Viral N(6)-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

N(6)-methyladenosine (m(6)A) 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 m(6)A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m(6)A methyltransferases decreases RSV replication and gene expression whereas knockdown of m(6)A demethylases has the opposite effect. The G gene transcript contains the most m(6)A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m(6)A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m(6)A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m(6)A methylation upregulates RSV replication and pathogenesis and identify viral m(6)A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.