Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance

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Abstract
Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18C61A/C61A) expressing enzymatically inactive USP18. USP18C61A/C61A mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18−/− mice, USP18C61A/C61A animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18C61A/C61A mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18C61A/C61A mice was completely reversed in USP18C61A/C61A mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects. Significance Viral infections still constitute a major health issue. Upon viral infection, interferon (IFN) elicits an antiviral response by activating multiple effector systems. The ubiquitin-like protein ISG15 is strongly induced by IFN and mediates its antiviral effect by being covalently conjugated to cellular and viral proteins. The protease USP18, which is also an important negative regulator of the IFN response, counteracts ISG15 conjugation. Within this study, we have generated knock-in mice expressing USP18, which selectively lacks protease activity. ISG15 modification was enhanced in these animals, but the IFN response was unaltered. This clearly shows that USP18 has enzymatic and nonenzymatic properties in vivo. Elevated ISGylation increased resistance to influenza B infections, qualifying USP18 protease inhibition as a potential antiviral strategy.

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