The Methyltransferase Domain Of The Respiratory Syncytial Virus L Protein Catalyzes Cap N7 And 2'-O-Methylation

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at mu M concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.Author summaryRespiratory syncytial virus (RSV) is responsible of infant bronchiolitis and severe lower respiratory tract infections in infants and young children, and the leading cause of hospitalization in children under one year of age. However, we still lack a vaccine and therapeutics against this important pathogen. The main enzymatic activities involved in RSV propagation are embedded in the Large (L) protein that contains the polymerase domain and also all the activities required for RNA cap structure synthesis and methylation. This post-transcriptional RNA modifications plays a key role in virus replication because cap N7-methylation is required for viral RNA translation into proteins, and 2'-O-methylation hides viral RNA from innate immunity detection. Viral methyltransferase (MTase) activities are now considered potential antiviral targets because their inhibition might limit the virus production and strengthen early virus detection by innate immunity sensors. In this work, we compared the enzymatic activities of the MTase expressed as a single domain or in the context of the full-length L protein. We demonstrated that the MTase protein catalyzes the specific methylation of the cap structure at both N7- and 2'-O-positions, and we obtained the proof of concept that a S-adenosylmethionine analogue can inhibit the MTase activity of the L protein.