Coronavirus NSP14 drives internal m7G modification to rewire host splicing and promote viral replication.

SARS-CoV-2 manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral non-structural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of GTP to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14's N7-methyltransferase (N7-MTase) domain, and the NSP10-NSP14 interaction increases cellular m7G levels primarily by increasing NSP14 protein abundance. NSP14-induced m7G modification is conserved across alpha-, beta-, and gamma-coronaviruses. Mechanistically, we show that this RNA modification is associated with altered splicing, particularly in genes regulating genome stability, RNA metabolism, and nuclear processes. Importantly, using SARS-CoV-2 infection models, we show that viral replication is associated with increased cellular m7G signal, supporting the relevance of this pathway during infection. Inhibition of NSP14 N7-MTase or RNA polymerase II reduces SARS-CoV-2 replication, consistent with a model in which NSP14-induced m7G modification may contribute to viral replication. Our findings reveal a previously unrecognized epitranscriptomic mechanism and suggest that NSP14-induced m7G modification may contribute to the remodeling of host gene expression during coronavirus infection.