key: cord-0883541-l5f89x93 authors: Labeau, Athéna; Lefevre-Utile, Alain; Bonnet-Madin, Lucie; Fery-Simonian, Luc; Soumelis, Vassili; Lotteau, Vincent; Vidalain, Pierre-Olivier; Amara, Ali; Meertens, Laurent title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome date: 2021-03-23 journal: bioRxiv DOI: 10.1101/2021.03.23.436611 sha: 9d57a862420a604362c2f7bc6a1b509b4714c020 doc_id: 883541 cord_uid: l5f89x93 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative 22 agent of COVID-19 pandemic, which has caused a devastating global health crisis. 23 The emergence of highly transmissible novel viral strains that escape neutralizing 24 responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 25 biology and to develop additional therapeutic strategies. Using a comprehensive 26 identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) 27 approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-28 2 viral genome during infection. By systematically knocking down their expression in a 29 human lung epithelial cell line, we found that the majority of the RBPs identified in our 30 study are proviral factors that regulate SARS-CoV-2 genome replication. We showed 31 that some of these proteins represented drug targets of interest for inhibiting SARS- raises the concern of future variants escaping from natural or vaccine-induced 49 neutralizing immunity. Furthermore, a recent study conducted in Manaus, Brazil, 50 suggested a potential antibody wane at 8 months post-exposure (Sabino et al., 2021) . 51 Thus, there is an urgent need to improve our understanding of SARS-CoV-2 replication 52 mechanisms to develop additional therapeutic strategies. 53 The genome of SARS-CoV-2 is composed of a single stranded positive RNA with 54 a length of approximatively 30 kb (Zhou et al., 2020) . In the cytoplasm of infected cells 55 the viral RNA is translated by the host machinery in two overlapping replicase 56 polyproteins that undergo subsequent viral-protease-mediated processing to generate 57 was unexpected. Pre-mRNA splicing is critical for mRNA nucleo-cytoplasmic export, 162 thereby contributing to the regulation of gene expression (Reed and Hurt, 2002) . It is 163 tempting to speculate that SARS-CoV-2 RNA may sequester host RBP involved in 164 splicing in order to hamper cellular mRNA nuclear export, thus favoring the cytoplasmic 165 translation of viral RNAs. This hypothesis is consistent with recent findings showing 166 that SARS-CoV-2 NPS16 disrupts mRNA splicing to antagonize innate immunity at a 167 post-transcriptional level (Banerjee et al., 2020) . To gain insights on the interactions between host factors and viral proteins we 173 compared our SARS-CoV-2 interactome with a reference coronavirus interactome, 174 which corresponds to a meta-analysis of the viral-host protein-protein interactions of 175 13 coronaviruses collected built upon 112 publications (Perrin-Cocon et al., 2020). Of 176 the 142 high-confidence hits identified by our ChIRP approach, 80 overlapped with the 177 1,140 host proteins (56%) of the reference coronavirus interactome. As illustrated in 178 Figure 3A , these host proteins captured by our ChIRP assay are known interactors of 179 one or several coronaviruses viral proteins. Interestingly, a majority of these proteins 180 (79%) were previously identified as N protein-binding partners. Shared interactors with 181 S, NSP1, NSP2, NSP9, NSP12, NSP13, NSP14, NSP15, ORF6 and ORF9b were also 182 identified ( Figure 3A ). In our interaction network, the ATP-dependent RNA helicase 183 DDX1 was the most connected factor interacting with both N and NSP14 from both 184 TGEV and PEDV, NSP14 from SARS-CoV-1 and NSP2 from MHV. Altogether, this 185 supports a key role of DDX1 in SARS-CoV-2 replication cycle, as previously reported 186 for other coronaviruses (Wu et al., 2014; Xu et al., 2010) . 187 We then analyzed the subset of cellular proteins that did not overlap with the 188 reference coronavirus interactome, which essentially corresponds to novel SARS-189 CoV-2 and coronavirus interactors. First, we compared the fold enrichment in the 190 ChIRP experiments for the 62 new and 80 known interactors and observed a similar 191 distribution, indicating that the two subsets were qualitatively equivalent and 192 correspond to robust interactions ( Figure 3B ). Next, we investigated distribution of Figure 4E ). Sunitinib malate inhibits also the N protein production in 267 infected cells, consistent with a role of EWSR1 in genome replication ( Figure S3B ). 268 Similar results were obtained in 293T-ACE2 cells ( Figure S3C ). 269 In conclusion, our data provides a landscape of functional interactions that the 270 SARS-CoV-2 RNA genome establishes with the host cell during infection. We provide 271 several evidences that SARS-CoV-2 exploit several cellular RBP to facilitate viral 272 replication and highlight host molecules that could be targeted for antiviral intervention. The authors declare no competing interest. 299 SARS-CoV-2 Disrupts Splicing Protein Trafficking to Suppress Host Defenses Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-600 transcriptional drivers of cancer progression? 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