Infection with cardiotropic Coxsackievirus B3 (CVB3) leads to activation of the innate immune system and production of type I Interferons (IFNs). Upon detection of IFNs, cells upregulate the expression of hundreds of downstream effector proteins called Interferon Stimulated Genes (ISGs) to establish the host cellular antiviral state. Among the ISGs, the small ubiquitin-like modifier ISG15 has pleiotropic antiviral function acting in its free form or covalently conjugated to other proteins. This so called ISGylation is mediated by an E1/E2/E3 enzyme cascade, peaking around day 3 of infection in cardiac tissue. Subsequent infiltration of innate and adaptive immune cells into the myocardium results in both, viral elimination as well as inflammation-induced cell death and thus causes acute viral myocarditis. Inefficient virus clearance and persistence of viral RNA in cardiac cells can then cause chronic cardiac inflammation. Previous work showed that ISGylation suppresses CVB3-mediated pathology, and enrichment of ISGylation by deactivating the deconjugating activity of the Ubiquitin-specific protease 18 (USP18) further enhances the antiviral host response. However, the manner in which ISGylation orchestrates resistance against CVB3 remains unclear. To investigate this, the cardiac proteome and ISGylome of infected wildtype, ISG15 / , and USP18C61A/C61A mice was analyzed by LC MS/MS. On the one hand, proteome analysis showed an infection-induced upregulation of the host-cellular immune response to establish a cardiac antiviral state. Additionally, IFIT1 and IFIT3, proteins with known antiviral effect against CVB3, were identified as ISG15-target proteins and ISGylation positively influenced their expression levels. On the other hand, infection led to a downregulation of proteins associated with metabolic pathways. Nonetheless, the cardiac mitochondrial energy production was upregulated in an ISGylation-dependent manner, indicating infection-induced increased energy demand. Concordantly, analysis of the cardiac ISGylome identified a variety of metabolic enzymes to be covalently modified by ISG15. Upregulation of glycolytic levels in primary cardiomyocytes was restricted by ISG15, which might be at least partly due to an attenuated activity of ISGylated hexokinase-2 and phosphofructokinase. Taken together, this study highlights the regulation of two key response pathways in viral infection by the ISG15 system. While the direct antiviral response might be supported by ISGylation-mediated stabilization of IFIT1/3, ISG15/ISGylation further ensures the efficient regulation of metabolic energy production during infection.
