Addition of m6A 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 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.

多瘤病毒科（Polyomaviridae）是一类小型DNA肿瘤病毒家族，包含多种可致病的人类病毒成员，例如默克尔细胞多瘤病毒、BK病毒与JC病毒。正如DNA肿瘤病毒的典型特征一样，多瘤病毒的基因表达呈时间调控模式，分为早期阶段与晚期阶段：早期阶段表达病毒调控蛋白，晚期阶段则表达病毒结构蛋白。此前有研究报道，原型多瘤病毒猿猴病毒40（SV40）表达的晚期转录本含有多个N6位甲基化腺苷（m6A），但尚未明确这些m6A位点的精确位置及其表型效应。本研究首先证实，在感染SV40的BSC40细胞中过表达关键m6A阅读蛋白YTHDF2，可加速病毒复制并形成更大的病毒噬斑；而对内源性YTHDF2基因或m6A甲基转移酶METTL3进行突变失活，则会产生相反效果，由此提示m6A在调控SV40基因表达中发挥正向作用。为直接验证这一假说，我们对SV40转录本上的m6A修饰位点进行了定位，共在病毒早期转录本上发现2个m6A位点，在晚期mRNA上发现11个m6A位点。我们通过同义突变使SV40晚期mRNA上的大部分m6A位点失活，结果发现由此获得的病毒突变株的复制速度慢于野生型SV40。SV40晚期mRNA的可变剪接不受m6A残基减少的影响，而我们的数据则表明m6A可增强病毒晚期转录本的翻译效率。综上，上述研究结果表明，SV40编码的晚期转录本上的m6A修饰，在增强病毒基因表达进而促进病毒复制方面发挥着重要作用。