Coronavirus NSP14 Drives Internal m ⁷ G Modification to Rewire Host Splicing and Promote Viral Replication

SARS-CoV-2, the causative agent of COVID-19, manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral nonstructural protein 14 (NSP14) in inducing N 7-methylguanosine (m ⁷ G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (GTP) to m ⁷ GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m ⁷ G modification. This activity is dependent on NSP14's N 7-methyltransferase ( N 7-MTase) domain and is enhanced by interaction with NSP10. Internal m ⁷ G modification by NSP14 predominantly occurs in the nucleus and is conserved across alpha-, beta- and gamma-coronaviruses. Mechanistically, we show that this RNA modification disrupts normal splicing by promoting intron retention and generating novel splice junctions, particularly in genes regulating genome stability, RNA metabolism and nuclear processes. Importantly, inhibition of m ⁷ G modification, through pharmacological targeting of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks host transcriptomic machinery to support infection. Our findings reveal a previously unrecognized epitranscriptomic mechanism by which coronaviruses reprogram host gene expression and suggest that NSP14-induced m ⁷ G modification is a potential therapeutic target.