AbstractThe newly identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a global health emergency because of its rapid spread and high mortality. The molecular mechanism of interaction between host and viral genomic RNA is yet unclear. We demonstrate herein that SARS-CoV-2 genomic RNA, as well as the negative-sense RNA, is dynamically N⁶-methyladenosine (m⁶A)-modified in human and monkey cells. Combined RIP-seq and miCLIP analyses identified a total of 8 m⁶A sites at single-base resolution in the genome. Especially, epidemic strains with mutations at these identified m⁶A sites have emerged worldwide, and formed a unique cluster in the US as indicated by phylogenetic analysis. Further functional experiments showed that m⁶A methylation negatively regulates SARS-CoV-2 infection. SARS-CoV-2 infection also triggered a global increase in host m⁶A methylome, exhibiting altered localization and motifs of m⁶A methylation in mRNAs. Altogether, our results identify m⁶A as a dynamic epitranscriptomic mark mediating the virus–host interaction.