In recent years, epitranscriptomic modifications have been detected in numerous viral RNA, but the physiological function of most of them remains barely understood. Among these modifications, RNA methylations are an important modification induced by specific viral or cellular RNA methyltransferases. The first part of this manuscript focuses on RNA 2'O-methylation. This post-transcriptional modification prevents viral RNA detection by RIG-like receptors (RIG-I and MDA5) that regulate type-1 interferon expression and is therefore considered a 'self' marker. In this work, we address the possibility that RNA 2'O-methylation may interplay with the antiviral action of an interferon-induced restriction factor, namely ISG20. Briefly, we show that ISG20 exonuclease activity is strongly impaired when it encounters a 2'O-methylation mark within the RNA. ISG20 stops two nucleotides upstream (N-2) and at the methylated residue (N0). Structure-function analyses revealed that ISG20 R53 and D90 residues play a key role in the RNA hydrolysis impairment, which results from a steric clash of these residues with the 2'O-methylated nucleotide. We next extrapolated our observations to HIV-1 which is naturally 2'O-methylated by the host FTSJ3. By comparing the sensitivity to ISG20 of HIV-1 RNAs extracted from hypo-methylated viruses (produced in FTSJ3-KO cells) and from normally methylated viruses, we showed that internal 2'O-methylation protects the HIV-1 genome from ISG20 degradation. We confirmed this observation in infected cells and showed that ectopically expressed ISG20 drastically reduces the replication of hypomethylated VSV-G pseudotyped HIV-1 virus, as a consequence of impaired reverse transcription. Altogether, our results shed light on a new pro-viral role of viral RNA 2'O-methylation. Indeed, we demonstrated that HIV-1 2'O-methylation promotes viral replication by limiting ISG20-mediated restriction. In the second part of the manuscript, we characterized the CoV nonstructural protein 14 (nsp14) which is a bifunctional protein harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal N7-MTase domain that is presumably involved in viral mRNA capping. We integrate structural, biochemical, and virological data to identify 4 conserved regions essential for nsp14's enzymatic activities and virus viability. We identified several residues involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases and we assess their importance for in vitro enzymatic activity and for virus replication. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.
