m6A Methylation of Transcription Leader Sequence of SARS‐CoV‐2 Impacts Discontinuous Transcription of Subgenomic mRNAs

The SARS‐CoV‐2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS−L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base‐paired in the short stem‐loop structure 5’SL3 that opens during discontinuous transcription to form long‐range RNA‐RNA interactions with nascent (−)‐strand transcripts at complementary TRS‐body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5’SL3 and the long‐range TRS‐leader:TRS‐body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5’SL3 and long‐range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization. Host‐mediated regulation: SARS‐CoV‐2 RNA at position A74 is methylated to m6A by the host METTL3/METTL14 complex. We show that this modification destabilizes the structure of stemloop 3 in the 5’ untranslated region. This modification of a transcription regulatory sequence has a significant effect on the stability of the duplexes that are formed during viral RNA synthesis, and regulate abundance of the subgenomic RNAs.