Single-RNA-level analysis of full-length HIV-1 RNAs reveals functional redundancy of m6As

Abstract HIV-1 exploits every aspect of RNA, a versatile macromolecule that undergoes various post-transcriptional modifications, to maximize its replication. Although the importance of chemical modifications on RNA has been recognized, their evolutionary benefits and precise roles in HIV-1 replication remain unclear. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of the phenotypic effects of perturbing host effectors, neglecting site-specificity and intra-RNA heterogeneity 1–9 . Here, we developed a new RNA-library-preparation method for full-length direct RNA sequencing and analyzed HIV-1-specific modifications at the single-RNA level. Our analysis revealed that the HIV-1 modification landscape is unexpectedly simple, showing three predominant N6-methyladenosine (m 6 A) modifications near the 3' end. More densely installed in viral mRNAs than in genomic RNAs, these m 6 As play a crucial role in maintaining normal levels of RNA splicing and translation. We also discovered that HIV-1 generates diverse RNA subspecies with distinct ensembles of the m 6 As and that these m 6 As regulate splicing independently of each other. Our single-RNA-level study demonstrates that HIV-1 tolerates functionally redundant m 6 As to provide stability and resilience to viral replication while minimizing the risk of unpredictable mutagenesis – a novel RNA-level strategy similar to bet-hedging in evolutionary biology.