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 heterogeneity1–9. 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 (m6A) modifications near the 3' end. More densely installed in viral mRNAs than in genomic RNAs, these m6As 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 m6As and that these m6As regulate splicing independently of each other. Our single-RNA-level study demonstrates that HIV-1 tolerates functionally redundant m6As 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.

Functional Impacts of Epitranscriptomic m6A Modification on HIV-1 Infection

“…Black boxes represent predicted peak locations based on various sequencing approaches using RNA purified from ( A ) HIV-1-infected cells or ( B ) HIV-1 virions. Where possible, boxes represent exact reported peak width [ 25 , 26 , 27 , 28 , 31 , 33 ]. Other boxes are drawn based on peak calling maps from individual publications [ 29 , 30 ].…”

“…Therefore, these data do not provide insight into whether there is differential m 6 A deposition among specific splice isoforms. The most recent analysis of m 6 A sites in HIV-1 RNA was performed using a modification of direct RNA sequencing (DRS) that resulted in a remarkable improvement in full-length genome reads from 0.01% to 34.9% [ 31 ]. This approach allowed for full-length, single-molecule analysis at single-nucleotide resolution for both viral gRNA and splice isoforms.…”

“…Four sites in the 1.2 kb of the 3′ HIV-1 genome coincide with the common peaks identified in env / rev and the 3′ UTR in each of the previous studies [ 26 , 27 , 28 , 29 ]. These m 6 A sites are located at positions 8079, 8110, 8975, and 8989 of HIV-1 NL4-3 [ 31 ].…”

“…These m 6 A sites represent three of the four most prominent sites of m 6 A modification, in agreement with other studies, and were mutated for functional studies. Interestingly, mutation of these sites individually had no significant effect on viral protein expression, the proportion of unspliced RNA, p24 release, or virion infectivity [ 31 ]. Only when all three adenosines were mutated was there a significant reduction in virus infectivity.…”

“…The reduction in HIV-1 replication is likely due to a decrease in the abundance of unspliced RNA, which is necessary to produce infectious virions. The triple mutation also results in modest but significant increases in the proportion of partially and completely spliced viral RNA relative to total RNA [ 31 ]. This suggests functional redundancy of the individual m 6 A and that the modifications are involved in the regulation of splicing.…”