Reversible ISG15 conjugation regulates antiviral innate immunity and intracellular metabolism 2305671

Abstract Introduction ISG15 is an interferon-induced modifier that regulates diverse cellular events through protein ISGylation, a process that is often reversed by viruses that encode deISGylating enzymes. However, the physiological consequences of dynamic ISGylation and deISGylation in host defense, particularly innate immunity and intracellular metabolism, remain largely elusive. Methods By integrating innate immune profiling, metabolomics, and ISGylome proteomics analyses, and through the use of a recombinant SARS-CoV-2 selectively deficient in the deISGylating activity of the papain-like protease (PLpro), we unveiled how ISGylation and viral deISGylation shape cellular immunometabolism. Results Infection studies in K18-hACE2 mice and human cells showed that the deISGylation-deficient virus elicited heightened innate immune responses, which correlated with virus attenuation. This augmented immune activation was driven by MDA5-MAVS or TRIF signaling in a cell type-specific manner. Global ISGylome proteomics analysis identified key innate immune proteins and several metabolic enzymes directly targeted by PLpro deISGylation. Untargeted metabolomics analysis revealed that viral deISGylation is a key driver of metabolic rewiring, particularly in pathways linked to carbon metabolism and redox homeostasis. Specifically, while ISGylation inhibited the enzymatic activities of ALDOA and PRDX1 by preventing their oligomerization/dimerization, viral deISGylation restored their activity, creating a milieu optimal for virus replication. Conclusion This work reveals previously unrecognized mechanisms by which virus- or inflammation-induced ISGylation regulates immunometabolism, and uncovers viral deISGylation as a key determinant of SARS-CoV-2 innate immune evasion and metabolic rewiring. Funding Source R37 AI087846 Topic Categories Viral Immunology (VIR)