Eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and RNAs. Whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone H3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in DNA virus-infected cells. However, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections. By contrast, the various covalent modifications added to RNAs, termed epitranscriptomic modifications, can positively regulate mRNA translation and/or stability, and both DNA and RNA viruses have evolved to utilize epitranscriptomic modifications as a means to maximize viral gene expression. As a consequence, both chromatin and RNA modifications could serve as novel targets for the development of antivirals. In this Review, we discuss how host epigenetic and epitranscriptomic processes regulate viral gene expression at the levels of chromatin and RNA function, respectively, and explore how viruses modify, avoid or utilize these processes in order to regulate viral gene expression.

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 N⁶ position (m⁶A), although the precise location of these m⁶A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m⁶A 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 m⁶A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m⁶A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m⁶A addition on SV40 transcripts and identified two m⁶A sites on the viral early transcripts and eleven m⁶A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m⁶A 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 m⁶A residues and our data instead suggest that m⁶A enhances the translation of viral late transcripts. Together, these data argue that the addition of m⁶A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication. All organisms, including all viruses, use mRNAs to express the proteins required for their viability and replication. Viruses, in particular, are under pressure to replicate rapidly in order to spread before the host immune response becomes effective. Previously, it was reported that several viruses, including polyomaviruses, express mRNAs that bear methylated adenosine (m⁶A) residues, but the functional relevance of this modification was unclear. Here, we demonstrate that addition of m⁶A to mRNAs encoded by the prototypic polyomavirus SV40 increases both viral structural protein expression and viral spread. Targeting the sites of addition of m⁶A on SV40 mRNAs, using mutagenesis, therefore reduces SV40 replication, and a drug that globally inhibits m⁶A addition has a similar effect. These data identify m⁶A addition as a key step, and potential Achilles heel, in the polyomavirus life cycle.