Coronavirus NSP14 Drives Internal m7G 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 N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (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 is enhanced by interaction with NSP10. Internal m7G 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. Importantly, inhibition of m7G 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 m7G modification is a potential therapeutic target. RNA-seq profiling was performed on HEK293T cells transfected with either empty vector (EV-pCAGGS) or with plasmids encoding either SARS-CoV-2 NSP14 (NSP14-HA-pCAGGS) or its mutant (M4-HA-pCAGGS). RNA-Seq libraries were prepared from poly(A)-enriched or m⁷G-immunoprecipitated RNA.