Influenza A viruses (IAVs) pose serious public health consequences, causing severe epidemics and occasional pandemics. Human IAV pandemics are associated with zoonotic spillover from animals to humans, especially from birds. Infection of IAV in chickens induces a range of transcriptional and epitranscriptional changes. Methylation at the N6-position of adenosines (m6A) is the most abundant chemical posttranscriptional modification deposited onto mRNA in eukaryotic species. The m6A regulates various RNA metabolic processes, including RNA structure, stability, protein translation, and splicing. Notably, the m6A has also been reported in viruses to play central regulatory roles in the viral lifecycle and host-pathogen interaction.

The m6A marks are installed onto mRNA by a complex group of methyltransferases (METTL3/14/WTAP complex), removed by a group of demethylases (ALKBH5 and FTO), and read by readers (YTHDF1-3, YTHDC1, and YTHDC2). The genetics and functions of m6A cellular machinery are well-characterized in humans; however, knowledge in animals, including birds, remained elusive.

This PhD project assessed the unique evolutionary patterns and genetic and structural alterations of chicken m6A machinery proteins compared to human orthologues. The conservation of m6A marks was also predicted in all IAV strains, and virus-specific m6A marks were highlighted and exploited to determine their roles in virus replication kinetics.

While IAV infection transcriptionally reduced m6A-associated genes, several m6Aassociated proteins, including chALKBH5, downregulated replication of IAVs and protein expression in a time-course manner. Mechanistic investigations revealed that middle and carboxy (C)-fragments were shown to be responsible for the antiviral effect of chALKBH5 against H9N2 and H1N1 influenza subtypes, whereas the nuclear localization signal located at C-fragment regulated the antiviral action of chALKBH5.

Using CRISPR/Cas13 editing technology, the chALKBH5 was tethered to a
catalytically inactive variant, dCas13b, to remove m6A marks from IAV transcripts. Targeted demethylation of the individual or multiple m6A modifications in the haemagglutinin (HA) gene of IAV H9N2 downregulated viral replication and protein expression. An in-house generated reporter chALKBH5 cell line revealed that chALKBH5 mediated inhibition of IAV is via interaction with the viral NP protein, but not NS1. Notably, in contrast to human WTAP, chWTAP failed to interact with the chMETTL3/14 complex, suggesting a differing mechanism in m6A methylation in chicken.

Using reverse genetics of IAV, several m6A sites were added or removed from the HA gene of H9N2. The presence of m6A marks promotes IAV replication and protein expression, whereas demethylation has the opposite effect. Ultimately, using m6A-seq, the alteration in the m6A methylome in virus-infected DF1 chicken cell line was mapped and H9N2 m6A peaks were identified. Using mass spectrometry, the chALKBH5 interactome was determined. Finally, it was shown that chALKBH5 exerts a pan-antiviral function against various RNA viruses. Taken together, viral and host m6A were epitrancriptomically investigated, which will unravel an array of future studies to examine the potential of m6A to regulate IAVs transmission across multiple susceptible species.