SARS-CoV-2 ORF9b Antagonizes Type I and III Interferons


Chinese scientist from Shandong University in Jinan-China have discovered that the SARS-CoV-2 ORF9b proteins are able to inhibit type I and III interferon (IFN) response and production by targeting various components of various immune signaling pathways.

The study findings were published in the peer reviewed Journal of Medical Virology. 

The assignment of the mutations in these ORFs may reveal further differences between SARS-CoV-2 variants and also constitute critical data, something that is significantly missing in the GISAID platform.
To date it has only been researchers from China that have been focused on the study and influence of these ORFs.
To date, various clinical findings indicated that the suppression of innate antiviral immunity, especially the type I and III interferon (IFN) production, contributes to the pathogenesis of COVID-19.
But how exactly the SARS-CoV-2 virus is able to evades antiviral immunity still remains unanswered with many hypothesis that are still not properly verified, hence needing further investigations.
The study team reported that the open reading frame 9b (ORF9b) protein encoded by the SARS-CoV-2 genome inhibits the activation of type I and III IFN response by targeting multiple molecules of innate antiviral signaling pathways.
According to the study findings, the SARS-CoV-2 ORF9b proteins impaired the induction of type I and III IFNs by Sendai virus or the dsRNA mimic poly (I:C).
It was found that the SARS-CoV-2 ORF9b proteins inhibits the activation of type I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε rather than IRF3-5D, the active form of IRF3.
Furthermore the SARS-CoV-2 ORF9b proteins also suppressed the induction of type I and III IFNs by TRIF and STING, the adaptor protein of endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively.
It should also be noted that another past study also by Chinese researchers published in the peer reviewed journal: Cellular and Molecular Immunology by Nature, also showed that the SARS-CoV-2 ORF9b suppresses interferon 1.
Mechanistically, SARS-CoV-2 ORF9b protein interacts with RIG-I, MDA-5, MAVS, TRIF, STING, TBK1, and prevents TBK1 phosphorylation, thus impeding the phosphorylation and nuclear trans-localization of IRF3 activation.

Another previous Chinese study has also showed that SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling.
Interestingly, it was found that the overexpression of SARS-CoV-2 ORF9b proteins facilitates the replication of the vesicular stomatitis virus.
Hence the study team concluded that SARS-CoV-2 ORF9b proteins negatively regulate antiviral immunity, facilitating virus replication.
It must also be noted that the SARS-CoV-2 is more sensitive to IFN treatment than other coronaviruses, multiple viral proteins become more critical to suppress IFN production at different steps to ensure the production and function of IFNs are minimized during SARS-CoV-2 infection.
The SARS-COV-2 ORF9b targets multiple proteins of the distinct immune signaling pathways, which may suppress IFN signaling at different steps.
Similarly, SARS-CoV- 2 ORF6 and MERS-CoV ORF4b are capable of perturbing multiple innate antiviral signaling pathways by target various components of these pathways.
Although cGAS-STING is cytosolic dsDNA sensing pathway, coronaviruses, a family of RNA viruses, also encode viral proteins such as papain-like protease to impair STING function; thus, this pathway is essential in defending against coronavirus infection.
The study team pointed out that since the inhibition of cGAS-STING pathway by SARS-CoV-2 5 ORF9b may suggest that this pathway may play a role in SARS-CoV-2 clearance, drugs or chemicals such as 2’-3’cGAMP that activates this pathway may be considered for use in COVID-19 treatments.
The study team added, “Although the administration of exogenous IFNs is show to be valid for SARS-CoV2 clearance on both SARS-CoV-2 patients and cell models, full evaluation of this treatment requires extensive studies on the relative importance of all IFN-antagonizing viral proteins encoded by SAR-CoV-2.

Thus, our findings that the SARS-COV-2 ORF9b suppresses type I and III IFN production contribute to our understanding of the pathogenesis of COVID-19, and the identification of multiple protein targets may provide more precise treatment of COVID-19.

The study findings contributes to our understanding of the molecular mechanism of how SARS-CoV-2 impairs antiviral immunity and providing an essential clue to the pathogenesis of COVID-19.”
The genome of SARS-CoV-2 is about 30 kb in length encoding 14 putative open reading frames, including the large replicase genes expressing two replicative polyproteins (pp1a and pp1ab) that would be cleaved into NSP1-16 by viral proteases, the structural genes expressing spike (S), membrane (M), envelop (E), and nucleocapsid (N) protein, and the accessory genes expressing ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10.
To date, we still do not know much about all these ORFs and also about the rest of the proteins on the genome besides the Spike proteins. The old school of virology which only focuses on certain proteins can no longer hold in the case of the SARS-CoV-2 virus when in comes to genomic surveillance, understanding  the  virus transmissibility, virulence, immune evasive characteristics, pathogenesis, influence on various human host cellular pathways and the development of therapeutics including vaccines.
Although the accessory proteins of coronaviruses are not essential for viral replication and virion assembly, they contribute to the virulence by affecting the virus release, stability, and pathogenesis. To date, the function of SARS-CoV-2 accessory proteins in immune evasion is still not properly understood and warrants more studies.

Although the SARS-CoV-2 infection-mediated dysregulation of the immune system, which involves the suppression of antiviral immunity and the elevation of inflammatory responses, contributes to the pathogenesis of COVID-19,6-11 the mechanism through which the recently emerged SARS-CoV-2 is recognized by innate immunity has not been clarified.

Double-stranded RNA (dsRNA), which is produced by many viruses during replication, is a common viral pathogen-associated molecular pattern that is sensed by pattern recognition receptors.12

Cytosolic retinoic acid-inducible gene (RIG)-I-like receptors, including RIG-I and MDA-5, and endosomal Toll-like receptor 3 (TLR3) recognize dsRNAs from intermediates generated during viral replication, and this recognition results in the serial activation of innate antiviral signaling cascades via induction of the production of types I and III IFNs.12, 13

The coronaviruses have homologous genomes, similar replication intermediates, and the same lifecycles; thus, it appears that SARS-CoV-2 can be recognized by RNA sensors similarly to other coronaviruses to elicit innate antiviral immunity.14

RIG-I participates in the immune sensing of murine coronavirus mouse hepatitis virus (MHV) in oligodendrocyte cells.15 MDA5 can recognize MHV in brain macrophages, microglial cells, and oligodendrocyte cells.15, 16 The sensing of cytosolic dsRNA by RIG-I/MDA-5 recruits the adaptor protein MAVS (also known as VISA, Cardiff, or IPS-1), which activates TANK-binding kinase 1 (TBK1)/inhibitor of κB kinase epsilon (IKKε) and then induces the phosphorylation and subsequent nuclear translocation of the transcription factor IFN regulatory factor 3 (IRF3), and nuclear IRF3 together with nuclear factor-κB (NF-κB), which is also activated by RIG-I/MDA-5 signaling, initiates the transcription of types I and III IFNs and other proinflammatory cytokines, which lead to antiviral immune responses.12

TLR3 is involved in the defense against SARS-CoV-1 infection.17 dsRNA-activated TLR3 activates IRF3 and NF-κB signaling via TIR-domain-containing adapter-inducing interferon-β (TRIF)-TBK1/IKKε signaling cascades, which results in the production of types I and III IFNs and other pro-inflammatory cytokines.12

Although the involvement of the cytosolic DNA-sensing pathway of cGAS-stimulator of IFN genes (STING) signaling in the recognition of coronaviruses has not been elucidated, the papain-like protease domain from SARS-CoV-1 can act as an antagonist of IFNs by targeting STING,18, 19 which suggests that the cGAS-STING pathway should play a vital role in the defense against certain coronaviruses. STING is activated by the second messenger 2ʹ−3ʹcGAMP produced by DNA-activated cGAS.20

Subsequently, STING recruits TBK1, which phosphorylates IRF3, and this phosphorylation leads to the translocation of IRF3 into the nucleus to induce the expression of types I and III IFNs and other proinflammatory cytokines.20 The RIG-I/MDA-5–MAVS, TLR3–TRIF, and cGAS–STING signaling pathways converge at TBK1/IKKε, which catalyzes IRF3 phosphorylation and the subsequent transcription of types I and III IFNs.21

Secreted type I and III IFNs bind to their receptors and then activate Janus kinase/signal transducers and activators of transcription signaling to drive the expression of IFN-stimulated genes (ISGs), which can initiate antiviral states by suppressing viral replication and spreading, activating immune cells, and causing the death of infected cells.13, 22

The types I and III IFN response is the essential action of host antiviral immunity in the clearance of virus infection.13, 22 To establish a successful infection of host cells, viruses, including coronaviruses, have developed various strategies to antagonize the IFN response.14 Previous studies have proposed that the accessory proteins of SARS-CoV-1, such as open reading frame 3b (ORF3b), ORF6, and ORF9b, inhibit the production of type I IFNs.14 In COVID-19 patients, the induction of types I and III IFNs is suppressed.7, 8, 23

The replenishment of types I or III IFNs can significantly contribute to the clearance of SARS-CoV-2 and to COVID-19 symptom relief.24-26 Compared with type I IFNs, type III IFNs exhibit some advantages in COVID-19 treatment regarding the induction of a longer-lasting antiviral state and a less proinflammatory response.27

Although SARS-CoV-2 infection impairs the antiviral immunity elicited by types I and III IFNs in COVID-19 patients and cell models,7, 8, 23 the mechanism through which the recently emerged SARS-CoV-2 blocks the induction of types I and III IFNs remains elusive.

Therefore, dissecting the molecular mechanism through which SARS-CoV-2 evades types I and III IFN responses will improve the understanding of the pathogenesis of COVID19 and provide therapeutic strategies for counteracting SARS-CoV-2 infections.

SARS-CoV-1 ORF9b was reported to suppress IFN production; however, whether SARS-CoV-2 ORF9b could evade host antiviral innate immunity is still unknown; thus, we explored the effect of SARS-CoV-2 ORF9b on IFN production and the potential mechanism.

We found that the SARS-CoV-2 accessory protein ORF9b, which is encoded by an alternative ORF within the N gene, can remarkably suppress RIG-I/MDA-5–MAVS, TLR3–TRIF, and cGAS–STING signaling-activated types I and III IFN production by targeting multiple molecules of these innate antiviral pathways.

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