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

Abstract

N⁶-methyladenosine (m⁶A) 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 m⁶A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m⁶A methyltransferases decreases RSV replication and gene expression whereas knockdown of m⁶A demethylases has the opposite effect. The G gene transcript contains the most m⁶A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m⁶A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m⁶A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m⁶A methylation upregulates RSV replication and pathogenesis and identify viral m⁶A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.