee et al. 2020)
	NSP9	Replication complex with NSP7, NSP8 and NSP12	Binds SRP and 7SL RNA with NSP8 to block IFN membrane transport (Banerjee et al. 2020)
ORF1b	NSP13	Helicase and triphosphatase that initiates the first step in viral mRNA capping.	Binds TBK1 (Xia et al. 2020)
	NSP14		Induces lysosomal degradation of IFNAR1 (Hayn et al. 2021)
ORF2	Spike (S)	ACE2 interaction, cell entry
ORF3a	ORF3a	Interacts with M, S, E and 7a; form viroporins; immune evasion	Binds STING (Rui et al 2021)
ORF4	Envelope (E)	Viral assembly and budding
ORF5	Membrane (M)	Viral assembly	Interacts with RIG-I and MAVS sensors of viral RNA (Fu et al 2020)
ORF6	ORF6	Viral pathogenesis and virulence; interacts with ORF8; promotes RNA polymerase activity	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
ORF7a	ORF7a	Interacts with S, ORF3a; immune evasion	Suppresses STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020).
ORF7b	ORF7b	Structural component of virion	Suppresses STAT1 and STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020)
ORF8	ORF8	Immune evasion	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)
ORF9	Nucleocapsid (N)	Stabilizes viral RNA	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)
ORF9b	ORF9b	Immune evasion	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)

How it is Measured or Detected

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:

Interferon I decrease (Xia et al. 2020):

• IFN-I production and signaling luciferase reporter assays
• Co-immunoprecipitation and western blot
• Indirect immunofluorescence assays
• DNA assembly and RNA transcription of a luciferase replicon for SARS-CoV-2
• Replicon RNA electroporation and luciferase reporter assay

SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling (Wu et al. 2021)

• Viral- and host-protein-specific antibodies
• Immunoprecipitation
• Immunofluorescent microscopy
• Dual-luciferase reporter assays
• Fluorescence quantification immunoblotting

SARS-CoV-2-Human Protein-Protein Interaction Map (Gordon et al. 2020)

• Cloning and expression of viral proteins via plasmid transfection into HEK293T cell line
• Protein affinity purification using MagStrep beads with detection by anti-strep western blot of cell lysate
• Global analysis of SARS-CoV-2 host interacting proteins using affinity purification-mass spectrometry

 

References

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

Andres et al. 2020. SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.256776

Banerjee et al. 2020. SARS-CoV-2 disrupts splicing, translation, and protein trafficking to supress host defenses. Cell 183, 1325–1339. https://doi.org/10.1016/j.cell.2020.10.004

Cascarina and Ross, 2020. A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates. The FASEB Journal, 34:9832–9842. DOI: 10.1096/fj.202001351

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.

Chen et al. 2021. SARS-CoV-2 Nucleocapsid Protein Interacts with RIG-I and Represses RIG-Mediated IFN-β Production. Viruses. 13(1):47. https://doi.org/10.3390/v13010047

Conceicao 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

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 www.pnas.org/cgi/doi/10.1073/pnas.2010146117 

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

Gordon et al. 2020. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 483:459-473. https://doi.org/10.1038/s41586-020-2286-9

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

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 https://doi.org/10.1101/2020.08.16.252973

Hayn et al. 2021. Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities. Cell Reports 35, 109126. https://doi.org/10.1016/j.celrep.2021.109126

Jiang et al. 2020. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cellular & Molecular Immunology 17:998–1000; https://doi.org/10.1038/s41423-020-0514-8

Kato et al. 2021. Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex. Biochemical and Biophysical Research Communications 536:59-66 https://doi.org/10.1016/j.bbrc.2020.11.115

Kim et al. 2019. The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-β response. J. Biol. Chem. 294(16): 6430–6438. DOI 10.1074/jbc.RA118.005868

Kim et al. 2020. The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914–921. https://doi.org/10.1016/j.cell.2020.04.011

Li et al. 2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Research vol. 286. https://doi.org/10.1016/j.virusres.2020.198074

Liu et al. 2021. ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity. Nature Microbiol 6: 467–478. https://doi.org/10.1038/s41564-021-00884-1

Moustaqil et al. 2021. SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species, Emerging Microbes & Infections, 10:1, 178-195. https://doi.org/10.1080/22221751.2020.1870414

Mu et al. 2020. SARS-CoV-2 N protein antagonizes type I interferon signaling by suppressing phosphorylation and nuclear translocation of STAT1 and STAT2. Cell Discov 6, 65. https://doi.org/10.1038/s41421-020-00208-3

O’Leary et al. 2020 Unpacking Pandora from Its Box: Deciphering the Molecular Basis of the SARS-CoV-2 Coronavirus. Int. J. Mol. Sci. 2021, 22, 386. https://doi.org/10.3390/ijms22010386

Quarleri and Delpino, 2020. Type I and III IFN-mediated antiviral actions counteracted by SARS-CoV-2 proteins and host inherited factors. Cytokine & Growth Factor Reviews, 58: 55-65. https://doi.org/10.1016/j.cytogfr.2021.01.003

Rashid et al. The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta. Virus Research 296, 198350. https://doi.org/10.1016/j.virusres.2021.198350

Ren et al. 2020. The ORF3a protein of SARS-CoV-2 induces apoptosis in cells. Cellular & Molecular Immunology 17:881–883; https://doi.org/10.1038/s41423-020-0485-9

Rui et al. 2021. Unique and complementary suppression of cGAS-STING and RNA sensing-triggered innate immune responses by SARS-CoV-2 proteins. Sig Transduct Target Ther 6, 123. https://doi.org/10.1038/s41392-021-00515-5

Schubert et al. 2020. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nature Structural & Molecular Bio. 27:959-966. https://doi.org/10.1038/s41594-020-0511-8

Shin et al. 2020. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587: 657–662. https://doi.org/10.1038/s41586-020-2601-5

Thoms et al. 2020. Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369(6508): 1249-1255. DOI: 10.1126/science.abc8665

Wu et al. 2021. SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Reports 34, 108761. https://doi.org/10.1016/j.celrep.2021.108761

Wu et al. 2020. Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2. Cell Discovery 6:68. https://doi.org/10.1038/s41421-020-00210-9

Xia et al. 2020. Evasion of Type I Interferon by SARS-CoV-2. Cell Reports 33, 108234. https://doi.org/10.1016/j.celrep.2020.108234

Xia and Shi, 2020. Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2. Journal of Interferon & Cytokine Research v.40, no. 12 DOI:10.1089/jir.2020.0214

Zhang et al. 2020. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.24.111823

Event: 1847: Increased SARS-CoV-2 production

Short Name: SARS-CoV-2 production

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Broad mammalian host range has been demonstrated 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]. 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.

Very broad mammalian tropism: human, bat, cat, dog, civet, ferret, horse, pig, sheep, goat, water buffalo, cattle, rabbit, hamster, mouse

Key Event Description

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. 

Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim et al.]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively [E. J. Snijder et al.] that contain cis-acting secondary RNA structures essential for RNA synthesis [N. C. Huston et al.]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx et al.]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder et al.]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di et al.]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [E. J. Snider et al.], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [M. D. Sacco et al.]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [S. Klein et al., E. J. Snijder et al., P. V'Kovski, et al.]. Besides replication, which yields the positive-sense gRNA, the replicase also 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. 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]. sgRNAs 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 [D. Kim et al.]. The gRNA is packaged by the structural proteins to assemble progeny virions.

Viral translation:

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 et al.]. 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 et al.]. Other elements of the genome include are shown below [V. B. O'Leary et al.]. The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).

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 et al.]. 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 et al.]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel et al.]. 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 pp1a is approximately 1.4–2.2 times more expressed than pp1ab. After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 protease can be referred to as 3C-like protease (3CL^pro) or as main protease (M^pro), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein et al.] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, suppressing host gene expression [M. Thoms et al.]. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors. Nsp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, whilst nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [P. V'Kovski et al.].  nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function. Nsp-10 composes the RNA capping machinery nsp-9. nsp13 provides the RNA 5′-triphosphatase activity. Nsp-14 is a N7-methyltransferase and nsp-16 the 2′-O-methyltransferase. 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 et al.]. Xia et al. [H. Xia et al.] 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.

 

Viral transcription and replication:

Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder et al.]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to 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  [S. Klein et al., E. J. Snijder et al.]. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-³[H]uridine) and detection by EM autoradiography [E. J. Snijder et al.].

Viral replicative proteins and specific host factors are recruited to ROs [E. J. Snijder et al.]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist et al.]. 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 et al.]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz et al.]. The intermediates comprise 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. The transcription of coronaviruses 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 Nidovirales 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 are unique features of the coronaviruses and arteriviruses [W. A. Miller and G. Koev.]. Of note, the produced genomic RNA represents a small fraction of the total vRNA [N. S. Ogando et al.].

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 [I. Sola, F. Almazan et al., I. Sola, J. L. Moreno, et al.]. 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 [H. Di et al.]. Therefore, the 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 [I. Sola, F. Almazan et al. I. Sola, J. L. Moreno, et al.]. 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 [X. Zhang et al.]. 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 [I. Sola et al. 2015, S. G. Sawicki et al., Y. Yang et al.]. 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 [Y. Yang]. The set of sg mRNAs is then translated to yield 29 identified different proteins [F. Wu et al.], although many papers have identified additional ORFs [D. Kim et al.. Y. Finkel et al., A. Vandelli et al.]. 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 et al.]. 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 5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [Y. Finkel et al.].

 

Viral assembly:

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 [J. R. Cohen et al., A. Perrier et al.] and viral 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, forming viral ribonucleoprotein (vRNPs) complexes, before being selectively packaged into progeny virions [P. V'Kovski et al.], a step in which vRNPs bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [N. S. Ogando et al.]. 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.

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 et al., D. E. Gordon et al., 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 et al.], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) in vitro [A. Savastano et al., H. Chen et al., C. Iserman et al., T. M. Perdikari et al., J. Cubuk et al.], dependent on its C-terminal domain (CTD) [H. Chen et al.]. 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 et al.], 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 et al., C. Iserman et al., C. R. Carlson et al.].

The ERGIC constitutes the main assembly site of coronaviruses [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.] and budding events have been seen by EM studies. For SARS-CoV-2, virus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.]. 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 [S. Klein et al.].

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 et al.]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh et al.].

How it is Measured or Detected

Viral translation:

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation [Schubert et al. 2020]

• Sucrose pelleting binding assay to verify Nsp1–40S complex formation
• In vivo translation assay
• Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay

SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee et al. 2020]

• SARS-CoV-2 viral protein binding to RNA
• Interferon stimulation experiments
• Splicing assessment experiments
• IRF7-GFP splicing reporter, 5EU RNA labeling, capture of biotinylated 5EU labeled RNA

Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface

Viral transcription:

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 et al.]. 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, et al.]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.

Viral replication:

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.

Viral production:

Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.

References

1.         D. Kim et al., The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914-921 e910 (2020).

2.         E. J. Snijder et al., Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage. Journal of Molecular Biology 331, 991-1004 (2003).

3.         N. C. Huston et al., Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms. Mol Cell 81, 584-598 e585 (2021).

4.         C. J. Budzilowicz, S. P. Wilczynski, S. R. Weiss, 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. J Virol 53, 834-840 (1985).

5.         H. Di, A. A. McIntyre, M. A. Brinton, New insights about the regulation of Nidovirus subgenomic mRNA synthesis. Virology 517, 38-43 (2018).

6.         M. D. Sacco et al., Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L. Sci Adv 6,  (2020).

7.         S. Klein et al., SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. BioRxiv,  (2020).

8.         E. J. Snijder et al., A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis. PLoS Biol 18, e3000715 (2020).

9.         P. V'Kovski, A. Kratzel, S. Steiner, H. Stalder, V. Thiel, Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol,  (2020).

10.       P. Liu, J. Leibowitz, in Molecular Biology of the SARS-Coronavirus. (2010),  chap. Chapter 4, pp. 47-61.

11.       V. B. O'Leary, O. J. Dolly, C. Hoschl, M. Cerna, S. V. Ovsepian, Unpacking Pandora From Its Box: Deciphering the Molecular Basis of the SARS-CoV-2 Coronavirus. Int J Mol Sci 22,  (2020).

12.       Y. Finkel et al., The coding capacity of SARS-CoV-2. Nature 589, 125-130 (2021).

13.       H. A. Hussein, R. Y. A. Hassan, M. Chino, F. Febbraio, Point-of-Care Diagnostics of COVID-19: From Current Work to Future Perspectives. Sensors (Basel) 20,  (2020).

14.       M. Thoms et al., Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369, 1249-1255 (2020).

15.       A. K. Banerjee et al., SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses. Cell 183, 1325-1339 e1321 (2020).

16.       H. Xia et al., Evasion of Type I Interferon by SARS-CoV-2. Cell Rep 33, 108234 (2020).

17.       P. Ahlquist, RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 296, 1270-1273 (2002).

18.       M. Schwartz et al., A Positive-Strand RNA Virus Replication Complex Parallels Form and Function of Retrovirus Capsids. Molecular Cell 9, 505-514 (2002).

19.       W. A. Miller, G. Koev, Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology 273, 1-8 (2000).

20.       N. S. Ogando et al., SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. J Gen Virol 101, 925-940 (2020).

21.       I. Sola, F. Almazan, S. Zuniga, L. Enjuanes, Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annu Rev Virol 2, 265-288 (2015).

22.       I. Sola, J. L. Moreno, S. Zuniga, S. Alonso, L. Enjuanes, Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. J Virol 79, 2506-2516 (2005).

23.       X. Zhang, C. L. Liao, M. M. Lai, Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. J Virol 68, 4738-4746 (1994).

24.       S. G. Sawicki, D. L. Sawicki, S. G. Siddell, A contemporary view of coronavirus transcription. J Virol 81, 20-29 (2007).

25.       Y. Yang, W. Yan, B. Hall, X. Jiang, Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination. bioRxiv,  (2020).

26.       F. Wu et al., A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020).

27.       A. Vandelli et al., Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. Nucleic Acids Res 48, 11270-11283 (2020).

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. J Virol 85, 5794-5803 (2011).

29.       A. Perrier et al., The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal. J Biol Chem 294, 14406-14421 (2019).

30.       D. E. Gordon et al., A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459-468 (2020).

31.       R. McBride, M. van Zyl, B. C. Fielding, The coronavirus nucleocapsid is a multifunctional protein. Viruses 6, 2991-3018 (2014).

32.       S. Alberti, S. Carra, Quality Control of Membraneless Organelles. Journal of Molecular Biology 430, 4711-4729 (2018).

33.       S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Biomolecular condensates: organizers of cellular biochemistry. Nature Reviews Molecular Cell Biology 18, 285-298 (2017).

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).

35.       H. Chen et al., Liquid-liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA. Cell Res,  (2020).

36.       C. Iserman et al., Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate. bioRxiv,  (2020).

37.       T. M. Perdikari et al., SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins. bioRxiv,  (2020).

38.       J. Cubuk et al., The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. bioRxiv,  (2020).

39.       C. Iserman et al. (Cold Spring Harbor Laboratory, 2020).

40.       C. R. Carlson et al., Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests abiophysical basis for its dual functions. Molecular Cell,  (2020).

41.       L. Mendonca et al., SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. bioRxiv,  (2020).

42.       S. Ghosh et al., beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. Cell 183, 1520-1535 e1514 (2020).

43.       Schubert, K., Karousis, E.D., Jomaa, A. et al. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nat Struct Mol Biol 27, 959–966 (2020). 

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 In Vitro and with Clinical Specimens. J Clin Microbiol. 2020:58(5)e00310-20. doi:10.1128/JCM.00310-20

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

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

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.

Event: 1848: Toll Like Receptor (TLR) Dysregulation

Short Name: TLR Activation/Dysregulation

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Cell applicability: TLRs are broadly expressed on various cell types. Examples include: epithelial cells, macrophages, neutrophils, platelets, dendritic cells, NK cells, Tcells, Bcells, neurons, Adipocytes.

Tissue/organ level : TLRs are broadly expressed in all vital tissues/organs: lung, heart, liver, spleen, kidney, brain, muscle, gut, skin

Taxonomic Applicability: TLRs are well conserved across species but between species variations are reported in terms of sensitivity towards stressors.  For instance certain CpG-ODNs have a stronger TLR9 activating potential in mice than in human and vice versa.

Life Stages: TLRs are expressed in all life stages but age variation of level of TLR activation/dysregulation are reported. In elderly immunoscenescence and inflammation are both linked to TLR dysregulation

Sex Applicability: Male and female subjects both express functionally active TLRs but sex differences have been reported. For instance certain TLR gain and/or loss of function polymorphisms have higher prevalence in men. Example of TLR7 loss of function (van der Made et al 2020) and TLR9 gain of function (Gao et al 2018, Traub et al 2012, Elsherif et al 2019). Higher testosterone in men has also been linked to higher TLR4 expression.

TLR7 is located in a region on the X-chromosome which have a high chance of escaping inactivation leading to higher expression levels in women. Estrogens trigger TLR7, which is higher in women. Exposure of Peripheral blood mononuclear cells (PBMC) to TLR7 ligands will cause a higher production of type I IFN (IFN-a) in female cells than male cells.  (Kovats, 2015;  Takahashi and Iwasaki, 2021; Libert et al.,  2010; Scully et al., 2020)

Key Event Description

Background

Toll-like receptors (TLRs) are a family of 13 conserved transmembrane receptors that are at the forefront of directing innate and adaptive immune responses against invading bacteria, fungi, viruses and parasites (Akira 2003, Takeda, Akira 2004, Pasare, Medzhitov 2005, Tal, Adini et al. 2020, van der Made, Simons et al. 2020). Upon activation TLRs initiate overlapping and distinct signaling pathways in various cell types such as macrophages (MP), conventinal DC (cDC), plasmacytoid DC (pDC), lamina propria DC (LPDC), and inflammatory monocytes (iMO). Engagement of TLR with specific stressors (e.g. PAMPs and DAMPs) induces conformational changes of TLRs that allow homo- or heterophilic interactions of TLRs and recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM to control intracellular signalling pathways leading to the synthesis and secretion of appropriate cytokines and chemokines by cells of the immune system. TLRs have various biological roles both in pathogen combat and tissue homeostasis.

This KE is first developed in context of COVID-19 CIAO project.

The key gatekeepers in detecting and combating viral infections are TLR3, TLR7, TLR8 and TLR9 and these are predominantly localized in intracellular compartments. In the setting of COVID-19, multiple TLRs are likely relevant in viral combat. Literature covering TLR triggering via SARS-CoV-2 derived PAMPS (Pathogen Associated Molecular Patterns) include:

• TLR7 and TLR8 (+TLR3, TLR4, TLR6)  (Khanmohammadi and Rezaei, 2021)
• TLR1, TLR4 and TLR6 activated by SARS-CoV-2 spike proteins (Choudhury A et al, 2020)
• TLR9: Less CpG suppression in coronavirus compared to other viruses, for SARS-CoV-2 in the Envelope (E) open reading frame (E-ORF) and ORF10 (Ng et al., 2004; Digard et al. 2020) and multidisciplinary links described in Bezemer and Garssen, 2021

TLR dysregulation can be multi-fold:

1. Underperformance of TLR function leading to poor pathogen combat. This is covered in AOP 378

• COVID-19 patients having poor TLR function (due to polymorphisms) could potentially have less viral clearance capability and more adverse events leading to more severe disease and mortality. This has been shown for TLR7 loss of function polymorphisms (van der Made et al 2020). Knowledge Gap: it is not known if loss of function of other TLRs has a worse outcome in COVID-19 patients.

2. Overperformance of TLR function contributing to exaggerated immune response/cytokine storm/thrombosis/progression into ARDS and MOD. This is covered in AOP377

• TLR7 and TLR9 expression, measured by RNAseq gene analysis, is more elevated in black Americans than white Americans, which is proposed to explain in part the racial disparity in Covid-19 mortality rates via TLR mediated DC activation (Tal et al. 2020)
• genetic mutations leading to TLR9 gain of function in human is associated with immune-mediated disease and with a higher incidence of ICU acquired infection (Chatzietal.,2018;Ng et al.,2010).
• Higher presence of host derived TLR stressors in vulnerable patients can contribute to TLR overstimulation/dysregulation. (Bezemer and Garssen, 2021)

Different classes of "stressors" act on TLR activation/dysregulation

1.  Pathogen associated molecular patterns (PAMPs). TLRs can sense PAMPS during infection or upon exposure to stressors containing micro-organisms or fragments thereof (e.g. cigarette smoke, bioaerosols, house dust mite)

• TLR1 is activated by bacterial Lipopeptides
• TLR2 is activated by bacterial lipoproteins and glycolipids, TLR2 can form conformations with TLR1 and TLR6 to distinguish between diacyl and triacyl lipopeptides.
• TLR3 is activated by viral double stranded RNA(dsRNA)
• TLR4 is activated by Bacterial LPS
• TLR5 is activated by Bacterial flagellig
• TLR6 is activated by Bacterial lipopeptides and Fungal zymosan
• TLR7 and 8 recognize viral single stranded RNA(ssRNA) and bacterial RNA.
• TLR9 recognizes RNA and DNAmotifs that are rich in unmethylated Cytosine-phosphate-Guanine (CpG) sequences. CpG-motifs are higher expressed in the bacterial and viral genome compared to the vertebrate genome (Hemmi et al., 2000).

2. host derived Damage-Associated Molecular Patterns (DAMPS). Note that in the context and nomenclature of AOP these DAMPS cannot be labeld as "stressors" since they are derived from inside and not from outside, however these "pseudostressors" do act on the TLR receptors in similar way as PAMPs

• TLR2 and TLR4 are activated by heat shock proteins 60 and 70  (HSP60 and HSP70); extracellular matrix components (ECM); oligosaccharides of hyaluronic acid (HA) and heparan sulfate (HS) (Piccinini AM and Midwood KS, 2010).

• high-mobility group protein B1 (HMGB1) triggers TLR2, TLR4 and TLR9

• Oxidative injury/Oxidized phospholipids  triggers TLR4 mediated NET formation
• Human mitochondrial DNA (mtDNA), evolutionary derived from endosymbiont bacteria, contains unmethylated CpG-motifs and triggers inflammatory responses directly via TLR9 during injury and/or infection (Zhang et al., 2010).
• Altered self-ligands, called carboxy-alkyl-pyrroleprotein adducts (CAPs), that are generated during oxidative stress, are known to aggravate TLR9/MyD88 pathway activation (Zhanget al., 2010;Panigrahi et al., 2013). CAPs have been shown to promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo (Panigrahi et al., 2013).

3. synthetic TLR triggers/blockers (agonists/antagonists) for therapeutic purposes. Examples include CpG-ODNs triggering TLR9 for vaccin adjuvants/cancer treatment/immuno-modulation

 

Several Modulating factors can contribute to TLR activation/dysregulation

• Co-infection and Trauma (for instance ventilator induced damage) can induce increased levels of TLR9 stressor, mtDNA, which is known to drive worse outcome at ICU in setting of other disorders.  
• High levels of Visceral Fat, can increase TLR9 expression levels ánd circulating mtDNA
• Aging triggers both immunosenescence and inflammaging in part via impaired TLR function versus inappropriate triggering via increases of circulating DAMPS (Shaw et al 2011)
• Genetic polymorphisms can lead to TLR dysregulation (TLR9 gain of function and TLR7 loss of function with worse outcome at ICU Chatzi et al 2018, van der Made et al 2020, Chen et al 2011, )
• Circulating DAMPS such as mtDNA levels increase with age which is a familiar trait contributing to chronic inflammation, so called“inflamm-aging”in elderly people (Pinti et al., 2014).
• Vitamin D inhibits expression levels of TLR9
• Men, higher testosterone, higher TLR4

How it is Measured or Detected

Patient specific Ex vivo analysis

• Levels of TLR specific stressors (for instance for TLR9, cell free DNA/RNA, mtDNA) are measurable in biological samples (serum, plasma)
• TLR gain of function and loss of function polymorphisms are measurable
• TLR expression levels on different cell types and different tissues are measurable by mRNA analysis and by protein analysis
• TLR function in response to stressors is measurable by analysing components of downstream cascades and read outs of inflammatory mediators (IL6, IL8, IL10, Il17, INF, TNFalpha, etc). This can be done by ex vivo stimulations of cells isolated from patients for instance PBMCs.

In vitro/ in vivo models

• TLR Reporter assays
• TLR knock-out mice

References

AKIRA, S., 2003. Toll-like receptor signaling. Journal of Biological Chemistry, 278(40), pp. 38105-38108.

BEZEMER, G.F.G. and GARSSEN, J., 2021. TLR9 and COVID-19: A Multidisciplinary Theory of a Multifaceted Therapeutic Target. Frontiers in pharmacology, 11, pp. 601685.

KAWAI, T. and AKIRA, S., 2011. Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity. Immunity, 34(5), pp. 637-650.

PASARE, C. and MEDZHITOV, R., 2005. Toll-like receptors: Linking innate and adaptive immunity. Mechanisms of Lymphocyte Activation and Immune Regulation X: Innate Immunity, 560, pp. 11-18.

Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm. 2010;2010:672395. doi:10.1155/2010/672395

Shaw AC, Panda A, Joshi SR, Qian F, Allore HG, Montgomery RR. Dysregulation of human Toll-like receptor function in aging. Ageing Res Rev. 2011;10(3):346-353. doi:10.1016/j.arr.2010.10.007

TAKEDA, K. and AKIRA, S., 2004. TLR signaling pathways. Seminars in immunology, 16(1), pp. 3-9.

TAL, Y., ADINI, A., ERAN, A. and ADINI, I., 2020. Racial disparity in Covid-19 mortality rates - A plausible explanation. Clinical immunology (Orlando, Fla.), 217, pp. 108481.

VAN DER MADE, C.I., SIMONS, A., SCHUURS-HOEIJMAKERS, J., VAN DEN HEUVEL, G., MANTERE, T., KERSTEN, S., VAN DEUREN, R.C., STEEHOUWER, M., VAN REIJMERSDAL, S.V., JAEGER, M., HOFSTE, T., ASTUTI, G., COROMINAS GALBANY, J., VAN DER SCHOOT, V., VAN DER HOEVEN, H., HAGMOLEN OF TEN HAVE, W., KLIJN, E., VAN DEN MEER, C., FIDDELAERS, J., DE MAST, Q., BLEEKER-ROVERS, C.P., JOOSTEN, L.A.B., YNTEMA, H.G., GILISSEN, C., NELEN, M., VAN DER MEER, J.W.M., BRUNNER, H.G., NETEA, M.G., VAN DE VEERDONK, F.L. and HOISCHEN, A., 2020. Presence of Genetic Variants Among Young Men With Severe COVID-19. Jama, .

Kovats, Cell Immunol. 2015 April; 294(2): 63–69;

Takahashi and Iwasaki, Science. 2021 Jan 22;371(6527):347-348

Scully EP, et al. Nat Rev Immunol. 2020. PMID: 32528136

 

Event: 1496: Increased, secretion of proinflammatory mediators

Short Name: Increased proinflammatory mediators

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat

Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as a diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.

Key Event Description

Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-3, IL-5 and Granulocyte-macrophage colony stimulating factor (GM-CSF) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (IL-1α , IL-1β, IL-1rα, IL-18, IL-36α, IL-36β, IL-36γ, IL-36Rα, IL-37), IL-6 family, Tumor necrosis factor (TNF) family, IL-17, and Interferon gamma (IFN-γ) (Turner et al., 2014). While IL-4 and IL-5 are considered T helper (Th) cell type 2 response, IFN-γ is suggested to be Th1 type response.

Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNF-α, IFN-γ are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002).

Literature evidence for its perturbation:

Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-α, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-κB], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-κB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte chemoattractant protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).

How it is Measured or Detected

The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.

Real-time reverse transcription-polymerase chain reaction (qRT-PCR) – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.

Enzyme-linked immunosorbent assays (ELISA) – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample.

Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).

Cell models - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used in vitro due to the large literature base, and ease of use, but do not adequately mimic the in vivo condition (Sharma et al., 2016; Sundarakrishnan et al., 2018). Recently, the EpiAlveolar in vitro lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNTs, and researchers noted increases in the pro-inflammatory molecules TNF-α, IL-1β, and the pro-fibrotic TGF-β using ELISA (Barasova et al., 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the in vivo pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments.

References

1. Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. 

2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

3. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. 

4. Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.

5. Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. 

6. Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.

7. Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.

8. Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.

9. Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.

10. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.

11. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.

12. Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P₂) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.

13. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.

14. Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. 

15. Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. 

16. Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013.

17. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. 

18. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.

Event: 1750: Increased inflammatory immune responses

Short Name: Increased inflammatory immune responses

AOPs Including This Key Event

Biological Context

Event: 1748: Increase, the risk of acute respiratory failure

Short Name: Increase, the risk of acute respiratory failure

AOPs Including This Key Event

Biological Context

List of Adverse Outcomes in this AOP

Event: 351: Increased Mortality

Short Name: Increased Mortality

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

All living things are susceptible to mortality.

Key Event Description

Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.

How it is Measured or Detected

Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.

Depending on the species and the study setup, mortality can be measured:

• in the lab by recording mortality during exposure experiments
• in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species
• in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.

Regulatory Significance of the AO

Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.

Appendix 2

List of Key Event Relationships in the AOP