Epitranscriptomic Addition of m 5C 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 many more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2’O-methyl modifications being particularly prevalent. The primary writer for m5C on HIV-1 RNAs was identified as NSUN2 and inactivation of NSUN2 inhibited not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition resulted from reduced HIV-1 protein, but not mRNA, expression, which in turn correlated with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulated the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both mRNA splicing and function.