Abstract
RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA.IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.