Virus-induced deconjugation of ISG15 dysregulates innate immunity and cellular metabolism.
Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that regulates diverse cellular responses, including antiviral immunity, through its conjugation to proteins in 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 genetically ablated the deISGylating activity of the SARS-CoV-2-encoded papain-like protease (PLpro) and found that loss of deISGylation boosted innate immune activation, attenuated viral replication, and promoted viral clearance in human cells and mice. Metabolomics, ISGylome proteomics, and functional analyses revealed that PLpro deISGylation relieved metabolic restriction of virus infection by directly regulating the activity of key enzymes controlling glycolysis, the pentose phosphate pathway, and redox homeostasis. These findings provide fundamental insight into how reversible ISGylation regulates immunity and metabolic processes at the molecular level and highlight viral deISGylation as a major strategy to overcome host immunometabolic defenses.