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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.

Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus