Viral N-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus.

N-methyladenosine (mA) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by mA within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of mA methyltransferases decreases RSV replication and gene expression whereas knockdown of mA demethylases has the opposite effect. The G gene transcript contains the most mA modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of mA display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the mA-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral mA methylation upregulates RSV replication and pathogenesis and identify viral mA methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.