PCIF1-mediated deposition of 5′-cap N(6),2′-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N(6),2′-O-dimethyladenosine (m(6)A(m)), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m(6)A(m)-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N(6)-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m(6)A(m)-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m(6)A(m) in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.