High-resolution HIV-1 m6A epitranscriptome reveals isoform-dependent methylation clusters and unique 2-LTR transcript modifications.

The N6 -methyladenosine (m6A) modification of HIV-1 has been widely studied but the number and precise positions of the m6A sites remain unclear due to the lack of precision of detection methods. Using the latest Nanopore chemistry and direct m6A base-calling, we identified 18 m6A: 14 at the 3' end and 4 in central regions of the genome. Our data reveal differential methylation of these positions between splicing isoforms. Eleven of these sites are clustered in two short segments with peak-shaped methylation profiles. Single-molecule analysis revealed that a very small number of transcripts were unmethylated in both clusters. We also identified a ∼732 nt RNA species resulting from the transcription of non-integrated viral DNA circles closed by two long terminal repeats. These transcripts started in the first LTR, terminated at the polyA site of the second LTR, and harbored six m6A sites. Five of these sites were present in other transcripts and, remarkably, had the highest methylation rates. The sixth site was methylated only in this transcript, suggesting a role for this RNA in HIV-1 infection. These findings reveal a new landscape of HIV m6A transcriptome modifications and pave the way for studies deciphering their role in the viral life cycle.