SARS-CoV-2 Nsp15 facilitates immune evasion and viral replication by limiting multiple host innate immune pathways,...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 15 (Nsp15) is a conserved uridine-specific endoribonuclease (EndoU) and is implicated in innate immune evasion, yet its precise molecular mechanism remains incompletely understood. Here, we demonstrate that Nsp15 limits antiviral innate immune responses in part by downregulating the cGAS-STING pathway. To investigate how Nsp15 antagonizes host innate immune responses, we engineered recombinant SARS-CoV-2 bearing WT or EndoU-inactive Nsp15 (H234A). Compared with the WT virus, the Nsp15-H234A mutant exhibited a 2-log decrease in peak viral titres in interferon (IFN)-competent A549-ACE2 cells, but not in their STAT1 knockout counterparts. This attenuation was partially reversed by STING knockout or STING inhibitors, highlighting STING's involvement in Nsp15-driven immune evasion. Transcriptomic analyses revealed the upregulation of IFNs and IFN-stimulated genes in cells infected with the Nsp15-H234A mutant virus. Notably, cGAS and STING transcripts and proteins were suppressed during WT but not mutant virus infections - even in STAT1-deficient cells - suggesting that Nsp15 contributes directly to their downregulation. In vitro, Nsp15 targeted cGAS and STING transcripts in an EndoU activity-dependent manner, thereby reducing cGAS-STING-driven IFN-β and NF-κB reporter activation. Infection of Syrian hamsters confirmed that the Nsp15-H234A mutant virus replicated to lower titres in respiratory tissues and elicited stronger expression of innate immune-related genes. Collectively, these findings define a key strategy by which SARS-CoV-2 Nsp15 subverts the cGAS-STING pathway to facilitate viral replication and immune evasion and underscore Nsp15 EndoU activity as a potential target for future coronavirus antiviral or vaccine design.