RABV L protein plays a role in immune escape through its methyltransferase activity

Viruses in the Mononegavirales order encode a large protein that orchestrates replication, transcription, and the capping of viral RNA. This protein, comprising over 2.000 amino acids, contains an RNA-dependent RNA polymerase, a capping domain, and a methyltransferase (MTase) domain involved in methylating the cap structure. The MTase domain features a conserved K-D-K-E catalytic tetrad -typical of 2'O-methyltransferases-which is essential for methylating viral mRNA caps at both the N7 and 2'O positions. However, the role of these residues in other epitranscriptomic modifications of rabies virus (RABV) RNAs remains poorly characterized. To further explore the role of mRNA cap methylation in the immune evasion strategies of RABV, we investigated the functional contribution of the K-D-K-E motif within the MTase domain, using the Thai isolate as a model. Using reverse genetics, we demonstrated that the mutation K1830R in the K-D-K-E tetrad of the Tha MTase domain induces changes in the methylation landscape of viral mRNAs and, intriguingly, of host mRNAs. In addition, viruses harbouring the K1830R mutation are more sensitive to interferon- and exhibit a less pathogenic phenotype in vitro and in vivo compared to the wild-type virus. Overall, these results suggest that the regulation of viral and cellular RNA methylation landscapes plays a crucial role in controlling RABV infection. Although the exact role of these epitranscriptomic modifications is not yet fully understood, some of these methylations appear to have proviral effects and enhance viral propagation by allowing RABV to efficiently evade the hosts antiviral response. ImportanceThis study highlights the pivotal role of the K-D-K-E catalytic domain included in the methyltransferase domain of the large protein of Rabies virus, by modelling viral RNAs with epitranscriptomic changes. For the first time, we identify specific methylations on the viral RNA, such as 2-O and m6A methylations, which seem to enable the virus to mask its RNA and evade detection by the hosts pattern recognition receptors. These epitranscriptomic modifications affect not only viral RNAs but also cellular RNAs, underscoring a complex interplay between viral and host mechanisms. We further demonstrate that RABV harbouring an altered K-D-R-E catalytic domain, exhibit differential methylation patterns correlated with increased sensitivity to IFN and lower pathogenicity. This emphasizes the importance of this domain in virulence and immune evasion.