Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m(6)A) sites among influenza A viruses

The addition of a methyl group to the N6-position of adenosine (m(6)A) is considered one of the most prevalent internal post-transcriptional modifications and is attributed to virus replication and cell biology. Viral epitranscriptome sequencing analysis has revealed that hemagglutinin (HA) mRNA of H1N1 carry eight m(6)A sites which are primarily enriched in 5′-DRACH-3′ sequence motif. Herein, a large-scale comparative m(6)A analysis was conducted to investigate the conservation patterns of the DRACH motifs that corresponding to the reference m(6)A sites among influenza A viruses. A total of 70,030 complete HA sequences that comprise all known HA subtypes (H1–18) collected over several years, countries, and affected host species were analysed on both mRNA and vRNA strands. The bioinformatic analysis revealed the highest degree of DRACHs conservation among all H1 sequences that clustered largely in the middle and in the vicinity to 3′ end with at least four DRACH motifs were conserved in all mRNA sequences. The major HA-containing subtypes displayed a modest DRACH motif conservation located either in the middle region of HA transcript (H3) or at the 3′ end (H5) or were distributed across the length of HA sequence (H9). The lowest conservation was demonstrated in HA subtypes that infect mostly the wild type avian species and bats. Interestingly, the total number and the conserved DRACH motifs in the vRNA were found to be much lower than those observed in the mRNA. Collectively, the identification of putative m(6)A topology provides a foundation for the future intervention of influenza infection, replication, and pathobiology in susceptible hosts.