The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive‐strand RNA viruses
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Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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We thank the Referees for their evaluation and their useful comments.
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
__ __ The MS from Bonaventure and colleagues used a CRISPR to identify novel IFN-induced antiviral effectors targeting HIV-1.
One hit, the DEAD Box helicase DDX42, while not itself part of the IFN response, exerts a substantial inhibitory effect on HIV-1 replication when over expressed, and gives a several fold boost to viral replication when knocked down in cells. …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Reply to the Reviewers
We thank the Referees for their evaluation and their useful comments.
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
__ __ The MS from Bonaventure and colleagues used a CRISPR to identify novel IFN-induced antiviral effectors targeting HIV-1.
One hit, the DEAD Box helicase DDX42, while not itself part of the IFN response, exerts a substantial inhibitory effect on HIV-1 replication when over expressed, and gives a several fold boost to viral replication when knocked down in cells. The effect of DDX42 KO or O/E is manifest at reverse transcription and PLA analysis suggests and interaction with incoming virions. Moreover, DDX42 appears to exert an inhibitory effect generally against retroviruses and retroelements, with evidence that it associates with viral/transposon RNA. The authors further show that DDX42 has antiviral against a range (but not all) RNA viruses, with very striking phenotypes seen especially with Zika, CHIKV and SARS CoV2, with DDX42 associating with dsRNA in infected cells. These data suggest DDX42 is a constitutively expressed a broad-spectrum inhibitor of a range of mammalian RNA viruses.
The manuscript is very well written, the data is of good quality and clearly DDX42 is having a general effect on viral replication. The results are novel, important and potentially of wide interest. Where the MS is somewhat lacking is understanding whether DDX42 has direct antiviral activity or is globally affecting cellular RNA metabolism. Some important areas for the authors to consider are:
DDX42 has a potential role in splicing and/or RNA metabolism so I think it would be important to see whether there is any clear global change in gene expression in knockout or knockdown cells cells vs control that might be suggestive of a generalized effect.
Responses
We thank the reviewer for this important question. Indeed, DDX42 didn’t impact the replication of 2 negative strand RNA viruses and this suggested that DDX42 didn’t have a global impact on the target cells, but we could not formally exclude a generalized effect. Therefore, we have performed RNA-seq analysis in order to evaluate the impact of DDX42 depletion (using 3 different siRNAs targeting DDX42 in comparison to a CTRL siRNA in U87-MG cells, and 2 different siRNA in comparison to a CTRL siRNA in A549-ACE2 cells, in samples obtained in 3 independent silencing experiments). The RNA-seq data (See Supplemental File 1 and Figure S5) showed that only 63 genes are commonly differentially expressed by the 3 siRNAs targeting DDX42 in U87-MG cells and only 23 of these genes were also found differentially expressed in A549-ACE2 cells depleted for DDX42. Importantly, the identity of these genes could not explain the observed antiviral phenotypes. These data are in favor of the absence of generalized effect on the target cells, which could have explained the antiviral phenotypes of the sensitive viruses.
• The HIV experiments in primary cells are only one round at present. Does the DDX42 knockdown enhance viral replication in multiround? Does it lead to more viral PAMPs for PRRs to induce IFN?
Responses
We agree with the reviewer that it would have been very informative to measure the impact of DDX42 knockdown in multiround infections in primary T cells. However, we tried several times to do this experiment (with primary T cells from several donors) and we were not successful: indeed, DDX42 KO appeared to slow down cell division, which could be taken into account for a short, one-cycle experiment (i.e. 24 h) 3 days post-Cas9/sgRNA electroporation by adjusting the number of cells at the time of infection. However, DDX42 KO appeared quite toxic in longer experiments, with cells stopping to grow.
The question regarding the generation of more viral PAMPs for PRRs to induce IFN is also very interesting. We know from published work (including ours) that primary T cells don’t normally produce IFN following HIV-1 infection (see for instance Bauby and Ward et al, mBio 2021). However, one can indeed hypothesize that as more viral DNAs are produced in the absence of DDX42, perhaps the primary T cells could detect them and produce IFN. To address this question in primary T cells, we would have needed to be able to perform multiround infections, which was not possible, as mentioned above. Moreover, we could not test this hypothesis in the cell lines that we used, such as U87-MG/CD4/CXCR4 cells, as they are unable to produce IFN following HIV-1 infection.
More could be made mechanistically of the lack of sensitivity of Flu and VSV to DDX42. In particular showing whether or not DDX42 interacts with the RNA of the insensitive virus, or whether DDX42/virus or dsRNA interactions by PLA occur with Flu would highlight the relevance of these observations to the antiviral mechanism.
Responses
This is an excellent remark. We have now performed RNA immunoprecipitation experiments using 2 viruses targeted by DDX42 (CHIKV and SARS-CoV-2) and 1 virus that is insensitive to DDX42 (IAV) (See New Figure 4J-L): whereas CHIKV and SARS-CoV-2 RNAs could be specifically pulled-down with DDX42 immunoprecipitation, this was not the case for IAV RNA. This strongly argues for a direct mechanism of action of DDX42 helicase on viral RNAs.
Reviewer #1 (Significance (Required)):
__ The role of helicases in host defence are of wide interest and importance. This has the potential to be a very important study that deserves a wide audience. However in my opinion it needs some further mechanistic insight along the lines I have suggested.
Responses
As mentioned above, we have now added important data: First, DDX42 is able to interact with RNAs from targeted viruses (and not from an insensitive virus); Second, we have checked that DDX42 didn’t have a substantial impact on the cell transcriptome. Taken together, these data are clearly in favour of a direct mode of action of DDX42.
__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __
In this brief report, the authors use a CRISPR screening approach to identify cellular proteins that limit HIV infection. The screen itself is elegantly designed and most of the top hits are components of the interferon signaling pathway that would be expected to emerge from such a screen, thus providing confidence in the results. The authors followed up on DDX42 as a new hit identified in their screen and confirmed that targeting DDX42 with distinct guide RNAs resulted in increased HIV infection in at least 3 cell lines. Conversely, DDX42 overexpression inhibited infection. They also confirmed a role for DDX42 in inhibiting HIV infection in primary macrophages and CD4 T cells using siRNA and CRISPR KO strategies, respectively. They also demonstrate that DDX42 inhibits several other divergent lentiviruses as well as Chikungunya virus and SARS-CoV-2, but not influenza virus. These data convincingly show that DDX42 plays a role in inhibiting many lentivirus and positive sense RNA virus infections. Using PCR assays for reverse transcription products they conclude that DDX42 inhibits an early process in the HIV life cycle occurring after virus entry, though the statistical significance of these differences is not clear. They further use proximity ligation assays to suggest that DDX42 is in proximity to HIV-1 and SARS-CoV-2 replication complexes. Mechanistically, these data are largely unsatisfying as they do not provide specific insight into how DDX42 so broadly inhibits virus replication. Overall, the manuscript presents a significant advance, it also has some weaknesses as listed below.
Statistical analysis is not included in any of the figures.
Response
Statistical analyses have now been included.
Many of the figure legends do not state how many independent biological replicates the figures are based on.
Response
The number of biological replicates for each panel is stated at the very end of each figure legend.
Detailed mechanistic understanding of DDX42 effects on virus replication is not provided by the manuscript.
Response
As mentioned in response to Reviewer 1, we have now added data showing that DDX42 could interact with RNAs from targeted viruses but not from an insensitive virus, arguing for a direct antiviral mode of action of this Dead-Box helicase.
__Reviewer #2 (Significance (Required)): __
__ __ DDX42 is a new antiviral protein identified and confirmed in this manuscript. It was also identified as one of many hits in a genome wide CRISPR screen for cellular proteins that regulate SARS-CoV-2 infections, but was not followed up. Thus, the identification and confirmation of DDX42 antiviral activity is highly significant for both the HIV and SARS-CoV-2 fields. This high significance may compensate to some extent for the lack of mechanistic insight contained in this initial report.
__**Referees Cross-commenting** __
__ __I find the comments of the other reviewers to be fair and reasonable, and I concur that the work is overall important and novel. It seems that reviewers generally agreed that some additional mechanistic insights would be desirable for publication in a high impact journal. Reviewer 1 makes some good suggestions in this regard. As for mouse experiments, I would reserve these for a follow up manuscript.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
__In this manuscript, Bonaventure et al report the results of a screen to identify cellular inhibitors of HIV-1 infection in IF treated cells. They identify DDX42 as such a factor though, unexpectedly, DDX42 did not turn out to be an ISG. Strikingly, DDX42 turns out to inhibit a wide range of retroviruses as well as retrotransposons and + sense, but not - sense, RNA viruses among which SARS-CoV2 turns out to be especially sensitive to DDX42, with siRNAs specific for SARS-CoV2 DDX42 increasing viral RNA expression by a startling 3 orders of magnitude, compared to only an 2-5 fold positive effect with HIV-1.
Response
We agree with the reviewer that DDX42’s impact on HIV-1 may appear as somewhat modest, however, it is highly reproducible across cell lines and primary cells and, more importantly, it is observed upon depletion of the endogenous protein (either by KO or silencing) in target cells that are highly permissive to viral replication, such as activated primary CD4+ T cells. We therefore believe that these findings, combined with the findings that other positive-strand RNA viruses are targeted, are of high interest.
Reviewer #3 (Significance (Required)):
__I found this paper generally convincing and technically sound though the emphasis was odd and clearly driven more by the history of how this work was done than by the actual results obtained. Specifically, the emphasis is on HIV-1 yet the most interesting data are the dramatic effects seen with Chikungunya and SARS2. If I was writing this paper, I would delete figure 4 and focus this paper entirely on retroviruses and retrotransposons. In that form, I think it would be competitive at PLoS Pathogens or perhaps EMBO Journal. The RNA virus work shown in figure 4 could then be figure 1 of a new, high impact, paper looking at the mechanism of action of DDX42 as an inhibitor of + sense, but not - sense, viral gene expression. Though Wei et al do mention DDX42 in their SARS-CoV2 screening paper this is certainly not a major theme of that paper so I don't think that would be a problem.
Responses
We thank the reviewer for this comment. We had hesitated to present the manuscript as suggested by the reviewer (i.e. focusing only on HIV-1, retroviruses and retroelements) and prepare a second manuscript with the remaining data. We’ve finally decided against it, as we believe that showing a broad antiviral effect of DDX42 on +strand RNA viruses increases the impact of our findings.
On another note, a conditional DDX42 KO mouse has been generated by the Wellcome trust Sanger institute and it would greatly improve this manuscript if they could show an in vivo a result similar to figure 3F using MLV.
Responses
We thank the reviewer for this information. We completely agree that in vivo work would be a massive plus and we will be planning to explore this in the future, but not at this stage as it would require specific funding and resources.
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Bonaventure et al report the results of a screen to identify cellular inhibitors of HIV-1 infection in IF treated cells. They identify DDX42 as such a factor though, unexpectedly, DDX42 did not turn out to be an ISG. Strikingly, DDX42 turns out to inhibit a wide range of retroviruses as well as retrotransposons and + sense, but not - sense, RNA viruses among which SARS-CoV2 turns out to be especially sensitive to DDX42, with siRNAs specific for SARS-CoV2 increasing viral RNA expression by a startling 3 orders of magnitude, compared to only an 2-5 fold positive effect with HIV-1.
Significance
I found this paper …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Bonaventure et al report the results of a screen to identify cellular inhibitors of HIV-1 infection in IF treated cells. They identify DDX42 as such a factor though, unexpectedly, DDX42 did not turn out to be an ISG. Strikingly, DDX42 turns out to inhibit a wide range of retroviruses as well as retrotransposons and + sense, but not - sense, RNA viruses among which SARS-CoV2 turns out to be especially sensitive to DDX42, with siRNAs specific for SARS-CoV2 increasing viral RNA expression by a startling 3 orders of magnitude, compared to only an 2-5 fold positive effect with HIV-1.
Significance
I found this paper generally convincing and technically sound though the emphasis was odd and clearly driven more by the history of how this work was done than by the actual results obtained. Specifically, the emphasis is on HIV-1 yet the most interesting data are the dramatic effects seen with Chikungunya and SARS2. If I was writing this paper, I would delete figure 4 and focus this paper entirely on retroviruses and retrotransposons. In that form, I think it would be competitive at PLoS Pathogens or perhaps EMBO Journal. The RNA virus work shown in figure 4 could then be figure 1 of a new, high impact, paper looking at the mechanism of action of DDX42 as an inhibitor of + sense, but not - sense, viral gene expression. Though Wei et al do mention DDX42 in their SARS-CoV2 screening paper this is certainly not a major theme of that paper so I don't think that would be a problem. On another note, a conditional DDX42 KO mouse has been generated by the Wellcome trust Sanger institute and it would greatly improve this manuscript if they could show an in vivo a result similar to figure 3F using MLV.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this brief report, the authors use a CRISPR screening approach to identify cellular proteins that limit HIV infection. The screen itself is elegantly designed and most of the top hits are components of the interferon signaling pathway that would be expected to emerge from such a screen, thus providing confidence in the results. The authors followed up on DDX42 as a new hit identified in their screen and confirmed that targeting DDX42 with distinct guide RNAs resulted in increased HIV infection in at least 3 cell lines. Conversely, DDX42 overexpression inhibited infection. They also confirmed a role for DDX42 in inhibiting HIV …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this brief report, the authors use a CRISPR screening approach to identify cellular proteins that limit HIV infection. The screen itself is elegantly designed and most of the top hits are components of the interferon signaling pathway that would be expected to emerge from such a screen, thus providing confidence in the results. The authors followed up on DDX42 as a new hit identified in their screen and confirmed that targeting DDX42 with distinct guide RNAs resulted in increased HIV infection in at least 3 cell lines. Conversely, DDX42 overexpression inhibited infection. They also confirmed a role for DDX42 in inhibiting HIV infection in primary macrophages and CD4 T cells using siRNA and CRISPR KO strategies, respectively. They also demonstrate that DDX42 inhibits several other divergent lentiviruses as well as Chikungunya virus and SARS-CoV-2, but not influenza virus. These data convincingly show that DDX42 plays a role in inhibiting many lentivirus and positive sense RNA virus infections. Using PCR assays for reverse transcription products they conclude that DDX42 inhibits an early process in the HIV life cycle occurring after virus entry, though the statistical significance of these differences is not clear. They further use proximity ligation assays to suggest that DDX42 is in proximity to HIV-1 and SARS-CoV-2 replication complexes. Mechanistically, these data are largely unsatisfying as they do not provide specific insight into how DDX42 so broadly inhibits virus replication. Overall, the manuscript presents a significant advance, it also has some weaknesses as listed below.
- Statistical analysis is not included in any of the figures.
- Many of the figure legends do not state how many independent biological replicates the figures are based on.
- Detailed mechanistic understanding of DDX42 effects on virus replication is not provided by the manuscript.
Significance
DDX42 is a new antiviral protein identified and confirmed in this manuscript. It was also identified as one of many hits in a genome wide CRISPR screen for cellular proteins that regulate SARS-CoV-2 infections, but was not followed up. Thus, the identification and confirmation of DDX42 antiviral activity is highly significant for both the HIV and SARS-CoV-2 fields. This high significance may compensate to some extent for the lack of mechanistic insight contained in this initial report.
Referees Cross-commenting
I find the comments of the other reviewers to be fair and reasonable, and I concur that the work is overall important and novel. It seems that reviewers generally agreed that some additional mechanistic insights would be desirable for publication in a high impact journal. Reviewer 1 makes some good suggestions in this regard. As for mouse experiments, I would reserve these for a follow up manuscript.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The MS from Bonaventure and colleagues used a CRISPR to identify novel IFN-induced antiviral effectors targeting HIV-1.
One hit, the DEAD Box helicase DDX42, while not itself part of the IFN response, exerts a substantial inhibitory effect on HIV-1 replication when over expressed, and gives a several fold boost to viral replication when knocked down in cells. The effect of DDX42 KO or O/E is manifest at reverse transcription and PLA analysis suggests and interaction with incoming virions. Moreover, DDX42 appears to exert an inhibitory effect generally against retroviruses and retroelements, with evidence that it associates with …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The MS from Bonaventure and colleagues used a CRISPR to identify novel IFN-induced antiviral effectors targeting HIV-1.
One hit, the DEAD Box helicase DDX42, while not itself part of the IFN response, exerts a substantial inhibitory effect on HIV-1 replication when over expressed, and gives a several fold boost to viral replication when knocked down in cells. The effect of DDX42 KO or O/E is manifest at reverse transcription and PLA analysis suggests and interaction with incoming virions. Moreover, DDX42 appears to exert an inhibitory effect generally against retroviruses and retroelements, with evidence that it associates with viral/transposon RNA. The authors further show that DDX42 has antiviral against a range (but not all) RNA viruses, with very striking phenotypes seen especially with Zika, CHIKV and SARS CoV2, with DDX42 associating with dsRNA in infected cells. These data suggest DDX42 is a constitutively expressed a broad-spectrum inhibitor of a range of mammalian RNA viruses.
The manuscript is very well written, the data is of good quality and clearly DDX42 is having a general effect on viral replication. The results are novel, important and potentially of wide interest. Where the MS is somewhat lacking is understanding whether DDX42 has direct antiviral activity or is globally affecting cellular RNA metabolism. Some important areas for the authors to consider are:
• DDX42 has a potential role in splicing and/or RNA metabolism so I think it would be important to see whether there is any clear global change in gene expression in knockout or knockdown cells cells vs control that might be suggestive of a generalized effect.
• The HIV experiments in primary cells are only one round at present. Does the DDX42 knockdown enhance viral replication in multiround? Does it lead to more viral PAMPs for PRRs to induce IFN?
• More could be made mechanistically of the lack of sensitivity of Flu and VSV to DDX42. In particular showing whether or not DDX42 interacts with the RNA of the insensitive virus, or whether DDX42/virus or dsRNA interactions by PLA occur with Flu would highlight the relevance of these observations to the antiviral mechanism.
Significance
The role of helicases in host defence are of wide interest and importance. This has the potential to be a very important study that deserves a wide audience. However in my opinion it needs some further mechanistic insight along the lines I have suggested.
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SciScore for 10.1101/2020.10.28.359356: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cell surface staining with anti-CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers. anti-CD4suggested: NoneCXCR4suggested: NoneLentiviral and retroviral infections: For infections with replication-competent HIV-1 Renilla or wild-type and/or VSV-G pseudotyped-HIV-1, target cells were plated at 2.5 x 104 cells per well in 96-well plates or at 2 x 105 cells per well in 12-well plates and infected for … SciScore for 10.1101/2020.10.28.359356: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cell surface staining with anti-CD4 and CXCR4 antibodies (Miltenyi Biotec) confirmed than more than 95% cells were positive for both markers. anti-CD4suggested: NoneCXCR4suggested: NoneLentiviral and retroviral infections: For infections with replication-competent HIV-1 Renilla or wild-type and/or VSV-G pseudotyped-HIV-1, target cells were plated at 2.5 x 104 cells per well in 96-well plates or at 2 x 105 cells per well in 12-well plates and infected for 24-48 h before lysis and Renilla (and Firefly) luciferase activity measure (Dual-Luciferase® Reporter Assay System Promega) or fixation with 2% paraformaldehyde (PFA)-PBS, permeabilization (Perm/Wash buffer, BDBiosciences) and intracellular staining with the anti-p24Gag KC57-FITC antibody (Beckman Coulter), as described previously (Goujon and Malim, 2010). anti-p24Gag KC57-FITCsuggested: NoneCells were incubated with mouse AG3.0 anti-HIV-1 Capsid antibody (National Institutes of Health (NIH) AIDS Reagent Program #4121), or J2 anti-dsRNA antibody (SCICONS), or anti-SARS-CoV-2 Nucleoprotein (N; BioVision), and rabbit anti-DDX42 antibody (HPA023571, Sigma-Aldrich) diluted in NGB buffer or in Duolink® blocking solution for 1h. anti-HIV-1suggested: (NIH AIDS Reagent Program Cat# 4121, RRID:AB_2734137)J2suggested: Noneanti-dsRNAsuggested: (Millipore Cat# MABE1134, RRID:AB_2819101)anti-SARS-CoV-2suggested: NoneHPA023571suggested: NoneImmunoblot analysis: Cell pellets were lysed in sample buffer (200 mM Tris-HCl, pH 6.8, 5.2% SDS, 20% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol), resolved by SDS–PAGE and analysed by immunoblotting using primary antibodies specific for human DDX42 (HPA023571, Sigma-Aldrich), Flag (M2, Sigma-Aldrich) and Actin (A1978, Sigma-Aldrich), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence (Bio-Rad). DDX42suggested: (Sigma-Aldrich Cat# HPA023571, RRID:AB_1847561)Actinsuggested: (Sigma-Aldrich Cat# A1978, RRID:AB_476692)anti-mousesuggested: Noneanti-rabbit immunoglobulinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Human ACE2 coding sequence (NM_021804) was amplified using the SuperScript® III One-Step RT-PCR System with Platinum® Taq (Invitrogen) from 500 ng RNA obtained from 293T cells, using primers 5’-AATTAATTTAGCGGCCGCATGTCAAGCTCTTCCTGGCTCC-3’ and 5’-AATTAATTTACTCGAGCTAAAAGGAGGTCTGAACATCATCAGTG-3, digested and inserted into NotI-XhoI-digested pRRL.sin.cPPT. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)T98G cells were a gift from Prof. G. Kochs (Freiburg University, Germany), A549 and MDCK cells were a gift from Prof. W. Barclay (Imperial College London, UK), Vero E6 cells were a gift from Christine Chable (CEMIPAI, CNRS). T98Gsuggested: NoneA549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)The crRNA sequences of the sgDDX42-1, -2, and -3 were identical to the ones cloned in pLentiguide-Puro, and the crRNA of the sgDDX42-4 and sgDDX42-5 were pre-designed by IDT®, as follow: sg4-DDX42 5’-CGGAGATCTATTAACTGCTG-3’, sg5-DDX42 5’-GAGTTGGTGAGTTTTCAGC-3’. siRNA transfection: DDX42 and control knockdowns were achieved by transfecting the indicated siRNAs at 44nM, 14.2nM, and 100nM final in U87-MG cells, TZM-bl cells and MDMs, respectively, with lipofectamine RNAimax (Thermofisher Scientific) according to the manufacturer’s instructions. U87-MGsuggested: RRID:CVCL_4V16)TZM-blsuggested: NIH-ARP Cat# 8129-442, RRID:CVCL_B478)For PLA with SARS-CoV-2, A549-ACE2 cells were plated in 24-well plates with coverslips and infected at an MOI of 0,1. 24 h later, the cells were fixed with 2-4% paraformaldehyde in PBS1X for 10 min, washed in PBS1X and permeabilized with 0.2% Triton X-100 for 10 min. A549-ACE2suggested: NoneStocks were titrated by plaque assays on MDCK cells. MDCKsuggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)After 7 days, supernatants were harvested, filtered and stock titers were determined by plaque assays on Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)The linearized plasmid coding CHIKV genome was transcribed with the T7 mMESSAGE mMACHINE kit (Thermofischer Scientific) and 5 x 105 HEK293T were transfected with 1-4 μg of transcribed RNA, using Lipofectamine 2000 (Thermofischer Scientific). HEK293Tsuggested: NoneAfter 24h, supernatants were harvested, filtered and viruses were then amplified on baby hamster kidney (BHK21) cells. BHK21suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)Viral supernatants were titrated by plaque assays in Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Briefly, 2 x 105 U87-MG/CD4/CXCR4 cells were plated in 24-well plates and incubated with BlaM-Vpr carrying NL4-3 particles (31, 62, 125 ng p24Gag) or mock-infected for 3 h at 37°C. U87-MG/CD4/CXCR4suggested: NoneSoftware and Algorithms Sentences Resources Plasmids: The pLentiCas9-Blast, pLentiGuide-Puro vectors and the GeCKO sub-library A and B plasmids were a gift from Prof. F. Zhang (Addgene #52962, #52963, and #1000000048, respectively (Sanjana et al., 2014)). GeCKOsuggested: (Gecko, RRID:SCR_009001)Control sgRNAs and sgRNAs targeting the candidate genes, MX2 and IFNAR1, were designed with the Optimized CRISPR Design tool (not available anymore), or with Chopchop (chopchop.cbu.uib.no). Chopchopsuggested: (CHOPCHOP, RRID:SCR_015723)Demultiplexing was performed using Illumina’s conversion software (bcl2fastq 2.20). Illumina’ssuggested: Nonebcl2fastqsuggested: (bcl2fastq , RRID:SCR_015058)The quality of the raw data was assessed using FastQC (v0.11.5) from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer). FastQCsuggested: (FastQC, RRID:SCR_014583)These retrieved sequences were then aligned to the GecKOv2 Human Library (A or B) reference sequences (keeping only non-duplicated sgRNA sequences, the duplicated ones being annotated) using Bowtie (Langmead et al., 2009) (v1.2), with options -v 2 -norc -S. Bowtiesuggested: (Bowtie, RRID:SCR_005476)Resulting bam files were sorted and indexed using Samtools (Li et al., 2009) (v1.5). Samtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Cells were incubated with mouse AG3.0 anti-HIV-1 Capsid antibody (National Institutes of Health (NIH) AIDS Reagent Program #4121), or J2 anti-dsRNA antibody (SCICONS), or anti-SARS-CoV-2 Nucleoprotein (N; BioVision), and rabbit anti-DDX42 antibody (HPA023571, Sigma-Aldrich) diluted in NGB buffer or in Duolink® blocking solution for 1h. AIDS Reagent Programsuggested: NoneBioVisionsuggested: (BioVision, RRID:SCR_005057)PLA punctae quantification was performed using the FIJI software (Schindelin et al., 2012). FIJIsuggested: (Fiji, RRID:SCR_002285)Processing of the raw Airyscan images was performed on the ZEN Black software. ZEN Blacksuggested: (Black Zen software, RRID:SCR_018163)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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