Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), and 2′O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m(6)A and m(5)C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m(6)A and m(5)C in viral replication, we also demonstrate that overexpression of the key m(6)A reader protein YTHDF2 enhances MLV replication, while downregulation of the m(5)C writer NSUN2 inhibits MLV replication.