Severe acute respiratory syndrome Corona virus 2 (SARS-CoV-2) is a newly emerged pathogen and is associated with Corona virus disease 19 (COVID-19) which can manifest into life threatening upper respiratory pathologies and lung dysfunctions in severely affected individuals (V’kovski et al., 2021). According to the World Health Organization, greater than 257 million people have been infected with SARS-CoV-2 worldwide resulting in more than 5.1 million deaths. SARS-CoV-2 is an enveloped, single stranded, positive sense RNA virus with a genome of nearly 30 kb that is about 79% similar to SARS-CoV and 50% similar to Middle east respiratory syndrome Corona virus (MERS-CoV) (Lu et al., 2020). The SARS-CoV-2 genome contains 23 open reading frames (ORF) encoding several accessory and non-structural proteins (NSP) as well as four conserved structural proteins: Spike, Envelope, Membrane, and Nucleocapsid (Finkel et al., 2021; Redondo et al., 2021).

Innate immunity provides a first line of defense against invading pathogens including viruses in which the induction of type I interferon (IFN-I) cytokines (IFNα/β) plays an essential role in limiting virus replication and spread (Gonzales-van Horn and Farrar, 2015; Ghosh et al., 2016). While IFN-I is regulated at the level of transcription, primarily by the interferon regulatory factor 3 (IRF3) transcription factor, IRF3 activation itself requires engagement of upstream pattern recognition receptors (PRRs) in cells of the innate immune system. In the context of RNA virus infections, viral RNA species are detected by the retinoic induced gene-I (RIG-I) like PRRs situated in the cytosolic compartment (Akira et al., 2006; Pandey et al., 2014; Roers et al., 2016). RIG-I receptor ligation results in its association with the mitochondrial antiviral signaling adaptor, MAVS, which subsequently recruits TNF receptor associated factor 3 (TRAF3) and TRAF6. TRAF3 serves as a scaffold in facilitating complex formations with the viral activated kinases, TANK binding kinase 1 (TBK1) and/or I kappa B kinase epsilon/inducible (IKKi), which activate IRF3 via c-terminal phosphorylation to induce the IFN-I response. Alternatively, TRAF6 activates IKKα/β as part of the classical NF-ĸB pathway, leading to the induction of pro-inflammatory cytokines (Liu et al., 2013; Fang et al., 2017). IFN-I elicits an antiviral state by binding to the IFNα/β receptor (IFNAR) on neighboring cells. IFNAR ligation results in the activation of a Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway that culminates in the formation of a STAT1/STAT2/IRF9 heterotrimeric transcription factor which binds to IFN stimulated response elements (ISRE) found on the promoters and enhancers of IFN stimulated genes (ISGs). ISGs operate as effectors and restriction factors that directly or indirectly target the virus or at various stages of its replication cycle (Akira et al., 2006; Pandey et al., 2014).

Many viruses that have co-evolved with their hosts have developed complex and multiple strategies to subvert or antagonize the host innate immune response as a mechanism to evade immune detection and elimination. Indeed, it has become increasingly clear that multiple SARS-CoV-2 encoded genes products target innate immune signaling pathways at various levels (Xia et al., 2020; Fung et al., 2021; Han et al., 2021; Kumar et al., 2021; Sui et al., 2021a; Vazquez et al., 2021; Zhao et al., 2021). Here we identify the Spike protein of SARS-CoV-2 as a key negative regulator of IFN-I activation in the RIG-I pathway that targets the IRF3 transcription factor and mediates its proteasomal degradation. Our findings provide key insight as to how SARS-CoV-2 may subvert innate antiviral immunity to instigate disease pathogenesis.

SARS-CoV-2 Spike is a large type I transmembrane fusion protein that is heavily glycosylated and plays a key role in facilitating virus infection compared to the other surface proteins, membrane and envelope, found on Coronaviruses. SARS-CoV-2 Spike binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and in conjunction with the transmembrane protease serine 2 (TMPRSS2) found on the host cell membrane, mediates viral entry into the cell (Huang et al., 2020; V’kovski et al., 2021). Upon viral entry, viral RNA is released, allowing for polyprotein translation as well as genome replication and transcription events yielding viral assembly and packaging (Hartenian et al., 2020). Viral RNA is further detected by germ-line encoded PRRs including RIG-I, which triggers the activation of the innate antiviral immune response coordinated by IFN-I. These cytokines activate an antiviral program resulting in the induction of nearly 300 ISGs, which collectively foster an antiviral state leading to viral inhibition and spread. Here we identified SARS-CoV-2 Spike as a key inhibitor of IFN-I in the RIG-I PRR pathway. While Spike suppressed the IFN-I response upon RIG-I stimulation and the downstream induction of ISGs, it did not block NF-ĸB and pro-inflammatory cytokine activation normally conferred during RIG-I signaling. Additional reports indicate the RIG-I like receptor, melanoma differentiation associated gene 5 (MDA5) can also detect SARS-CoV-2 genomic RNA to trigger the host IFN-I response in human lung epithelial cells (Rebendenne et al., 2021; Sampaio et al., 2021; Yin et al., 2021). As MDA5 utilizes the same adaptor (MAVS), kinases (TBK1/IKKi), and transcription factor (IRF3) as RIG-I to activate IFN-I, it may be likely SARS-CoV-2 Spike suppresses MDA5 signaling via a similar mechanism. SARS-CoV-2 encodes a variety of other genes that antagonize innate immunity, suggesting SARS-CoV-2 employs a network of genes to collectively circumvent innate immune activation (Lei et al., 2020; Beyer and Forero, 2021).

In contrast to blocking the innate IFN-I response, we found Spike elevated a separate viral triggered cell stress pathway (UPR), suggesting that SARS-CoV-2 Spike does not globally antagonize broad antiviral programs. As the UPR pathway can initiate autophagic or cell death outcomes, it will be of future interest to determine the overall impact of SARS-CoV-2 Spike activation of this pathway and its impact on COV1D-19 pathology.

Consistent with our findings, it was recently reported that recombinant SARS-CoV-2 Spike protein, when incubated with primary cells from lung bronchoalveolar lavage from rhesus macaques, was able to suppress IFN-I levels (Sui et al., 2021b). Our data, in part, attempts to uncover the mechanism by which Spike suppresses the IFN-I response by revealing that SARS-CoV-2 Spike targets the IRF3 transcription factor. Recent studies found SARS-CoV-2 NSP6 inhibited IRF3 by suppressing its phosphorylation mediated by the upstream kinase TBK1 (Xia et al., 2020). Additionally, SARS-CoV-2 ORF6 was reported to limit IRF3 signaling by targeting host factors that facilitate IRF3 translocation into the nuclear compartment where it binds the IFNβ promoter after phosphorylation dependent activation (Xia et al., 2020). Similarly, SARS-CoV-2 NSP12 was shown to block IRF3 nuclear translocation, however a new report found NSP12 was incapable of inhibiting IFNβ activation (Li et al., 2021; Wang et al., 2021).

We found SARS-CoV-2 Spike interacted with IRF3 and further instigated its proteasomal mediated degradation to terminate IFN-I activation. SARS-CoV-2 Spike is comprised of an S1 and S2 domain where SARS-CoV-2 entry requires proteolytic cleavage at these sites (Huang et al., 2020). In many of our immunoblot experiments that examine ectopic Spike expression, we noted the detection of both the full length Spike as well as the faster migrating, but lower intensity S2 domain. However, in some of our immunoblots, (particularly relating to Spike-IRF3 characterizations, Figures 4A, D ), while the full- length Spike was detected, the S2 domain was below detection capabilities. This may possibly be due to technical circumstances in our experimental design where we reduced cell harvest times post Spike transient transfections in order to minimize excessive or complete IRF3 suppression. Nevertheless, our data suggests that proteasomal inhibition countered the inhibitory effect of Spike on IRF3 in terms of expression and IFN-I activation. Interestingly, we found proteasomal inhibition to not only restore IRF3 expression in the presence of Spike, but also to increase Spike expression and minimize its proteolytic cleavage ( Figure 4F ). Thus, it may be possible that SARS-CoV-2 Spike is subjected to host cell mediated proteasomal degradation. A recent study identified the 3C-like protease of SARS-CoV-2 to also inhibit IRF3 in a similar fashion, while another report indicated it cleaves IRF3 (Shin et al., 2020; Moustaqil et al., 2021; Zhang et al., 2021). Thus, additional studies will be needed to clarify exactly how the 3C-like protease inhibits IRF3. Nevertheless, if this protease indeed is involved in the proteasomal degradation of IRF3, it is tempting to speculate 3C-like protease and Spike may be operating together to subvert IFN-I activation. It is also noteworthy that Spike (nor 3C-like protease) is not known to confer E3 ubiquitin ligase function. As multiple E3 ligases have been described in fostering the degradation of IRF3, further investigation will be required to determine which E3(s) and potential co-factors cooperate with Spike to degrade IRF3 (Higgs et al., 2008; Zhang et al., 2008; Yu and Hayward, 2010; Lei et al., 2013; Wang et al., 2015, Chen et al., 2021).

Taken altogether, our work identified a novel mechanism by which the Spike protein of SARS-CoV-2 operates beyond its currently understood role in host cell attachment and entry. Accordingly, we found Spike antagonized the host IFN-I antiviral innate immune response in the RIG-I signaling pathway by targeting IRF3 for degradation. These findings shed new light into the potential mechanisms of SARS-CoV-2 pathophysiology and magnify the importance of Spike as a key target for therapeutic intervention against COVID-19.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

KP conceptualized the project and supervised the research. RF, TC, and KP performed the experiments and analyzed the data. KP prepared the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the Louisiana Board of Regents Support Fund LEQSF(2021-24)-RD-A-33 and Tulane SOM pilot funds awarded to KP.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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