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

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. Here, Xue et al. identify N⁶-methyladenosine (m⁶A) modification sites in RNAs of respiratory syncytial virus (RSV) and show that these sites, particularly sites in the transcript encoding for the viral glycoprotein, affect virus replication in primary human cells and cotton rats. [ABSTRACT FROM AUTHOR]