Conserved phosphorylatable residues in motif G of positive-stranded virus RdRps regulate polymerase activity and suggest targets for drug design.

RNA viruses rely on an RNA-dependent RNA polymerase (RdRp) to replicate their genome. RdRps share a conserved core replicase structure described as a right hand within which RNA replication occurs. RNA polymerases contain a series of conserved motifs (A-G) that are essential for catalysis. Motif G, located at the RNA entry channel, guides the incoming RNA into the catalytic centre and and holds it in place during catalysis. Although RdRp phosphorylation has been reported, it has been scarcely studied. In most studied cases, phosphomimetic mutations reduced viral replication. In this study, we identified Theilers murine encephalomyelitis virus (TMEV) polymerase (3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 located in motif G are highly conserved in the sequences of picornavirus polymerases and in the structure of many positive-stranded virus polymerases, including nsp12 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using mutagenesis and reporter viruses, we show that phosphomimetic mutation of either residue abrogates viral replication, for both TMEV and SARS-CoV-2. Mutations of 3Dpol residues 109 and 110 into all other possible residues shows that, besides negatively charged phosphomimetic residues, bulky residues strongly inhibit replication, suggesting that phosphorylation inhibits polymerase activity by steric hindrance and/or through charge repulsion with RNA entering the catalytic core. Because these phosphorylatable residues are surface-exposed and conserved among viral polymerases, they represent promising targets for the rational design of broad-spectrum antiviral agents. ImportanceRNA viruses require an RNA-dependent RNA polymerase to replicate their genome. We identified in Theilers murine encephalomyelitis virus polymerase (TMEV 3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 are located in the entry channel of the polymerase, a region important for directing the RNA into the polymerase and locking it in place during catalysis. Incidentally, Thr109 and Ser110 are highly conserved in the sequences of picornavirus polymerases and in the structures of many positive-stranded virus polymerases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp12. Our study revealed that, in both TMEV 3Dpol and SARS-CoV-2 nsp12, mutation of either residue into a negatively charged amino acid that mimics phosphorylation abrogates viral replication, suggesting phosphorylation would block polymerase activity. As these phosphorylated residues are accessible and conserved, they provide important candidate targets for the design of antiviral molecules.