Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

Abstract

The addition of a methyl group to the N6-position of adenosine (m⁶A) 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 m⁶A sites which are primarily enriched in 5'-DRACH-3' sequence motif. Herein, a large-scale comparative m⁶A analysis was conducted to investigate the conservation patterns of the DRACH motifs that corresponding to the reference m⁶A 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 m⁶A topology provides a foundation for the future intervention of influenza infection, replication, and pathobiology in susceptible hosts.

Figure 1

Locations and conserveness of the DRACH motifs utilized in this study. Schematic representation of locations of the putative motifs on the IAV-PR8 HA mRNA (A,B) and vRNA (C,D), accession no. AF389118, which coincident with the 8/9 m⁶A peaks identified in that are indicated by numbers. Motifs contain the complete, partial, and novel DRACH are indicated by coloured circles. (B,D) WebLogo-based diversity and/or conserveness of nucleotides in the proposed DRACH motifs in PR8 HA mRNA (B) and vRNA (D), one stack for each position in the sequence, the height of the stack indicates the sequence conservation at that position, the height of symbols within the stack indicates the relative frequency of each nucleotide at that position. The percent of each nucleotide in each position are indicated by coloured pie charts.

Figure 2

Summary of conserved DRACHs among all HA subtypes of IAVs. From the centre, the HA cladding system is represented by a maximum-likelihood tree contain representing sequences of each subtype. The numbers of conserved DRACH motifs to each HA subtype that located on either vRNA or mRNA are indicated. The highly susceptible species affecting each subtype are indicated.

Figure 3

Summary of conserved DRACH motifs among some IAVs HA mRNAs. Conserved motifs, virus, host species, and the number of sequences listed on IRD are indicated. Conservation percent is indicated on the upper side.

Figure 4

Summary of conserved DRACH motifs among the H5 mRNA. Conserved motifs, virus, host species, and the number of sequences listed on IRD are indicated. The conservation percent is indicated by coloured circles.