SARS-CoV-2 shortens the lifespan


A new study by researchers in the field of molecular biology, genetics and oncology from the Laboratoire de Biologie Structurale de la Cellule (BIOC), CNRS, Ecole Polytechnique- France has found that the NSP2 proteins of the SARS-CoV-2 coronavirus is able to bind to the human 4EHP-GIGYF2 complex, impairing microRNA-mediated silencing.

The study findings were published in the peer reviewed journal: i Science by Cell Press.

The repercussions from the impairment of such an important cellular process is varied and the inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could lead to many health disorders ranging from neurodevelopmental and neuropsychiatric disorders, heart disorders, immune issues, cancers and a host of health issues which shorten a person’s lifespan.
Hence this new study also adds to the growing evidence and validation of our hypothesis that all those who got exposed to the SARS-CoV-2 virus would have shortened lifespans. 

Upon infection, SARS-CoV-2 impairs splicing, export, translation and degradation of host mRNAs (Finkel et al., 2021, Banerjee et al., 2020). Here, we present evidence to support a new layer of complexity in the post-transcriptional alteration of the host transcriptome by SARS-CoV-2. We propose that SARS-CoV-2 NSP2 directly targets the 4EHP-GIGYF2 complex to decrease its silencing capacity (Figure 4D).

While our model lacks the context of other viral proteins that would be present in a bona fide infection, this mechanism could nonetheless unveil the impact of NSP2 on the post-transcriptional silencing of gene expression of human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses. Further studies in a more physiological context, such as lung/airway cell lines or SARS-CoV-2 infected samples, should help resolve this conundrum.

How does NSP2 impair 4EHP-GIGYF2 function? Combining co-IP experiments and in vitro binding assays with recombinant proteins, we concluded that NSP2 uses its N-terminal region encompassing its conserved zinc finger domain, to interact with the 4EHP-GIGYF2 complex.

Our pull-down assays indicate the direct interaction of NSP2 with both 4EHP and two domains from GIGYF2, confirming a sophisticated mode of binding in cellulo. While we searched for the minimal region of NSP2 required for these interactions, we

failed to narrow down a fragment smaller than the 1-350 region since truncations at both extremities of this domain abrogate its binding to 4EHP-GIGYF2 (Figures 1E and S2D). Nevertheless, this remains in agreement with Gupta et al. who pointed out that the G262V and G265V mutations located within this region of NSP2 reduced binding to 4EHP-GIGYF2 (Gupta et al., 2021).

This natural variation occurs in a poorly conserved patch in NSP2 that is subsequently becoming more hydrophobic due to the G to V substitution. It is worth noting that G262 and G265 are not conserved across the SARS-CoV-1 and MERS-CoV (Supplementary Figure S3C), while the NSP2/4EHP-GIGYF2 interaction exists among these viruses.

The direct contribution of the G262/G265 residues of NSP2 in binding 4EHP-GIGYF2 is therefore questionable. It is tempting to speculate that the G to V variation could rather increase NSP2’s affinity for host interactors that outcompete 4EHP- GIGYF2. This point is supported by the affinity purification mass spectrometry made by Gupta et al., showing that the G262V/G265V variation increases the affinity of NSP2 for factors such as the mitochondrial protein UQCRC1, or the actin-nucleation-promoting protein WASHC5 (Gupta et al., 2021).

This could explain why the G262V/G265V variation reduces the NSP2/4EHP-GIGYF2 interaction in cellulo, but not in our in vitro pull-down assay lacking the context of other host interactors of NSP2. Further investigation will be needed to fully resolve the structural basis of the NSP2/4EHP-GIGYF2 complex and thus elucidate NSP2 action on translation silencing.

In particular, it now remains to be determined whether NSP2 binding induces conformational changes in 4EHP-GIGYF2 that impair either the cap-binding pocket of 4EHP, or influence the recruitment of GIGYF2’s co-factors such as CCR4-NOT and DDX6.

Global measurement of miRNA action showed that translational repression accounts for 6–26% of the silencing of each mRNA target in mammalian cells, and 4EHP-GIGYF2-mediated translational repression is observed at early time points of the silencing process (Schopp et al., 2017, Eichhorn et al., 2014). Consistent with these observations, the impact of NSP2 remains mild on the let7a-targeted RLuc reporter (Figure 4A).

The latter is also known to underestimate the contribution of the translational repression to the silencing process since mRNA destabilization is the dominant effect of miRNA-mediated silencing at steady state (Eichhorn et al., 2014, Bethune et al., 2012). Our tethering assays with GW182SD have proven helpful to overcome this limitation.

The derepression of the GW182SD-induced silencing of RLuc-5BoxB-A114-N40-HhR upon deletion of the PPGL motif supports the fact that our tethering assay faithfully recapitulates the contribution of GIGYF2 into miRNA-induced translation repression. Consistently, these tethering assays showed that the silencing capacity of GW182SD upon NSP2 expression equals the one of its ΔPPGL version in control cells (Figures 4B and 4C), indicating that the contribution of 4EHP-GIGYF2 into GW182SD- mediated translation repression is fully targeted by NSP2. Indeed, this impact of NSP2 will need to be investigated in further more physiological studies using endogenous miRNAs and transcripts.

At the moment it is uncertain what the functional interplay between SARS-CoV-2 infection and miRNA-mediated silencing is in human cells. Host miRNAs are known to be produced as a part of antiviral response to counteract the infection by targeting viral transcripts, although SARS-CoV-2 infection was recently shown to have minimal impact on the miRNA repertoire of its host cell (Cullen, 2006, Bruscella et al., 2017, Pawlica et al., 2021).

Computational analyses have predicted the presence of many putative miRNA- binding sites on the SARS-CoV-2 genome, suggesting that the SARS-CoV-2 genome could be actively targeted by host miRNAs (Xie et al., 2021, Arisan et al., 2020, Chow and Salmena, 2020).

Particularly worth mentioning is the work of Xie et al. which recently identified let-7 binding sites in the coding sequence of S and M proteins of SARS-CoV-2 genome, and experimentally confirmed that let-7 blocks SARS-CoV-2 replication by targeting S and M proteins (Xie et al., 2021).

Through the NSP2/4EHP-GIGYF2 axis, SARS- CoV-2 could therefore escape from the host defense system by impairing the function of the effector machinery of miRNAs. The recent discovery that 4EHP and GIGYF2 are needed for infection by SARS-CoV-2 could reinforce this idea, although further research will be required to test this possibility (Hoffmann et al., 2021).

While the silencing capacity of miRISC is partially impeded upon NSP2 expression, there is no guarantee that miRNA action is the prime target of NSP2. The activity of 4EHP-GIGYF2 is mobilized by several pathways, one of which may be more affected than miRNAs. These pathways include TTP- and ZNF598-mediated mRNA silencing, as well as the repression of mRNAs with altered ribosome activity or premature termination codons as part of the nonsense-mediated mRNA decay pathway (Christie and Igreja, 2021).

Future studies are thus mandatory to evaluate the potential impact of NSP2 in modulating these processes in human cells. In the case of miRNA, the alteration of let-7a-mediated inhibition by NSP2 could be extrapolated to other miRNAs whose action relies on 4EHP, such as miR-145 or miR-34a (Jafarnejad et al., 2018, Zhang et al., 2021).

Recent evidence demonstrated that the 4EHP/miR-34a axis is required for the translational repression of mRNAs encoding IFN-β through targeting the 3’UTR of Ifnb1 mRNA (Zhang et al., 2021). Beyond miRNA, 4EHP-GIGYF2 also controls the production of TTP-targeted mRNAs that encode inflammatory cytokines such as TNF-α and IL-8 (Morita et al., 2012, Fu et al., 2016, Villaescusa et al., 2009, Cho et al., 2006, Cho et al., 2005).

In this context, a possible consequence of NSP2 expression could be the overproduction of early response pro-inflammatory cytokines. Exploring this point would be of utmost importance since impaired type I interferon activity and inflammatory responses are detected in severe COVID-19 patients (Hadjadj et al., 2020). To examine whether NSP2 could impact the function of 4EHP in regulating IFN-β expression, we expressed NSP2 along with  a reporter  construct  containing the 3′ UTR    of Ifnb1 mRNA into HEK293T cells (Supplementary Figure S4A).

Remarkably, the reporter expression was repressed ∼2.9-fold in control cells, but only ∼1.6-fold in NSP2-expressing cells, indicating that NSP2 could potentially unbalance the production of IFN-β through the Ifnb1 3′ UTR (Supplementary Figure S4B). With this in mind, further investigations into whether the NSP2/4EHP-GIGYF2 axis can dysregulate sustained cytokine production may therefore prove useful.

In conclusion, our study raises the possibility that SARS-CoV-2 could target the human 4EHP- GIGYF2 complex to selectively modulate its capacity to effect translation repression. Our model may represent a novel framework to investigate the mechanisms underlying the pathogenicity of SARS-CoV-2 based on the interaction of NSP2 with 4EHP-GIGYF2. Ultimately, we hope that this study will be a primer for further more physiological research to evaluate the generalizability of our model.


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