A team of researchers used co-immunoprecipitation (co-IP) and in vitro interaction assays to identify the molecular basis of interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 2 (NSP2) with eIF4E-homologous protein (4EHP)-GRB10-interacting glycine-tyrosine-phenylalanine domain protein 2 (GIGYF2) complex, involved in microRNA(miRNA)-mediated gene silencing in human cells.
The study can be found on the bioRxiv* preprint server before undergoing peer review.
Study: The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells. Image Credit: Kateryna Kon/Shutterstock
A better understanding of the influence of SARS-CoV-2 on the host machinery would help develop coronavirus disease 2019 (COVID-19) treatment strategies. The SARS-CoV-2 genome encodes 29 proteins, including 16 NSPS, with multiple functions in virus replication, but their specific roles in SARS-CoV-2 replication, dissemination, and impact on viral pathogenicity are poorly understood.
Previous proteomic-based screening studies found a close connection of SARS-CoV-2 proteins with multiple biological processes, such as protein trafficking, transcription, and mRNA translation. Among these, interactions of NSP2 with 4EHP and GIGYF2 could be of therapeutic importance. More specifically, the cap-binding activity of 4EHP in complex with GIGYF2 plays a significant role in the optimal translational repression by microRNAs (miRNAs) and the RNA-binding proteins ZNF598 and Tristetraprolin (TTP).
A recent genetic screen has also revealed that both 4EHP and GIGYF2 are necessary for infection by SARS-CoV-2 in vitro, most likely as they interact with NSP2, thus serving as a potential drug target for anti-SARS-CoV-2 drug development.
About the study
In the present study, the researchers used interaction assays and reporter-based approaches to illustrate the impact of NSP2 on the 4EHP-GIGYF2-mediated translational silencing of gene expression complex in human cells.
They validated the physical association of NSP2 with the GIGYF2-4EHP complex using co-IP. Subsequently, they performed a Western blot (WB) analysis of the co-IP fractions to show the efficiency of binding of NSP2 with endogenous GIGYF2 and 4EHP.
Before performing coIPs, the Flag-NSP2 expression was induced in the HEK293 Flp-In T-REX cells. As controls, lysates prepared from non-induced and induced cells were immunoprecipitated with an anti-Flag antibody. Knockout (KO) HEK293 cells were used to conduct co-IPs with Flag-NSP2 to determine which protein was involved in the formation of the 4EHP-GIGYF2 complex. Vectors expressing Flag-NSP2, or anti-Flag antibody, were transiently transfected in KO and their wild-type (WT) populations.
Finally, an in situ proximity ligation assay (PLA) examined the subcellular localization of NSP2, and confocal imaging illustrated the spatial proximity between Flag-NSP2 and GIGYF2.
Findings
Previous studies have shown that SARS-CoV-2 infection impairs splicing, export, translation, and degradation of host mRNAs. The current study results unveiled a new layer of complexity in the post-transcriptional alteration of the host transcriptome by SARS-CoV-2, wherein NSP2 directly targeted the 4EHP-GIGYF2 complex to impair the silencing capacity of miRNAs, thereby suppressing host defenses.
Using both co-IP experiments and in vitro binding assays, the researchers identified the role of the N-terminal region encompassing a conserved zinc finger domain within NSP2 that interacted with the 4EHP-GIGYF2 complex.
Upon investigating Flag IP in the 4EHP KO cells, the NSP2/GIGYF2 interaction was detectable, while ZNF598 co-IP was reduced, indicating a contribution of 4EHP in NSP2 binding. In the absence of GIGYF2, NSP2 did not react either with 4EHP or ZNF598, supporting a central role of GIGYF2 in NSP2 binding.
No spot-like PLA signal was observed in control cells transfected with a control plasmid. However, the confocal live imaging showed the sub-cellular/cytoplasmic distribution of NSP2, wherein both the expression of both eGFP-tagged GIGYF2 and mCherry-NSP2 resulted in a diffused cytoplasmic signal when vectors expressing these proteins were individually co-transfected in HEK293T cells.
The findings of the pull-down assays indicated direct interaction of NSP2 with both 4EHP and two domains from GIGYF2, confirming the mode of in cellulo binding largely attributable to the 1-350 fragment of these domains.
Future studies will have to determine whether NSP2 binding induces conformational changes in 4EHP-GIGYF2 that in turn either impairs the cap-binding pocket of 4EHP or influence the role of GIGYF2 co-factors CCR4-NOT and DDX6. With this information, the structural basis of the NSP2/4EHP-GIGYF2 complex will be fully resolved, thus confirming the role of NSP2 in miRNA-mediated silencing.
4EHP-GIGYF2 also controls the production of TTP-targeted mRNAs that encode proinflammatory cytokines such as interleukin 8 (IL-8). Hence, NSP2 expression could lead to an overproduction of these cytokines, investigating which would help understand the dynamics of COVID-19 in severely ill patients as impaired type I interferon activity and inflammatory responses have been observed in such patients.
To evaluate how NSP2 impacted 4EHP function in regulating interferon β (IFN-β) expression, NSP2 was expressed along with a reporter construct into HEK293T cells. The reporter expression was repressed ∼1.6-fold in NSP2-expressing cells, indicating that NSP2 could potentially unbalance the production of IFN-β.
Conclusions
The biochemical approaches and reporter-based assays used in the current study represent a novel framework to imagine an innovative therapeutic approach based on the interaction of NSP2 with 4EHP-GIGYF2.
Thus, the study raises the possibility that SARS-CoV-2 could target the human 4EHP-GIGYF2 complex to selectively modulate the expression of host or viral transcripts, opening new avenues to investigate the mechanisms underlying the pathogenicity of SARS-CoV-2, making NSP2 a therapeutic intervention target.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
