key: cord-0788753-hp1ptiyi
authors: Zou, Limei; Moch, Clara; Graille, Marc; Chapat, Clément
title: The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells
date: 2022-01-26
journal: bioRxiv
DOI: 10.1101/2022.01.25.477753
sha: 05177698418b619f0448c150da107309adfc8bc7
doc_id: 788753
cord_uid: hp1ptiyi

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in 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.

Beta-coronaviruses (β-CoVs) are enveloped RNA viruses that infect a variety of vertebrate hosts, including humans (1). In the last decades, two β-CoVs have caused epidemic diseases of the respiratory tract: severe acute respiratory syndrome (SARS-CoV-1) in 2002 (2, 3) and Middle East respiratory syndrome (MERS) in 2012 (4) . A new β-CoV (SARS-CoV-2) emerged in 2019 that is the causative agent of coronavirus disease 2019 (COVID-19) pandemic (5) . These three viruses possess single-stranded, positive-sense RNA genomes of nearly 30 kb in length (6) . The SARS-CoV-2 genome encodes 29 proteins with multiple functions in virus replication and packaging, including 4 structural proteins (the nucleocapsid N, envelope E, membrane M, and spike S proteins), 7 accessory proteins (ORF3a-ORF8) whose functions in SARS-Cov-2 pathogenesis remain largely unknown, and 16 non-structural proteins (NSP1-NSP16) that encode the RNA-directed RNA polymerase, helicase, protease, and other components required for virus replication (for review, see (7)).

Due to the urgent need to better understand SARS-CoV-2 biology, several CRISPR-Cas9 and proteomic-based screening campaigns investigated the landscape of host factors which are targeted by the virus proteome (8) (9) (10) (11) (12) . These screens identified more than 300 high-confidence protein-protein interactions between human and SARS-CoV-2 proteins, highlighting the intimate connection of SARS-CoV-2 proteins with multiple biological processes, including protein trafficking, transcription and mRNA translation. Among these interactions, the Non-Structural Protein 2 (NSP2) has been found to interact with key host proteins involved in vesicle trafficking (FKBP15, WASHC) and mRNA translation (4EHP, GIGYF2) which could be of therapeutic importance (10, 12) .

NSP2 exists in all coronaviruses studied to date, including SARS-CoV-1, SARS-CoV-2, MERS, and in their closely related β-CoVs infecting mammals. Although its role in the SARS-CoV-2 pathogenicity has not been fully elucidated, the deletion of NSP2 in SARS-CoV-1 attenuates viral growth and RNA synthesis (13) . Recent findings showed that SARS-CoV-2 NSP2 is undergoing positive nature selection and could be thus essential to the virus (14, 15) . At the structural level, NSP2 has a complex multi-domain topology including an N-terminal domain with a highly-conserved zinc binding site, and a C-terminal region rich in β-strands.

With the exception of the zinc binding site, NSP2 displays a rapidly evolving surface with the presence of natural variations that could impact host-virus interactions (16) (17) (18) (19) . Among its host interactors, SARS-CoV-2 NSP2 interacts with the 4EHP-GIGYF2 complex, a key machinery in translational silencing and mRNA decay (10) (11) (12) 20) . It is worth noting that the NSP2/4EHP-GIGYF2 association is conserved across SARS-CoV-1 and MERS, corroborating a functional importance for β-CoV infection in general (11, 20) .

The cap-binding eIF4E-Homologous Protein (4EHP) is an integral component of post-transcriptional silencing mechanisms through competing with the eIF4F complex for binding to the mRNA 5´cap (21) . In complex with the GRB10-Interacting GYF (glycine-tyrosine-phenylalanine domain) protein 2 (GIGYF2), the cap-binding activity of 4EHP is required for the optimal translational repression by microRNAs (miRNAs), as well as the RNA-binding proteins ZNF598 and Tristetraprolin (TTP) (22) (23) (24) (25) (26) (27) (28) . In the case of miRNA-driven silencing, the recruitment of 4EHP-GIGYF2 is initiated by the miRNA-induced silencing complex (miRISC), an assembly of argonaute and TNRC6/GW182 proteins. 4EHP can be physically mobilized through the interaction of the GYF domain of GIGYF2 with a proline-proline-glycine-leucine (PPGL) motif in TNRC6/GW182 (29, 30) . At the functional level, this 4EHP/miRNA axis is required to control ERK signaling, as well as to suppress IFN-β production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA (31, 32) .

Beyond miRNA action, 4EHP-GIGYF2 also forms a translation inhibitory complex with the RNAbinding protein ZNF598, which functions in ribosome stalling on internally polyadenylated mRNAs during ribosome quality control (22, 33) . ZNF598 is also necessary for the repression of TTP-targeted mRNAs that encode inflammatory cytokines. The 4EHP-GIGYF2-ZNF598 complex binds TTP during an innate immune response in mouse macrophages to control the production of TTP-targeted mRNAs such as TNF-α, Ier3, Csf2, and Cxcl10. In all cases, TNRC6/GW182, ZNF598 and TTP display a comparable binding mode to GIGYF2, namely via the recognition of a proline stretch by the GYF domain of GIGYF2 (23, 34) .

A recent genetic screen has revealed that both 4EHP and GIGYF2 are necessary for infection by SARS-CoV-2 in vitro, while dispensable for seasonal coronaviruses (8) . This contribution of 4EHP-GIGYF2 could likely originate from their interaction with NSP2, and thus serves as a potential drug target for anti-SARS-CoV-2 drug development. In the present report, we focused on the physical and functional interplays existing between the 4EHP-GIGYF2 complex and the SARS-CoV-2 protein NSP2 in human cells. Combining interaction assays and reporter-based approaches, our data shed light on the profound impact of NSP2 on the 4EHP-GIGYF2-mediated translational silencing of gene expression.

Early large scale studies reported the capacity of SARS-CoV-2 NSP2 to bind the 4EHP-GIGYF2 complex using affinity-purification mass spectrometry (10, 11) . To validate the physical association of NSP2 with the GIGYF2-4EHP complex, we first sought to detect their interaction using co-immunoprecipitation (co-IP). An inducible Flag-tagged version of NSP2 was stably expressed in HEK293 Flp-In T-REX cells and co-IPs were performed following tetracycline-induced expression of Flag-NSP2. Lysates prepared from control, non-induced, and induced cells were immunoprecipitated with anti-Flag antibody. Subsequent Western blot (WB) analysis of the co-IP fraction showed that Flag-NSP2 efficiently binds both endogenous GIGYF2 and 4EHP (Fig. 1A) . This interaction was similarly detected in RNase A-treated lysates, indicating that the NSP2/4EHP-GIGYF2 interaction occurs in an RNA-independent manner. The GIGYF2-associated protein ZNF598 was also found along with NSP2, while CNOT9, a subunit of the CCR4-NOT complex known to bind GYGYF2, was not detected (35) (Fig. 1A) . Together, these data demonstrate that NSP2 physically associates with the 4EHP-GIGYF2 complex, as well as their interacting protein ZNF598.

To elucidate which protein is involved in the formation of this complex, GIGYF2-and 4EHP-Knock-Out (KO) HEK293 cells were used to conduct co-IPs with Flag-NSP2. Vectors expressing Flag-NSP2, or Flag as a control, were then transiently transfected in these KO populations, as well as in their Wild-Type (WT) counterpart. Following Flag IP in the 4EHP KO cells, we observed that the NSP2/GIGYF2 interaction was still detectable, while ZNF598 co-IP was reduced, indicating a plausible contribution of 4EHP in NSP2 binding. By contrast, in the absence of GIGYF2, we could not detect any interaction of NSP2 with either 4EHP or ZNF598 (Fig. 1B) , supporting a central role of GIGYF2 in NSP2 binding. However, a contribution of 4EHP cannot be excluded since its level is decreased in the GIGYF2 KO cells due to a co-stabilization effect ( Fig.   1B and (22)).

The sub-cellular localization of NSP2 was then examined using an in situ proximity ligation assay (PLA). PLA was conducted in Flag-NSP2-expressing cells and antibodies raised against the Flag sequence and endogenous GIGYF2 were used. Following confocal imaging, a spot-like signal was abundantly detected in the cytoplasm of HEK293T cells expressing Flag-NSP2, indicating the spatial proximity between endogenous GIGYF2 and Flag-NSP2 (Fig. 1C ). This PLA signal was not observed in cells transfected with a control plasmid, supporting the specificity of this interaction. We then performed confocal live imaging to analyze the sub-cellular distribution of NSP2. Vectors expressing mCherry-tagged NSP2 fused either at the Nor C-terminal extremity were individually co-transfected along with an eGFP-tagged GIGYF2-encoding vector in HEK293T cells. A diffuse cytoplasmic signal was detected upon the expression of both eGFP-tagged GIGYF2 and mCherry-NSP2, indicating their cytoplasmic distribution (Supplementary Fig. 1A and B). The eGFP-GIGYF2 pattern was also punctuated by distinct condensates, in agreement with the presence of GIGYF2 in P-bodies, a subclass of RNA granules enriched in translationally repressed mRNAs and silencing factors (36) . We did not find any co-localization between mCherry-fused NSP2 and the eGFP-GIGYF2 foci (Supplementary Fig. 1A and B), indicating that NSP2 could preferentially bind the diffuse form of GIGYF2 instead of its P-body-associated counterpart. To confirm this point, we took advantage of the properties of biotinylated-isoxazole (b-isox), a compound which selectively precipitates proteins located in RNA granules such as P-bodies and stress granules (37, 38) (Supplementary Fig. 1C ). Extracts of HEK293T expressing Flag-NSP2 were exposed to 100 μM of b-isox, or DMSO as a mock control. By comparing the level of proteins left in the soluble fraction after precipitation (unbound) with the precipitated fractions (pellet), we confirmed that Flag-NSP2 was not recovered in the pellet whereas a significant proportion of endogenous GIGYF2 was selectively enriched in the b-isox precipitate, alongside DDX6, a known resident of P-bodies ( Supplementary   Fig. 1D ). Since NSP2 could not be precipitated by b-isox along with GIGYF2, we concluded that it only binds the diffuse form of GIGYF2 in the cytoplasm. We next investigated which part of NSP2 binds the 4EHP-GIGYF2 complex. For this purpose, we generated a collection of vectors encoding Flag-tagged truncated versions of NSP2 which were expressed in HEK293T for Flag IP ( Fig. 1D and E). Incremental terminal deletions revealed that the N-terminal half of NSP2 is required for the maximal co-IP of the endogenous 4EHP-GIGYF2 complex as well as ZNF598. While weakly expressed, the NSP2 1-350 fragment remained the minimal segment which was able to bind 4EHP-GIGYF2. Interestingly, deleting either the N-terminal extremity (fragment NSP2 107-350 ) or the middle region (fragment NSP2 1-212 ) impaired the NPS2/4EHP-GIGYF2 interaction, indicating a large interaction surface involving an intact N-terminal half of NSP2 (Fig. 1E) .

We then sought to delineate which part of the 4EHP-GIGYF2 complex is targeted by NSP2. Human GIGYF2 is a 150 kDa scaffolding protein composed of several domains interspaced by intrinsically disordered regions. These include the 4EHP-binding domain at the N-terminus, the so-called GYF domain, a putative Single Alpha-Helix (SAH) and many glutamine-rich stretches (polyQ) at the C-terminus ( Fig. 2A ) (22, (39) (40) (41) .

V5-tagged fragments of GIGYF2 were designed to isolate these features, and expressed along with Flag-NSP2

for Flag IP ( Fig. 2A and B) . Our WB analysis of the IP fractions revealed that NSP2 binds two distinct segments of GIGYF2, namely the central putative SAH region (residues: 742-1,085), and to a smaller extent, the Nterminal extremity containing the 4EHP-binding motif (residues: 1-267; Fig. 2B ). The strong interaction between NSP2 and GIGYF2 742-1,085 prompted us to test their direct association. Full-length hexahistidine (His6)-tagged NSP2 and Glutathione S-transferase (GST)-fused GIGYF2 742-1,085 were recombinantly expressed in Escherichia coli (E. coli), and individually purified to perform a His pull-down assay. Following incubation of His6-NSP2 with untagged GIGYF2 742-1,085 on a Ni-NTA resin, analysis of the eluates by SDS-PAGE and Coomassie blue staining revealed a specific retention of GIGYF2 742-1,085 with His6-NSP2, confirming their direct interaction in vitro (Fig. 2C) . GST alone was used as control and did not show any pull-down by His6-NSP2, supporting the specificity of the NSP2-GIGYF2 interaction. We also successfully detected this interaction when both His6-NSP2 and GST-GIGYF2 742-1,085 were co-expressed in E.coli. In this case, a GST pull-down assay was performed using an E. coli lysate and a specific His6-NSP2 retention on GST-GIGYF2 742-1,085 -bound beads was detected by both Coomassie blue staining and WB ( Supplementary Fig. 2 ). Similarly, we sought to test the capacity of His6-NSP2 to bind the N-terminal region of GIGYF2 (residues: 1-267), but failed to obtain this recombinant GIGYF2 fragment with a sufficient yield and solubility (data not shown).

Since an interaction was detected between NSP2 and the 4EHP-binding region of GIGYF2 (residues: 1-267) by co-IP, we speculated that 4EHP could also bind NSP2 independently of GIGYF2. To test this, we used a V5-tagged version of 4EHP carrying the W95A substitution which is known to disrupt its interaction with GIGYF2 (39) . HEK293T cells were co-transfected by vectors encoding Flag-NSP2 and V5-4EHP W95A , or its WT counterpart. Using Flag IPs, we found that both WT and W95A versions of 4EHP were coimmunoprecipitated by NSP2 to a comparable extent (Fig. 2D) , indicating that 4EHP can bind NSP2 independently of GIGYF2. This result prompted us to evaluate their direct association in vitro. As performed with GIGYF2 742-1,085 , full-length GST-fused 4EHP was expressed in E. coli, and the purified untagged protein was incubated with His6-NSP2 to perform a His pull-down assay. In agreement with our co-IP results, we found a specific binding of 4EHP to His6-NSP2, thus confirming their interaction in a GIGYF2-independent manner (Fig. 2E ).

The role of 4EHP-GIGYF2 as a translational repressor has been described in various contexts (for review, see (21)). Having shown that NSP2 directly targets the 4EHP-GIGYF2 complex through at least two contact points, namely the region 742-1,085 of GIGYF2 and 4EHP itself, we wished to assess whether the silencing capacity of 4EHP-GIGYF2 could be altered by NSP2. To address this point, we used the λN-BoxB tethering approach in HEK293T cells (42) . A Renilla luciferase reporter mRNA containing five BoxB sequences (RLuc-5BoxB) in the 3′ untranslated region (3'UTR) was expressed with a plasmid expressing V5tagged GIGYF2 fused to a λN peptide, which has a high affinity for the BoxB sequences (Fig. 3A) . The silencing capacity of GIGYF2 was assessed following the measurement of luciferase activity in HEK293T expressing Flag-NSP2 or Flag as control. The expression of Flag-NSP2 and λN-GIGYF2 was also verified by WB (Fig. 3C ). As expected, we observed that the recruitment of GIGYF2 to the 3′ UTR markedly reduced luciferase activity in control cells (5-fold repression). Interestingly, this λN-GIGYF2-mediated repression was reduced upon expression of Flag-NSP2 (3-fold repression), indicating an inhibitory effect of NSP2 on GIGYF2 function in silencing (Fig. 3A ).

Several studies have reported that GIGYF2 has two distinct mechanisms of repression: one is 4EHPdependent and affects translation; the other is 4EHP-independent and involves the deadenylase activity of the CCR4-NOT complex (36) . Our co-IP data showed that NSP2 did not interact with CCR4-NOT ( Fig. 1A ), suggesting that it should preferentially impair the 4EHP-dependent activity of GIGYF2. To test this, λN-GIGYF2 was tethered to a RLuc-5BoxB mRNA containing a self-cleaving hammerhead ribozyme (HhR) at the 3´-end to generate a poly(A) stretch of 114 nucleotides, followed by 40 nucleotides to block CCR4-NOTdependent deadenylation (RLuc-5BoxB-A114-N40-HhR), as previously described (18) . When tethered to this reporter, GIGYF2 induced a 2.2-fold repression of this reporter in cells transfected with a control vector (Fig.   3B ). By contrast, we observed that NSP2 expression decreased the GIGYF2-mediated repression of the reporter to 1.4-fold, indicating a specific role of NSP2 in blocking the deadenylation-independent role of GIGYF2.

The miRNA-induced translational repression is effected by the 4EHP-GIGYF2 complex in human cells (27, 28, 30) . To evaluate the impact of NSP2 in miRNA-mediated silencing, we transiently transfected HEK293T cells with a Renilla luciferase construct either lacking (RLuc), or containing six bulged let7a miRNA binding sites in its 3′ UTR (RLuc-6let7a), together with a firefly luciferase construct (FLuc) as a transfection control (Fig. 4A ). Normalized RLuc activity was markedly reduced by the presence of let7abinding sites, with ∼40-fold repression in cells expressing a control vector. By contrast, expression of NSP2 resulted in a decreased repression magnitude of the RLuc-6let7a reporter compared to RLuc (∼30-fold), indicating a reduced let7a-mediated silencing in the presence of NSP2 (Fig. 4A ).

Evidence suggests that the 4EHP-GIGYF2 complex contributes to miRNA action through the binding of GIGYF2 to a proline-rich motif (PPGL) in the silencing domain (SD) of GW182, the scaffolding protein of miRISC (30) . To investigate whether NSP2 could impair GW182-mediated silencing, the silencing domain of GW182 was artificially tethered to the RLuc-5BoxB reporter in control and Flag-NSP2-expressing cells. We found that tethering GW182 SD induced a ∼40-fold repression of the RLuc-5BoxB level in the control cells, while this silencing effect was reduced in NSP2-expressing cells (∼30-fold; Fig. 4B ). As a control, a GW182 SD mutant carrying a deletion of the GIGYF2-binding motif (ΔPPGL) was used to evaluate the contribution of GIGYF2. Tethering this ΔPPGL mutant on RLuc-5BoxB in control and Flag-NSP2 cells showed that its silencing activity is not affected by NSP2 expression, indicating that NSP2 action on GW182 SD -mediated silencing is exerted through targeting GIGYF2 (Fig. 4B ). We detected a comparable result when GW182 SD was tethered on the RLuc-5BoxB-A114-N40-HhR reporter (Fig. 4C) . Similarly, the silencing activity of its ΔPPGL version was unchanged upon NSP2 expression, confirming a specific alteration of the deadenylationindependent role of GIGYF2 by NSP2. Altogether, these results indicate that the GIGYF2-dependent repressive activity of miRISC is altered upon expression of NSP2 in human cells.

Upon infection, SARS-CoV-2 impairs splicing, export, translation and degradation of host mRNAs (43, 44) . 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 impair the silencing capacity of miRNAs (Fig. 4D ). This mechanism therefore unveils the potential 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.

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 (Fig. 1E) . Nevertheless, this remains in agreement with Gupta et al. who pointed out that the G262V and G265V mutations located within this region of NSP2 disrupted binding to 4EHP-GIGYF2 (18) . 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 co-factors such as CCR4-NOT and DDX6. Further investigation will be needed to fully resolve the structural basis of the NSP2/4EHP-GIGYF2 complex and thus elucidate NSP2 action on miRNA-mediated silencing. replication by targeting S and M proteins (48) . 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 (8) .

The silencing capacity of miRISC is partially impeded upon NSP2 expression, as demonstrated by the de-repression of GW182-or let-7a-mediated inhibition of a reporter mRNA in NSP2-expressing cells ( Fig.   4A-C) . This alteration can be extrapolated to other miRNAs whose action relies on 4EHP, such as miR-145 or miR-34a (31, 32) . 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 (31) . Beyond miRNA, 4EHP-GIGYF2 also controls the production of TTP-targeted mRNAs that encode inflammatory cytokines such as TNF-α and IL-8 (22) (23) (24) (25) (26) . 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 (51) . 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 Fig. 3A) . 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 Fig. 3B ). 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 the expression of host and/or viral transcripts. These results open new directions to investigate the mechanisms underlying the pathogenicity of SARS-CoV-2 and how NSP2 could become a target for therapeutic intervention. Thus, our biochemical approaches and reporter-based assays may represent a novel framework to imagine innovative therapeutic approaches based on the interaction of NSP2 with 4EHP-GIGYF2. 

For Flag-NSP2 IP, pcDNA5-FRT-TO-FH-Nsp2 (Addgene plasmid 157683) was used. Truncated versions of NSP2 were generated by replacing the sequence encoding full-length NSP2 by PCR-amplified fragments at the BamHI-XhoI sites of the pcDNA5-FRT-TO-FH vector. The mCherry2-NSP2 expressing plasmids were created by cloning a PCR-amplified NSP2 fragment into the mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 vectors (Addgene plasmid 54517) using EcoRI and BamHI restriction enzymes. To generate the pCI-λNV5-GIGYF2 vector, a fragment containing the GIGYF2 sequence was obtained by PCR from the pcDNA4/TO/GFP-GIGYF2 vector (Addgene plasmid 141189) (34) and inserted into the pCI-λNV5 at the XhoI-NotI sites. A similar strategy was used to generate the V5-tagged fragments of GIGYF2 using primers to isolate the following domains encompassing residues: 1-267; 257-495; 485-752; 742-1,085; 1,075-1,320. pCI-eGFP-GIGYF2 was generated by inserting the eGFP sequence, PCR-amplified from pEGFP-C1 (Clontech), at the NheI-XhoI sites of the pCI-Neo vector and then by adding the fragment encoding GIGYF2 at the XhoI-NotI sites. The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites. To generate the pCI-λNV5-GW182 SD vector, a fragment encoding the residues 1382-1690 was cloned by PCR as a XhoI-NotI fragment from the pFRT/TO/FLAG/HA-DEST TNRC6C vector (Addgene plasmid 19885) (52) and inserted into the pCI-λNV5 at the XhoI-NotI sites. For recombinant GST-fused protein expression, full-length 4EHP and GIGYF2 742-1,085 coding sequences contained in PCR-amplified BamHI-NotI and XhoI-NotI fragments, respectively, were cloned into a pGEX-6P-1 vector (Amersham) in frame with the GST coding sequence. The pET-28b vector (EMD Biosciences) was used to express NSP2 as a recombinant protein fused to an His6 tag at the N-terminus. For this, a PCR-amplified fragment encoding NSP2 was inserted at the XhoI-BamHI sites of pET-28b. Amino acid substitutions and deletions were introduced by site-directed mutagenesis using the QuikChange Kit (Agilent). 

HEK293T cells were resuspended in a lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 μM DTT) supplemented with cOmplete EDTA-free Protease inhibitor, and phosphatase inhibitors, and incubated for 20 min on ice. The lysate was clarified by centrifugation at 10,000 × g for 10 min at 4 °C. 50µg of the sample were mixed with 100 μM of b-isox (Sigma-Aldrich) and rotated at 4°C for 90 min. The incubated reaction was then centrifuged at 10,000 × g for 10 min to pellet the precipitates. The pellet was washed twice in the lysis buffer and resuspended in protein sample buffer for Western blot analysis. Proteins in the supernatant fractions were precipitated by addition of four volumes of cold acetone, incubated for 1 h at −20°C and centrifuged at 15,000 × g for 10 min to pellet the precipitates.

Generation of 4EHP KO cells were described in Jafarnejad et al. (32) . CRISPR-Cas9-mediated genome editing of HEK293 cells was performed according to Ran et al. (53) . After centrifugation for 30 min at 20,000 g, 4℃, clarified samples were transferred to batch-bind with Glutathione SepharoseTM 4B (Cytiva) resin for ~1 hr at 4℃ followed by a washing step with 30 mL of washing buffer (20 mM HEPES pH 7.5, 1 M NaCl and 5 mM β-mercaptoethanol) supplemented with 10 mM ATP, followed by an overnight 3C protease digestion in order to remove GST. The untagged GIGYF2 742-1,085 domain was present in the flowthrough and further purified on an HiTrap S FF column (Cytiva) using a linear gradient of 92.5% Hitrap buffer A (20 mM HEPES pH7.5, 5 mM β-mercaptoethanol) to 100% Hitrap buffer B (20 mM HEPES pH 7.5, 1 M NaCl and 5 mM β-mercaptoethanol). The peak fractions corresponding to 4EHP and GIGYF2 742-1,085 were pooled, concentrated and used for pull-down assays.

Pull-down experiments were performed by incubating 1 nmol of His6-NSP2 with equimolar amount of untagged GIGYF2 742-1,085 or 4EHP, or GST used as control. All proteins were free of nucleic acids according to the OD280 nm/OD260 nm ratio. Binding buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 50 mM imidazole, 10% glycerol and 0.1 % triton X-100) was added to a final volume of 60 μL. The reaction mixtures were incubated on ice for 1 h. 10 μL was withdrawn and used as an input fraction for SDS-PAGE analysis. The remaining 50 μL were incubated at 4 °C for 2 h with 40 μg of HisPur Ni-NTA magnetic beads (Thermo Scientific) preequilibrated in binding buffer, in a final volume of 200 μL. After binding, beads were washed three times with 500 μL of binding buffer. Bound proteins were eluted with 50 μl of elution buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 250 mM imidazole, 10 % glycerol and 0.1 % triton X-100). Samples were resolved on SDS-PAGE and visualized by Coomassie blue staining.

Full-length His6-NSP2 and GST-GIGYF2 742-1,085 proteins were co-expressed in BL21 (DE3) Gold E. coli (Agilent technologies). Small-scale expression was done in 5 ml of auto-inducible terrific broth media (ForMedium, AIMTB0260) supplemented with ampicillin (100 μg/ml) and kanamycin (50 µg/mL), first at 37°C for 3 h and then at 18°C overnight. The cells were harvested by centrifugation at 4,000 rpm for 30 min and the pellets were resuspended in 750 μl of lysis buffer (50 mM HEPES pH 7.5, 200mM NaCl, 5 mM βmercaptoethanol). The cells were lysed by sonication on ice and the lysate clearance was performed by centrifugation at 13,000 g for 30 min. The supernatant was applied on Glutathione SepharoseTM 4B resin (Cytiva) pre-equilibrated with the lysis buffer, incubated at 4°C on a rotating wheel for 1 h, followed by a washing step with 1 ml of lysis buffer (50 mM HEPES pH 7.5, 200mM NaCl, 5 mM β-mercaptoethanol).

Retained proteins were eluted by 50 μl of elution buffer (50 mM HEPES pH 7.5, 200mM NaCl, 5 mM βmercaptoethanol, 20mM GSH), followed by SDS-PAGE and Coomassie blue staining or Western blot.

Tethering assays were performed as previously described (27, 54) . (C) Artificial tethering of GW182 SD to the 3′ UTR of a reporter mRNA which is refractory to deadenylation.

The upper panel shows a schematic of the RLuc-5boxB-A114-N40-HhR reporter. Transfections were performed as in (B), except that GW182 SD was tethered on the RL-5boxB-A114-N40-HhR reporter. Data are presented as mean ± SD (n = 3). (***) P < 0.001 (two-tailed Student's t test). GST pull-down assay showing the interactions between recombinant GST-fused GIGYF2 742-1,085 and His6-NSP2. Both His6-NSP2 and GST-GIGYF2 742-1,085 were co-expressed in E.coli and GST pull-down assays were performed using an E. coli lysate. The starting material (Input) and bound fractions were analyzed by SDS-PAGE followed by Coomassie blue staining and Western blot with the indicated antibodies. GST served as negative control. 

University of Cologne) are acknowledged for their gifts of pCI-RL (5BoxB or 6let7a) and pCI-λN

Addgene plasmid 19885), pcDNA5-FRT-TO-FH-Nsp2 from David Tollervey (Addgene plasmid 157683) and pcDNA4/TO/GFP-GIGYF2 from Simon Bekker-Jensen (Addgene plasmid 141189). For mCherry2 fusion, mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 (Addgene plasmid 54517) were gifts from Michael Davidson. The pSpCas9(BB)-2A-Puro vector was a gift from Feng Zhang (Addgene plasmid 62988

Origin and evolution of pathogenic coronaviruses

A novel coronavirus associated with severe acute respiratory syndrome

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Coronavirus biology and replication: implications for SARS-CoV-2

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

Exploring the SARS-CoV-2 virus-host-drug interactome for drug repurposing

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms

Comparative Multiplexed Interactomics of SARS-CoV-2 and Homologous Coronavirus Nonstructural Proteins Identifies Unique and Shared Host-Cell Dependencies

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication

COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis

Molecular Epidemiology Surveillance of SARS-CoV-2: Mutations and Genetic Diversity One Year after Emerging

Structure and Function of N-Terminal Zinc Finger Domain of SARS-CoV-2 NSP2

Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein

Severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling

2021) eIF4E-homologous protein (4EHP): a multifarious cap-binding protein

A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development

Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements

Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation

Capdependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos

A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP

Cap-binding protein 4EHP effects translation silencing by microRNAs

MicroRNAs recruit eIF4E2 to repress translation of target mRNAs

Post-transcriptional gene silencing activity of human GIGYF2

Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

2021) microRNA-induced translational control of antiviral immunity by the capbinding protein 4EHP

Translational control of ERK signaling through miRNA/4EHP-directed silencing

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

GIGYF1/2-Driven Cooperation between ZNF598 and TTP in Posttranscriptional Regulation of Inflammatory Signaling

Involvement of RQCD1 overexpression, a novel cancer-testis antigen, in the Akt pathway in breast cancer cells

4EHP-independent repression of endogenous mRNAs by the RNA-binding protein GIGYF2

Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

Charged single alpha-helix: a versatile protein structural motif

The GYF domain

Using the lambdaN peptide to tether proteins to RNAs

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

Viruses and microRNAs

Viruses and miRNAs: More Friends than Foes

SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes

Therapeutic potential of C1632 by inhibition of SARS-CoV-2 replication and viral-induced inflammation through upregulating let-7

The Prediction of miRNAs in SARS-CoV-2 Genomes: hsa-miR Databases Identify 7 Key miRs Linked to Host Responses and Virus Pathogenicity-Related KEGG Pathways Significant for Comorbidities

Prediction and Analysis of SARS-CoV-2-Targeting MicroRNA in Human Lung Epithelium

Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients

Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs

Genome engineering using the CRISPR-Cas9 system

Alternative splicing of CNOT7 diversifies CCR4-NOT functions

We thank N. Ulrich for helpful technical assistance, and A. Garcia (Rockefeller University, New York) for sharing information on sgRNA sequences targeting the Gigyf2 gene. W. Filipowicz (FMI, Basel) and N.

Flag-NSP2Repression fold