key: cord-0753580-i35pic5o authors: Bonaventure, Boris; Rebendenne, Antoine; de Gracia, Francisco Garcia; McKellar, Joe; Gracias, Ségolène; Labaronne, Emmanuel; Tauziet, Marine; Valadão, Ana Luiza Chaves; Bernard, Eric; Briant, Laurence; Gros, Nathalie; Djilli, Wassila; Arnaud-Arnould, Mary; Courgnaud, Valérie; Parrinello, Hugues; Rialle, Stéphanie; Ricci, Emiliano; Jouvenet, Nolwenn; Schulz, Reiner; Moncorgé, Olivier; Goujon, Caroline title: The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses date: 2021-09-24 journal: bioRxiv DOI: 10.1101/2020.10.28.359356 sha: 1b3fcbfe2352ad385365b1b7834e391bf6b3a4b1 doc_id: 753580 cord_uid: i35pic5o Genome-wide screens are powerful approaches to unravel new regulators of viral infections. Here, we used a CRISPR/Cas9 screen to reveal new HIV-1 inhibitors. This approach led us to identify the RNA helicase DDX42 as an intrinsic antiviral inhibitor. DDX42 was previously described as a non-processive helicase, able to bind RNA secondary structures such as G-quadruplexes, with no clearly defined function ascribed. Our data show that depletion of endogenous DDX42 significantly increased HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant increased infection. Importantly, DDX42 also restricted retrotransposition of LINE-1, infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 did not inhibit infection with three negative-strand RNA viruses, arguing against a general, unspecific effect on target cells, which was confirmed by RNA-seq analysis. DDX42 was found in the vicinity of viral elements by proximity ligation assays, and cross-linking RNA immunoprecipitation confirmed a specific interaction of DDX42 with RNAs from sensitive viruses. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results show for the first time a new and important role of DDX42 in intrinsic antiviral immunity. The intrinsic and innate immunity are at the frontline against viral invasion and provide a rapid 46 and global defence. The innate immunity relies on viral sensing by Pathogen Recognition of the hostile environment induced by IFN to develop a whole-genome, CRISPR/Cas9 screen endogenous DDX42 is antiviral in various cell types, including primary targets of HIV-1, and CTRL IFNAR1 MX2 WARS2 DDX42 CLDN12 REEP1 IKBIP TOPBP1 COPG1 FGF4 SPRYD7 RDH8 DCTPP1 PRLR SUN3 CXorf27 TMEM161A KCN6 LSM5 LOC339862 PKD2 NDPC1 KIAA1549 mir-4737 VSTM2A CALU SMARCA2 DNAJB2 FAM212A ETNPPL RHOC mir-105-1 CXorf56 CCSER1 SLC25A2 OR6N2 UBA1 HSPA1B SIAE KRT222 ADRA1A LIN54 TECTB CDRT1 TCEAL7 TRERF1 CD14 HSP90AA1 CTNNA2 PLAC1L populations was analysed by next-generation sequencing (NGS) and the MAGeCK algorithm 123 was used to rank the candidate genes (Fig. 1B ). An enrichment was observed for 200 genes 124 (RRA score > 0,01), with the best hits being IFNAR1, JAK1 and STAT2 (Fig. 1B) . The crucial 125 mediators of type 1 IFN signalling cascade were among the top hits in both screens (with the 126 notable exception of STAT1), validating our approach and confirming the identification of 7 infection in another model cell line, two additional sgRNAs were designed (sgRNA-2 and -3) and 150 used in parallel to the one identified in the GeCKO screen (sgDDX42-1) ( Fig. 2A) . U87-151 MG/CD4/CXCR4 cells were used here, as we previously extensively characterized the IFN 152 phenotype in these cells (11). Control and DDX42 KO cell populations were generated and pre-153 treated or not with IFN prior to infection with HIV-1 Renilla. Of note, CRISPR/Cas9 KO of 154 DDX42 induced only a partial decrease of DDX42 protein levels ( Fig. 2A) and cell populations 155 tended to derive (not shown), suggesting a potential role for DDX42 in cell proliferation or long-156 term survival. We observed however that DDX42 partial depletion with all 3 sgRNAs improved The immunoprecipitation eluates were then divided in two; the immunoprecipitated proteins 295 were analysed by immunoblot (Fig. 3I ) and their associated RNAs were extracted and analysed 296 by RT-qPCR using LINE-1 specific primers (Fig. 3J) . A significant enrichment of LINE-1 RNAs was observed with DDX42 immunoprecipitation as compared to the Firefly negative control, showing that DDX42 could interact with LINE-1 RNAs. Finally, we sought to determine whether DDX42's inhibitory activity was specific towards Here, we identified for the first time the RNA helicase DDX42 as an intrinsic inhibitor of HIV-1, In conclusion, this work shows for the first time an antiviral role for DDX42 and highlights the Interferon-Stimulated Genes: What Do They All Do? MAGeCK enables robust identification of essential genes from genome-946 scale CRISPR/Cas9 knockout screens Am I ready for CRISPR? A user's guide to genetic screens MOV10 RNA Helicase Is a Potent 1069 Inhibitor of Retrotransposition in Cells Robust hepatitis C virus infection in 1072 vitro Growth of human 1074 hepatoma cells lines with differentiated functions in chemically defined medium FastQ Screen: A tool for multi-genome mapping and 1077 quality control Cutadapt removes adapter sequences from high-throughput sequencing 1079 reads Ultrafast and memory-efficient 1081 alignment of short DNA sequences to the human genome Characterization of the alpha interferon-induced postentry 1086 block to HIV-1 infection in primary human macrophages and T cells A molecularly cloned Schwarz strain of 1107 measles virus vaccine induces strong immune responses in macaques and transgenic 1108 mice 1110 (2017) Reverse genetic system We wish to thank Tom Doyle and Chad Swanson for their useful comments on the manuscript, 865 and Matthieu Lewis, Nadine Laguette, Nathalie Arhel, Juliette Fernandez, Jean-Luc Battini,