All hands on deck: SARS-CoV-2 proteins that block early anti-viral interferon responses

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is responsible for the current pandemic coronavirus disease of 2019 (COVID-19). Like other pathogens, SARS-CoV-2 infection can elicit production of the type I and III interferon (IFN) cytokines by the innate immune response. A rapid and robust type I and III IFN response can curb viral replication and improve clinical outcomes of SARS-CoV-2 infection. To effectively replicate in the host, SARS-CoV-2 has evolved mechanisms for evasion of this innate immune response, which could also modulate COVID-19 pathogenesis. In this review, we discuss studies that have reported the identification and characterization of SARS-CoV-2 proteins that inhibit type I IFNs. We focus especially on the mechanisms of nsp1 and ORF6, which are the two most potent and best studied SARS-CoV-2 type I IFN inhibitors. We also discuss naturally occurring mutations in these SARS-CoV-2 IFN antagonists and the impact of these mutations in vitro and on clinical presentation. As SARS-CoV-2 continues to spread and evolve, researchers will have the opportunity to study natural mutations in IFN antagonists and assess their role in disease. Additional studies that look more closely at previously identified antagonists and newly arising mutants may inform future therapeutic interventions for COVID-19.

Keywords: 3CLpro, 3-chymotrypsin like protease; COVID-19, coronavirus disease of 2019; IFN, interferon; IFNAR, interferon alpha/beta receptor; IFNLR, interferon lambda receptor; IRF, interferon response factor; ISRE, interferon stimulated response element; Immune evasion; MAVS, mitochondrial antiviral-signaling protein; MDA-5, melanoma differentiation-associated protein 5; ORF, open reading frame; ORF6; PLpro, papain-like protease; RIG-I, retinoic acid-inducible gene I; SARS-CoV-2; SARS-CoV-2, SARS coronavirus 2; SRP, signal recognition particle; STAT, signal transducer and regulator of transcription; SeV, Sendai virus; TAB1, TGF-beta activated kinase 1 binding protein 1; TAK1, TGF-beta activated kinase 1; TBK1, TANK-binding kinase 1; TLR, toll-like receptor; TRIF, TIR domain-containing adapter-inducing interferon beta; Type I interferon; UTR, untranslated region; eIF, eukaryotic initiation factor; nsp, non-structural protein; nsp1.

Graphical abstract

Fig. 1

Early response to SARS-CoV-2 infection can influence the course of COVID-19. The diagram represents the model that the efficiency of the early type I IFN response to infection may influence the outcome of disease.

Fig. 2

Viral proteins disrupt the interferon response at many different points A) SARS-CoV-2 proteins antagonize interferon induction 1. ORF9b inhibits IFN induction via MDA5 and RIG-I by binding with TOM70 and inhibiting MAVS activation (Gao et al., 2021; Jiang et al., 2020). 2. The N protein binds to TRIM25, inhibiting RIG-I activation (Gori Savellini et al., 2021). 3. Nsp5 viral protease 3CLpro cleaves TAB1, inhibiting the activation of NFkB (Moustaqil et al., 2021) 4. ORF9b binds to NEMO and blocks NFkB signaling (Wu et al., 2021) 5. Nsp13 binds TBK1 to prevent its phosphorylation (Vazquez et al., 2021; Xia et al., 2020). 6. Nsp6 binds to TBK1 preventing the phosphorylation of transcription factor IRF3 (Xia et al., 2020). 7. Nsp3 viral protease PLpro cleaves IRF3 prior to phosphorylation (Moustaqil et al., 2021). 8. ORF6 binds to the Nup98-Rae1 complex in the nuclear pore complex and prevents nuclear translocation of transcription factors and nuclear export of mRNAs and mRNA transporters (Kimura et al., 2021; Addetia et al., 2021; Kato et al., 2021; Miorin et al., 2020) 9. SARS-CoV-2 nsp1 prevents host translation by binding to the 40s ribosomal unit and blocking the mRNA entry channel (Banerjee et al., 2020; Schubert et al., 2020; Thoms et al., 2020; Yuan et al., 2020). B) SARS-CoV-2 proteins inhibit interferon response and ISG production 10. Nsp14 marks the interferon receptor IFNAR1 for lysosomal degradation (Hayn et al., 2021). 11. STAT1 and/or STAT2 phosphorylation after type I IFN stimulation is inhibited by nsp1, nsp6, nsp13, ORF3a, ORF7b, M, and N (Xia et al., 2020). 12. Nsp16 binds to U1 and U2 small nuclear RNAs and blocks splicing of pre-mRNA (Banerjee et al., 2020) 13. Nsp8 and 9 bind 7SL in the signal recognition particle (SRP) complex, disrupting protein trafficking and resulting in the degradation of newly translated proteins (Banerjee et al., 2020) 14. Viral proteases have been found to cleave interferon-stimulated antiviral proteins after they are formed. PLpro cleaves ISG15 (Shin et al., 2020), and 3CLpro cleaves RNF20 (Zhang et al., 2021).

Fig. 3

Multiple SARS-CoV-2 proteins inhibit type I IFN induction and response. Charts of studies that have tested the role of SARS-CoV-2 proteins on type I IFN (A) or interferon-stimulated gene (ISG) induction (B). Proteins that showed an effect in each study are in red, proteins that did not have an effect in grey and proteins that were not tested in white. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Nsp1 binds to ribosomal subunit 40s and blocks the mRNA entry channel. Structural and protein-RNA interactions study show that two helices at the C terminus of nsp1 bind to the mRNA channel on the 40S subunit blocking mRNA access to it and inhibiting translation (Banerjee et al., 2020; Schubert et al., 2020; Thoms et al., 2020; Yuan et al., 2020). The C-terminal nsp1 helices and the hairpin between them make contact with the uS3, uS5 and uS30 ribosomal proteins, and with the helix h18 of the 18S rRNA (Banerjee et al., 2020; Schubert et al., 2020; Thoms et al., 2020; Yuan et al., 2020). The residues highlighted in the figure mediate key interactions and are required for translation inhibition by nsp1 (Vazquez et al., 2021; Banerjee et al., 2020; Schubert et al., 2020; Shen et al., 2021; Thoms et al., 2020; Yuan et al., 2020). As shown in the panel on the right, the 5′ UTR sequence of SARS-CoV-2 can restore translation of transcripts. This is mediated by the SL1 stem loop. SL1 needs to be located at the very 5′ end of the transcript to promote translation, suggesting that it does not simply function to recruit ribosomes, like an IRES (Banerjee et al., 2020).

Fig. 5

ORF6 binds to the Nup98-Rae1 complex and blocks bidirectional nucleocytoplasmic transport. ORF6 obstructs nuclear pore trafficking by binding to mRNA export proteins Nup98 and Rae1 (Kimura et al., 2021; Kato et al., 2021; Miorin et al., 2020; Gordon et al., 2020). ORF6 binding to the Nup98-Rae1 complex prevents nuclear translocation of transcription factors including IRF3 and STATs (Kimura et al., 2021; Addetia et al., 2021; Kato et al., 2021; Miorin et al., 2020). Mutation of methionine 58 to an arginine (M58R) prevents ORF6 binding to Nup98-Rae1, but does not affect interactions with importins (KPNA1 and KPNA2) (Miorin et al., 2020). The M85R mutation rescues the effect of ORF6 on STAT1/2 translocation to the nucleus, demonstrating that SARS-CoV-2 ORF6 activity does not depend on interactions with importins, unlike that of its SARS-CoV ortholog (Frieman et al., 2007; Miorin et al., 2020). In addition to preventing nuclear entry of proteins, ORF6/Nup98-Rae1 binding inhibits nuclear export of mRNAs and results in an accumulation of polyA + mRNAs in the nucleus (Addetia et al., 2021).