SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression

  1. Parisa Naeli
  2. Xu Zhang
  3. Patric Harris Snell
  4. Susanta Chatterjee
  5. Muhammad Kamran
  6. Reese Jalal Ladak
  7. Nick Orr
  8. Thomas Duchaine
  9. Nahum Sonenberg
  10. Seyed Mehdi Jafarnejad

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Abstract

microRNAs (miRNAs) inhibit mRNA translation initiation by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5’ cap structure. Viruses utilise miRNAs to impair the host antiviral immune system and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-ß, and thereby impairs the host antiviral immune response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) and enhances the translational repression mediated by natural miRNA binding sites in the 3’ UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.

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  1. Review Commons

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Naeli et al., presents study on effects of SARS-CoV-2 NSP2 protein on miRNA mediated translational repression. Authors show in multiple ways using reporter mRNAs with various miRNA sites that NSP2 protein stimulates miRNA mediated repression. The increase in miRNA repression is likely due to the interaction of NSP2 with either GIGYF2 or Argonaute protein directly thus making more stable repressive complex on mRNA. The manuscript is written clearly and methods provide enough details for reproducibility. Only major comment would be that authors could have tested multiple endogenous targets of miRNAs for extent of miRNA mediated expression in the presence of NSP2. This could be achieved using either western blots for known targets (looking at protein levels) or using targeted qRT-PCR or distribution of mRNAs in polysome fractions (mRNA translational repression. An alternative would be Ribo-Seq experiment.

    Minor comment:

    Line in introduction arguing: "The GIGYF2/4EHP complex is recruited by a variety of factors including miRNAs" should state miRISC instead of miRNAs.

    Significance

    General assessment: The study presents sold evidence that NSP2 protein interacts with miRISC complex and increases miRNA-mediated translational repression of reporter mRNAs. Multiple target sites for miR20, let7 and miR92 are tested as well as two different human cell lines (Hek293 and U87) which gives strength to study and reproducibility of the results. Focus on the endogenous miRNA targets in the presence or absence of the NSP2 protein would make study even stronger.

    The advance of the study is more rigorous analyses of NSP2 protein effects on miRNA-mediated gene expression regulation with some novel mechanistic insights. The study will be of interest for specialized and basic research audience with potential impact on translational research.

    My field of expertise covers mechanisms of gene expression regulation by miRNAs and RBPs as well as impact of mRNAs, nascent peptides and ribosomes on protein synthesis.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Naeli and collaborators propose a role for the SARS-CoV2 protein NSP2 in the miRNA-mediated translational repression. Their data show that NSP2 co-immunoprecipitate with the Argonaute AGO2 and the previously reported translation modulator GIGYF2. Using different reporters sensitive to miRNA repression, they show that overexpressing NSP2 enhances miRNA-mediated translational repression in different cell lines as well as one endogenous miRNA target CDK1 and thus without affecting the stability of targeted mRNAs. Interestingly, their data show that the effect of NSP2 depends on the number of miRNA binding sites suggesting that highly repressed mRNAs, due to the presence of several miRNA binding sites, are not affected by NSP2. Finally, using an AGO2 tethering reporter assay that does not rely on miRNA binding but needs two NSP2 interactors GIGYF2 and 4EHP, they demonstrate that overexpressing NSP2 still stimulates mRNA repression, suggesting that NSP2 can have a broader impact on miRISC-dependent silencing.

    Overall, this is an interesting follow-up study from their previous paper (Xu et al., 2022) that have the potential to add another dimension to the modulation of mRNA translation by the SARS-CoV2 protein NSP2. But unfortunately, in its current form, the work falls short of supporting their claims appropriately and providing sufficient and relevant insights about the role of NSP2 in regulating the miRNA-mediate gene regulation. To overcome this, the authors should perform and add the following experiments to strengthen their study.

    1. Interaction of NSP2 with the miRISC: With the data presented here, we can only conclude that NPS2 forms a complex with AGO2. Is their interaction direct or indirect? Is miRNA- and TNRC6-bound AGO2 (and thus miRISC) associated with NSP2? To address those important questions, the authors should perform NSP2 immunoprecipitations in GIGYF2 (and 4EHP) KO cell lines and monitor the presence of AGO2 and miRNAs in the immunoprecipitated complex. Those experiments will define the type of interaction NSP2 has with the miRISC (GIGYF2 dependent or not).
    2. Along this line, is NSP2 action on miRNA-mediated gene regulation dependent or not of GIGYF2? Again, testing this by monitoring the repression of the different reporters upon NSP2 overexpression in the presence or not of GIGYF2 in cells will precise the contribution of NSP2 in miRNA-mediated gene silencing.

    Besides these two sets of critical experiments that will define the relationship between NSP2 mRNA modulation and the microRNA pathway, it would be interesting for this study to readily test the effect of NSP2 on miRNA targets related to immune-regulatory processes and antiviral response. As the authors pointed out in their discussion, several mRNAs are involved in the control of genes found in those pathways. Demonstrating the contribution of NSP2 in the regulation of a few of them will strengthen their study's significance and interest by providing a possible mechanism at play during a SARS-CoV2 infection. Also, it would be interesting to test if the modulation of translation inhibition by NSP2 occurs in cells infected by the SARS-CoV2 virus. For instance, is NSP2 and miRISC interaction enhances during the viral infection? As the authors propose, NSP2 could also have improved antiviral microRNA repression, so it would be interesting to characterize this interplay in a relevant biological context.

    Minor comment:

    From the data presented in Figure S1A, it is impossible to conclude "that miR-20a represses the expression of the target mRNA in a GIGFY2-dependent manner" as its KO also affect the level/stability of 4EHP. Therefore, we cannot distinguish the contribution of GIGFY2 and 4EHP in this context as both protein levels decrease. The authors should tone down this statement on page 3.

    Significance

    With the addition of the proposed experiments, the revised study will better define the direct contribution of NSP2 with the miRISC. This work has the potential to provide another aspect of the mRNA translation modulation by the SARS-CoV2 protein NSP2 with the interesting angle of miRNA-mediated gene silencing.

    As mentioned by the authors, another recent paper reports the potential impact of NSP2 on post-transcriptional silencing (Zou et al., iScience 2022). However, in contrast to the current study, this previous work did not directly test the interaction of NSP2 with the miRISC. Furthermore, it only used a single miRNA reporter (let-7) to support NSP2 contribution in miRNA-mediated gene silencing, which does not demonstrate the broader impact of NSP2 on this gene regulatory mechanism as tested in this current study. Upon revision, this study will provide more definitive proof of the involvement of NSP2 in miRNA-mediated gene regulation and thus will be of interest to experts in the miRNA field and scientists interested in understanding viruses/hosts interaction.

    Although not essential, if the authors want to add data that addresses the function of NSP2 on this regulatory pathway in the context of viral infection (which seems feasible for this group), that will definitely increase the broader significance of their work.

    I am an expert in molecular biology and molecular genetics, miRNA-mediated gene regulation, and small non-coding RNA biology.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript the authors examine whether SARS-CoV-2 protein, NSP2 mediate translation repression of cellular transcripts by increasing miRNA mediated suppression. The same authors previously demonstrated NSP2 interacts with GIGYF2 and this interaction suppresses the translation of the Ifnb mRNA. Here they extend this finding and using a series of reporters they illustrate that at least part of NSP2 translation suppression is mediated by increasing GIGYF2 mediated miRNA translation suppression. Overall the manuscript is clearly written and the experiments well executed.

    Major comments:

    Optional- The authors show that the ability of NSP2 to enhance miRNA-mediated repression is dependent on the extent of the initial repression and suggest this dependency on the initial repression levels may explain the discrepancy with published work showing NSP2 actually impairs (and not increase) microRNA-mediated silencing . Therefore, an important addition could be to examine what happen to translation in cells that express NSP2 and whether generally translation repression is significant in transcripts (native) that are enriched in miRNA binding site. This experiment will help to show NSP2 effect on native transcripts and potentially help to understand how much of these changes are likely explained by miRNAs.

    Minor comments:

    1. Personally I found the term repression fold quite confusing and unintuitive. Why not to present the actual measurements so that the control (blue bars) have high value and the red bars (representing repression) have lower values?
    2. It seems inadequate to present on the same graph two values that are normalized to 1. For example, in figure 1D it will be important to show the mir20-Mut real values (at least mir20-Mut in NSP2 cells should not be normalized to 1). This will allow to show that the differences are indeed mediated by stronger translation repression of miR-20 WT luciferase in the presence of NSP2 and not by unexplained differences in mir20-mut luciferase expression. This is true for almost all the figures in the manuscript. Correspondingly, the statistical test should be two-factor ANOVA test examining if NSP2 expression significantly increase the difference between Luciferase miR20-WT and luciferase miR20-mut.

    Significance

    There is an urgent need for better molecular understanding of how SARS-CoV-2 proteins influence the machineries of the host cell.

    This study investigates how NSP2 interaction with GIGYF2 mediate translation repression of cellular transcript. The authors also address the discrepancy with previously published work that showed NSP2 actually impairs (and not increase) microRNA-mediated silencing.

    The paper would be of interest to RNA biologists and for molecular virologists that study SARS-CoV-2

  5. Version published to 10.1101/2023.01.01.522328v1 on bioRxiv