Title: Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication
Abstract: Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein–mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs. Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein–mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs. The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus that belongs to the family Coronaviridae of genus Betacoronavirus and has a positive-sense strand RNA genome of ∼30 kb (1Kim D. Lee J.Y. Yang J.S. Kim J.W. Kim V.N. Chang H. 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Recent studies show that intrinsically disordered region 1 and NTD regulate N protein condensates affecting nucleic acid annealing and potentially implicated in viral packaging and assembly (20Iserman C. Roden C.A. Boerneke M.A. Sealfon R.S.G. McLaughlin G.A. Jungreis I. Fritch E.J. Hou Y.J. Ekena J. Weidmann C.A. Theesfeld C.L. Kellis M. Troyanskaya O.G. Baric R.S. Sheahan T.P. et al.Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid.Mol. Cell. 2020; 80: 1078-1091.e6Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21Savastano A. Ibanez de Opakua A. Rankovic M. Zweckstetter M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.Nat. Commun. 2020; 11: 6041Crossref PubMed Scopus (177) Google Scholar, 24Lu S. Ye Q. Singh D. Cao Y. Diedrich J.K. Yates J.R. Villa E. Cleveland D.W. Corbett K.D. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.Nat. Commun. 2021; 12: 502Crossref PubMed Scopus (194) Google Scholar). The SR linker region is phosphorylated by SRSF protein kinase (SRPK) (29Heaton B.E. Trimarco J.D. Hamele C.E. Harding A.T. Tata A. Zhu X. Tata P.R. Smith C.M. Heaton N.S. SRSF protein kinases 1 and 2 are essential host factors for human coronaviruses including SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.08.14.251207Crossref Scopus (0) Google Scholar), glycogen synthase kinase 3 (GSK-3) (30Wu C.H. Yeh S.H. Tsay Y.G. Shieh Y.H. Kao C.L. Chen Y.S. Wang S.H. Kuo T.J. Chen D.S. Chen P.J. Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.J. Biol. 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Arginine methylation of prototype foamy virus Gag in its glycine–arginine–rich box by PRMT1 regulates its nucleolar localization during replication (46Paris J. Tobaly-Tapiero J. Giron M.L. Burlaud-Gaillard J. Buseyne F. Roingeard P. Lesage P. Zamborlini A. Saïb A. The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag.Retroviralogy. 2018; 15: 48Crossref PubMed Scopus (7) Google Scholar). In the present study, we report that PRMT1 methylates SARS-CoV-2 N protein within its RGG/RG motifs to regulate the RNA-binding activity of the N protein toward its 5'-UTR genomic RNA. Moreover, arginine methylation modulates the role of N protein to inhibit SG formation. Our findings show for the first time that inhibition of type I PRMTs decreased SARS-CoV N methylation within virions and that arginine methylation is required for viral production. We noted that the SARS-CoV-2 N protein harbors two RGG (Fig. 1A) and three RG motifs like SARS-CoV, but unlike Middle East respiratory syndrome coronavirus (MERS-CoV). As RGG/RG motifs are preferred sites of PRMT1, PRMT5, and PRMT6 (31Thandapani P. O'Connor T.R. Bailey T.L. Richard S. Defining the RGG/RG motif.Mol. Cell. 2013; 50: 613-623Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), we tested whether they could be methylated. We first expressed and purified glutathione S-transferase (GST)-fusion proteins of the SARS-CoV-2 N protein fragments (GST-N 1–150, GST-N 150–262, and GST-N 263–419, Fig. 1A) and performed in vitro arginine methylation assays. Both the N-terminal fragment (amino acid residues 1–150) and the central region (amino acid residues 150–262) were arginine-methylated by PRMT1 (Fig. 1B). By contrast, the N protein fragments were not methylated by PRMT5 or PRMT6 (Fig. 1, C and D). We then substituted arginines in the RGG/RG motifs to lysines to identify the methylated sites and maintain the charge. Mutation of arginine 68 (R68K) in the N-terminal fragment had no significant effect on arginine methylation, whereas mutation of arginine 95 (R95K) completely abolished PRMT1 methylation (Fig. 1E), suggesting that R95 was the methylated residue in the GST-N 1 to 150 fragment. Similarly, mutation analysis identified R177 as the methylated residue in the central fragment (Fig. 1F). Taken together, R95 and R177 within the RGG/RG motifs of the SARS-CoV-2 N were methylated in vitro by PRMT1. We then determined whether the SARS-CoV-2 N protein was methylated in cells. HEK293 cells were transfected with a plasmid expressing Flag-epitope N protein (Flag-N). The cells were lysed, and the N protein was immunoprecipitated with anti-Flag antibodies and its methylation detected by Western blotting using the ASYM26 antibody, which specifically recognizes asymmetrically dimethylated arginine residues within RGG/RG motifs. Importantly, the asymmetrical dimethylarginine methylation of the Flag-N (N-me2) was significantly reduced by treatment of the cells with the type I PRMT inhibitor MS023 (Fig. 1G) and transfection with siPRMT1 (Fig. 1H). We next monitored patient data to identify modulation of PRMT1 expression during SARS-CoV-2 infection. Single-cell RNA sequencing analysis of nasopharyngeal and bronchial samples from 19 clinically well-characterized SARS-CoV-2 patients and five healthy controls was performed (47Chua R.L. Lukassen S. Trump S. Hennig B.P. Wendisch D. Pott F. Debnath O. Thürmann L. Kurth F. Völker M.T. Kazmierski J. COVID-19 severity correlates with airway epithelium–immune cell interactions identified by single-cell analysis.Nature biotechnology. 2020; 38: 970-979Crossref PubMed Scopus (618) Google Scholar). Importantly, analysis of their data showed that PRMT1 was significantly upregulated in infected patients (Fig. S1). These data suggest PRMT1 may play a role during the SARS-CoV-2 life cycle. We then performed MS analysis to identify SARS-CoV-2 N-interacting proteins in the absence or presence of MS023. Flag-tagged SARS-CoV-2 N protein was expressed in HEK293 cells and a pull-down performed using anti-Flag affinity resin. Co-purified cellular proteins were subsequently analyzed by affinity-purification (AP) LC-MS/MS. We identified 119 cellular proteins interacting with SARS-CoV-2 N protein (peptide count >2, fold change >2 between Flag-N and empty vector transfected, 0.1% false discovery rate, Fig. 2, A and B, Supplementary Dataset 1). Importantly, we identified several protein components of SGs such as G3BP1 and G3BP2 (Ras-GTPase–activating protein SH3 domain-binding protein 1 and 2) (48Tourrière H. Chebli K. Zekri L. Courselaud B. Blanchard J.M. Bertrand E. Tazi J. The RasGAP-associated endoribonuclease G3BP assembles stress granules.J. Cell Biol. 2003; 160: 823-831Crossref PubMed