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.

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