N6-Adenosine Methylation of a Single Site in SARS-CoV-2 5'-UTR Promotes Translation

The coronavirus disease 2019 (COVID19) led to devastating health outcomes and has continued to spread despite global vaccination efforts1. 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)2-7. The viral genome is a single positive RNA strand divided into a genomic and a subgenomic segment. All 16 non-structural viral proteins are translated from the genomic polycistronic ORFs 1a, and 1b using a single 5'-UTR leader8,9. To our surprise, the full length 5'-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of biochemical assays and bioinformatic analyses, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5'-UTR regulates the rate of translation initiation. We demonstrate that m6A likely exerts this effect by destabilizing the third stem loop (SL3) and increasing overall accessibility to protein complexes. Our discovery provides a foundational insight into the biology of SARS-CoV-2 by linking m6A modification to its translational regulation and, thus, opens a new avenue for potential novel therapeutic strategies.