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

The N6-methyladenosine (m6A) modification of HIV-1 RNAs plays essential roles in regulating viral infection. This modification has been extensively studied but due to a lack of precision of the detection methods, the number and precise positions of m6A sites remain unclear. Here, using the latest Nanopore chemistry and direct m6A base-calling option, we identify 18 m6As among which 14 are located in the 3' genome extremity and four in central regions. In addition, our data show that these positions are differently methylated among splicing isoforms. Among these sites, eleven are clustered in two short segments (197 nt and 43 nt) presenting peak-shaped methylation profiles. Single molecule analysis reveals a very small number of transcripts which are unmethylated in both clusters (= 1.5% of spliced transcripts and = 5.4% of unspliced viral RNA). We also identify 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 start in the first LTR and terminate at the polyA site of the second LTR and harbour six m6A sites. Five of them are shared with the other transcripts and remarkably, they present the highest methylation rates. The sixth site is uniquely methylated in this transcript suggesting that this RNA plays a function in HIV-1 infection. Together, these results reveal a new landscape of HIV m6A transcriptome modifications and open the route to decipher their role in the viral life cycle. Overall design: CD4+ T and supT1 cells infected by the virus HIV NL4-3 XCS. Cells are harvested 20h, 30h post-infection for SupT1 cells and 48h and 72h for CD4+ T cells. Two independant infections have been done, which were sequenced in duplicate for supT1.