High-resolution HIV-1 m ⁶ A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

The N ⁶ -methyladenosine (m ⁶ A) modification of HIV-1 RNAs plays an essential role in regulating viral infection. This modification has been widely studied but the number and precise positions of the m ⁶ A sites remain unclear due to the lack of precision of detection methods. We used the latest Nanopore chemistry and direct m ⁶ A base-calling option to identify 18 m ⁶ As, 14 of which were located at the 3’ end of the genome, the other four being located in central regions. Our data reveal differential methylation of these positions between splicing isoforms. Eleven of these sites are clustered in two short segments (197 nt and 43 nt) with peak-shaped methylation profiles. Single-molecule analysis revealed that a very small number of transcripts were unmethylated in both clusters (≤ 1.5% of spliced transcripts and ≤ 5.4% of unspliced viral RNA). We also identified a ∼732 nt RNA species resulting from the transcription of non-integrated viral DNA circles closed by two long terminal repeats (2-LTR circles). These transcripts started in the first LTR, terminated at the polyA site of the second LTR and harbored six m ⁶ A 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 m ⁶ A transcriptome modifications and pave the way for studies deciphering their role in the viral life cycle.