Addition of m(6)A to SV40 late mRNAs enhances viral structural gene expression and replication

Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N(6) position (m(6)A), although the precise location of these m(6)A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m(6)A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m(6)A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m(6)A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m(6)A addition on SV40 transcripts and identified two m(6)A sites on the viral early transcripts and eleven m(6)A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m(6)A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m(6)A residues and our data instead suggest that m(6)A enhances the translation of viral late transcripts. Together, these data argue that the addition of m(6)A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication.