How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m 5C) and 2’O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m 5C on HIV-1 RNAs. NSUN2 inactivation not only inhibits m 5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m 5C dysregulates the alternative splicing of viral RNAs. These data identify m 5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.
Courtney et al. report that HIV-1 transcripts are modified by the addition of 5-methylcytosine (m 5C) residues. The nuclear methyltransferase NSUN2 is the primary m 5C writer and is required for appropriate HIV-1 translation. NSUN2 deficiency, and concomitant loss of m 5C residues, inhibits ribosomal recruitment to and alternative splicing of HIV-1 mRNAs.