Targeting the m(6)A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication

N(6)-methyladenosine (m(6)A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m(6)A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m(6)A-modified. How the cellular m(6)A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human β-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m(6)A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m(6)A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m(6)A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m(6)A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m(6)A-modified and host m(6)A pathway components control β-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m(6)A pathway to restrict coronavirus reproduction.