Gene expression profiling of HBEpCs and Caco-2 cells during infection with WT SARS-CoV-2 or a recombinant SARS-CoV-2 deficient in PLpro deISGylation activity

Interferon-stimulated gene 15 (ISG15) regulates diverse cellular processes, including antiviral immunity, through its conjugation to proteins, a process known as ISGylation. Several pathogens, including SARS-CoV-2, subvert ISGylation by encoding deISGylating enzymes. However, the direct targets and physiological consequences of coronaviral deISGylation remain poorly defined. Here, we engineered a recombinant SARS-CoV-2 harboring a mutant Nsp3 (Nsp3mut) with impaired papain-like protease (PLpro) deISGylating activity. Caco-2 and human bronchial epithelial (HBEpC) cells were infected with either wild-type or Nsp3mut SARS-CoV-2. Comparative transcriptomic profiling revealed that Nsp3mut infection led to a stronger induction of IFN- and ISG-related gene signatures compared to wild-type virus infection in both cell types. These results show that loss of PLpro-mediated deISGylation restores efficient antiviral innate immune responses during SARS-CoV-2 infection.