N 6 -methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA

Abstract Previous studies have found widespread N 6 -methyladenosine (m 6 A methylation) on all forms of Influenza A virus (IAV) RNA, with m 6 A found critical for viral replication, pathogenicity as well as viral RNA packaging. Here we applied the latest quantitative technologies to revisit the methylation landscape on the anti-sense genomic RNA of IAV. Unexpectedly, upon Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) analysis of IAV virion -extracted genomic RNA, we detected very little m 6 A regardless of production from human cells or chicken eggs. Concordantly, Nanopore direct RNA sequencing also detected an overall low occurrence and stoichiometry (generally <5%) of m 6 A across all viral genomic RNA segments, compared with abundant m 6 A sites on viral mRNAs at ∼20-30% m 6 A. Cross validation with glyoxal- and nitrite-mediated deamination of unmethylated adenosines (GLORI) confirmed multiple m 6 A sites on viral mRNA yet very few m 6 A on the genomic RNA. This paucity of m 6 A on genomic RNA makes it unlikely that m 6 A contributes to viral RNA packaging. Knockdown or pharmacological inhibition of the m 6 A methyltransferase METTL3 as well as the reader protein YTHDF2 both reduced viral mRNA levels and infectious viral particle production, with YTHDF2 promoting viral mRNA stability. Thus, the presence of m 6 A on IAV transcripts is indeed proviral, yet it is the mRNAs instead of genomic RNAs that are methylated at functionally relevant levels. Lastly, we provide proof of concept that a METTL3 small molecule inhibitor can be antiviral, and propose that m 6 A-targeted antivirals would mainly impact the intracellular gene expression phase of IAV replication. Importance Influenza A virus (IAV) is a contagious pathogen that cause seasonal epidemics, and with a large reservoir among birds, has historically switched hosts on multiple occasions and contributed to several pandemics. The rapid evolution of influenza viruses necessitates the development of antivirals that work across virus strains, and one approach is to target host mechanisms that are needed for the replication of a wide range of viruses. The adenosine methylation m 6 A, is present on the RNA of a wide range of viruses including IAV, where it mostly supports viral replication. However, it remains unclear how abundant is m 6 A on IAV RNA. Here, we utilize the latest RNA modification detection methods and found that IAV mRNAs are m 6 A methylated, with little m 6 A on the genomic RNA. We provide evidence that m 6 A on IAV mRNAs may prevent degradation of IAV mRNAs, and provide proof of concept that an m 6 A modification inhibitor can be antiviral against IAV.