N6-methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA
- 1Institute of Biomedical Sciences Academia Sinica
Abstract
Previous studies have found widespread N6-methyladenosine (m6A methylation) on all forms of Influenza A virus (IAV) RNA, with m6A 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 m6A 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 m6A across all viral genomic RNA segments, compared with abundant m6A sites on viral mRNAs at ~20-30% m6A. Cross validation with glyoxal- and nitrite-mediated deamination of unmethylated adenosines (GLORI) confirmed multiple m6A sites on viral mRNA yet very few m6A on the genomic RNA. This paucity of m6A on genomic RNA makes it unlikely that m6A contributes to viral RNA packaging. Knockdown or pharmacological inhibition of the m6A 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 m6A 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 m6A-targeted antivirals would mainly impact the intracellular gene expression phase of IAV replication.
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BibTeX
@misc{wu2026n6methyladenosine,
title = {{N6-methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA}},
author = {H.-Y. Wu and W.-J. Tung and R. Wungmaiwo and P.-Y. A. Su and H.-W. Hsu and A. Gupta and B. N. Papas and M. Morgan and W.-C. Liu and I.-H. Wang and K. Tsai},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.01.748482},
url = {https://www.biorxiv.org/content/10.64898/2026.09.01.748482v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - N6-methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA
AU - H.-Y. Wu
AU - W.-J. Tung
AU - R. Wungmaiwo
AU - P.-Y. A. Su
AU - H.-W. Hsu
AU - A. Gupta
AU - B. N. Papas
AU - M. Morgan
AU - W.-C. Liu
AU - I.-H. Wang
AU - K. Tsai
PY - 2026
DA - 2026/09/01
PB - bioRxiv
DO - 10.64898/2026.09.01.748482
UR - https://www.biorxiv.org/content/10.64898/2026.09.01.748482v1
AB - Previous studies have found widespread N6-methyladenosine (m6A methylation) on all forms of Influenza A virus (IAV) RNA, with m6A 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 m6A 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 m6A across all viral genomic RNA segments, compared with abundant m6A sites on viral mRNAs at ~20-30% m6A. Cross validation with glyoxal- and nitrite-mediated deamination of unmethylated adenosines (GLORI) confirmed multiple m6A sites on viral mRNA yet very few m6A on the genomic RNA. This paucity of m6A on genomic RNA makes it unlikely that m6A contributes to viral RNA packaging. Knockdown or pharmacological inhibition of the m6A 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 m6A 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 m6A-targeted antivirals would mainly impact the intracellular gene expression phase of IAV replication.
ER -