Interferon-I antiviral response, antagonized by SARS-CoV-2

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.

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

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).

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