Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance Lars Ketschera,b,1, Ronny Hannßc,d,1, David J. Moralese, Anja Bastersa,b, Susana Guerraf, Tobias Goldmanna, Annika Hausmanna, Marco Prinza,g, Ronald Naumannh, Andrew Pekoszi, Olaf Utermöhlenj,k, Deborah J. Lenschowe, and Klaus-Peter Knobelocha,2 aInstitute of Neuropathology, University of Freiburg, 79106 Freiburg, Germany; bFaculty of Biology, University of Freiburg, 79104 Freiburg, Germany; cDepartment of General, Visceral, Vascular and Thoracic Surgery, Division of Molecular Biology, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany; dFachbereich Biologie, Chemie, Pharmazie, Free University Berlin, 14195 Berlin, Germany; eDepartment of Medicine and Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; fDepartment of Preventive Medicine, Public Health and Microbiology, Universidad Autónoma Madrid, 28029 Madrid, Spain; gCentre of Biological Signalling Studies (BIOSS), University of Freiburg, 79106 Freiburg, Germany; hMax Planck Institute of Molecular Cell Biology and Genetics, Transgenic Core Facility, 01307 Dresden, Germany; iW. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; jInstitute for Medical Microbiology, Immunology and Hygiene, Medical Center, University of Cologne, 50935 Cologne, Germany; and kCenter for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved December 23, 2014 (received for review July 9, 2014) Protein modification by the ubiquitin-like protein ISG15 is an is also conjugated to cellular proteins. For example, ISGylation interferon (IFN) effector system, which plays a major role in antiviral increases the stability of the IFN regulatory factor 3 (IRF3), defense. ISG15 modification is counteracted by the isopeptidase resulting in enhanced activation of IRF3 target genes (11, 12). USP18, a major negative regulator of IFN signaling, which was also Consistent with the antiviral function, mice lacking ISG15 are shown to exert its regulatory function in an isopeptidase-indepen- more susceptible to influenza A and B, Sindbis, and herpes virus dent manner. To dissect enzymatic and nonenzymatic functions of infections (13). However, antiviral responses against vesicular USP18 in vivo, we generated knock-in mice (USP18C61A/C61A) express- stomatitis virus (VSV) and lymphocytic choriomeningitis virus C61A/C61A ing enzymatically inactive USP18. USP18 mice displayed in- (LCMV) are not altered (14). For both influenza B and Sindbis creased levels of ISG15 conjugates, validating that USP18 is a major virus infection, the antiviral function of ISG15 is dependent on its −/− C61A/C61A ISG15 isopeptidase in vivo. Unlike USP18 mice, USP18 ability to form conjugates because mice lacking the ISG15 E1 animals did not exhibit morphological abnormalities, fatal IFN hy- enzyme, UbE1L, are also susceptible to these infections (15, 16). persensitivity, or increased lethality, clearly showing that major Several viruses including influenza B virus, vaccinia virus (VACV), USP18 functions are unrelated to its protease activity. Strikingly, C61A/C61A and Crimean Congo hemorrhagic fever virus (CCHFV) have elevated ISGylation in USP18 mice was accompanied evolved strategies to target ISG15 conjugation, further suggesting by increased viral resistance against vaccinia virus and influenza B an important antiviral role of ISGylation (3, 17–19). virus infections. Enhanced resistance upon influenza B infection in C61A/C61A C61A/C61A Conjugation of ISG15 to its substrates is counteracted by the USP18 mice was completely reversed in USP18 mice, activity of ubiquitin-specific protease 18 (USP18/UBP43) (20). which additionally lack ISG15, providing evidence that the observed In vitro studies also provided evidence for other USPs to be reduction in viral titers is ISG15 dependent. These results suggest cross-reactive for ISG15, but the relevance of these isopeptidases that increasing ISGylation by specific inhibition of USP18 protease for deISGylation in vivo is unclear (21). Beyond enhanced activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects. Significance ISG15 | UBP43 | interferon | ubiquitin isopeptidase | influenza Viral infections still constitute a major health issue. Upon viral MICROBIOLOGY infection, interferon (IFN) elicits an antiviral response by acti- rotein modification by the ubiquitin-like (UBL) modifier in- vating multiple effector systems. The ubiquitin-like protein Pterferon (IFN)-stimulated gene 15 (ISG15) is strongly induced ISG15 is strongly induced by IFN and mediates its antiviral ef- by type I IFNs and represents one of the major antiviral IFN ef- fect by being covalently conjugated to cellular and viral pro- fector systems (1). ISG15 has weak sequence identity to ubiquitin teins. The protease USP18, which is also an important negative and is structurally characterized by two ubiquitin-like domains regulator of the IFN response, counteracts ISG15 conjugation. connected by a short linker region (2). In analogy to ubiquitin, Within this study, we have generated knock-in mice expressing ISG15 is covalently conjugated to protein targets via the consecutive USP18, which selectively lacks protease activity. ISG15 modifi- action of an E1-activating enzyme (Ube1L) (3), an E2-conjugating cation was enhanced in these animals, but the IFN response enzyme (UbcH8) (4), and a few E3 ligases. All enzymes involved in was unaltered. This clearly shows that USP18 has enzymatic the conjugation process are themselves strongly inducible by type I and nonenzymatic properties in vivo. Elevated ISGylation in- IFNs. The major E3 ligases for human and murine ISG15 modifi- creased resistance to influenza B infections, qualifying USP18 cation (ISGylation) are hHERC5 (5, 6) and mHERC6 (7, 8), re- protease inhibition as a potential antiviral strategy. spectively. hHERC5 is associated with polyribosomes and newly synthesized proteins are modified by ISG15 in a cotranslational Author contributions: S.G., A.P., O.U., D.J.L., and K.-P.K. designed research; L.K., R.H., D.J.M., A.B., S.G., T.G., A.H., R.N., O.U., D.J.L., and K.-P.K. performed research; A.P. con- manner (9). Thus, it was suggested that ISG15 modification of virus- tributed new reagents/analytic tools; L.K., R.H., D.J.M., A.B., S.G., T.G., M.P., O.U., D.J.L., derived proteins, which are the most prevalent translational prod- and K.-P.K. analyzed data; and L.K., S.G., O.U., D.J.L., and K.-P.K. wrote the paper. ucts in a virus-infected cell, mediates the antiviral activity of the The authors declare no conflict of interest. ISGylation system. In line with this suggestion, ISG15 modification This article is a PNAS Direct Submission. of the human papilloma virus protein HPV16 L1 interfered with 1L.K. and R.H. contributed equally to this work. virus assembly and decreased the infectivity of HPV in a dominant 2To whom correspondence should be addressed. Email: klaus-peter.knobeloch@uniklinik- manner (9). Moreover, ISGylation of the influenza virus protein freiburg.de. α NS1A prevents interaction with importin- and thereby interferes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with the capability of NS1A to counteract host defense (10). ISG15 1073/pnas.1412881112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1412881112 PNAS | February 3, 2015 | vol. 112 | no. 5 | 1577–1582 Downloaded by guest on September 25, 2021 − − ISGylation, USP18 knockout (USP18 / ) mice develop severe brain a protease-independent manner (28). Thus, the enhanced malformations and have a shortened lifespan (22). In addition, ISGylation seen in the USP18-deficient mice could have been due these animals fail to properly terminate IFN-signaling, exhibit to either the enhanced IFN signaling or impaired deISGylation. In enhanced IFN target gene expression, and show lethal hyper- addition, previous in vitro studies have provided evidence for other sensitivity to poly(I:C) injections (23). Furthermore, IFN de- USPs that exhibit cross-reactivity to ISG15 (21, 29). Therefore, we sensitization, which assures long-term refractoriness of the IFN − − evaluated whether the loss of USP18 isopeptidase activity is suffi- signaling pathway, is abrogated in USP18 / mice (24), under- cient to augment ISG15 modification in vivo. USP18C61A/C61A or lining the role of USP18 as a critical negative regulator of the − − WT mice were stimulated with poly(I:C), and ISG15-conjugated IFN pathway. USP18 / mice also exhibit increased resistance to proteins were monitored in lysates from liver, lung, lymph nodes, intracranial (i.c.) infections with VSV and LCMV (25). The spleen, and thymus (Fig. 1A and Fig. S3A). In addition, ISG15 −/− phenotypic alterations seen in USP18 mice were first exclu- modification was analyzed in BMMs treated with IFN-α,IFN-β, sively ascribed to enhanced ISG15 modification. However, sub- poly(I:C), or LPS (Fig. 1B and Fig. S3B). Inactivation of the USP18 sequent analysis of mice doubly deficient for either USP18 and isopeptidase activity significantly increased levels of ISGylated ISG15 (26) or USP18 and Ube1L (27) surprisingly revealed that substrates upon IFN-β, poly(I:C), or LPS stimulation and in all the lack of ISGylation cannot rescue the brain