SARS-CoV-2 papain-like protease (PLpro) employs deISGylation as a critical mechanism to simultaneously suppress innate immune signaling through MDA5-MAVS and TRIF pathways and reprogram cellular metabolism by reversing ISGylation-mediated inhibition of key glycolytic and pentose phosphate pathway enzymes, while maintaining antioxidant capacity to protect viral components from oxidative damage, thereby establishing deISGylation as a multifaceted viral strategy for immune evasion and metabolic hijacking.

Interferon-stimulated gene 15 (ISG15) regulates diverse cellular responses 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 ablated the deISGylating activity of the SARS-CoV-2 papain-like protease (PLpro) and found that loss of deISGylation boosted innate immune activation, attenuated virus replication, and promoted viral clearance in human cells and in mice. Through untargeted metabolomics and ISGylome proteomics analyses, combined with functional studies, we discovered in molecular detail how the activities of key metabolic enzymes in glycolysis, the pentose phosphate pathway, and oxidative stress are controlled by PLpro deISGylation. These findings provide fundamental new insight into how reversible ISGylation regulates immunity and metabolic processes at the molecular level and highlight viral deISGylation as a major viral tactic for rewiring immunometabolism.