N6-Adenosine Methylation of SARS-CoV-2 5-UTR Regulates Translation

The coronavirus disease 2019 (COVID19) continues to spread despite global vaccination efforts (1). This, alongside the rapid emergence of vaccine resistant variants, creates a need for orthogonal therapeutic strategies targeting more conserved facets of severe acute respiratory syndrome coronavirus (SARS-CoV-2) (2-4). One conserved feature of all coronaviruses is their ability to undergo discontinuous transcription wherein individual open reading frames fuse with the 5-UTR leader sequence during negative-strand RNA synthesis (5). As such all viral protein coding genes use the same 5-UTR for translation (6). Using in vitro reporter assays, we demonstrate that the SARS-CoV-2 5-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of bioinformatic and biochemical assays, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5-UTR regulates the rate of translation initiation. We show that m6A likely exerts this effect by destabilizing secondary structure in the 5-UTR, thereby facilitating access to the ribosomal pre-initiation complex. This discovery opens new avenues for novel therapeutic strategies aimed at controlling the ability of SARS-CoV-2 to replicate in host cells.