5-methylcytosine RNA modifications promote retrovirus replication in an ALYREF reader protein-dependent manner.

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 (m(6)A)-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 characterize the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We find that 5-methylcytosine (m(5)C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we map at single-nucleotide resolution the m(5)C sites, which are located in multiple clusters throughout the MLV genome. Further, we show that the m(5)C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m(5)C 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 identify unexpectedly high levels of m(5)C in the murine leukemia virus RNA, precisely map its location, and show that ALYREF, a "reader" protein that specifically recognizes m(5)C, regulates viral production. Together, our findings provide a high-resolution atlas of m(5)C in murine leukemia virus and reveal a functional role of m(5)C in viral replication.