Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression

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(5)C) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m(5)C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m(5)C 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(5)C dysregulates the alternative splicing of viral RNAs. These data identify m(5)C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.