Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species’ ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species’ ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.

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Nairoviruses are arthropod-borne viruses with a nearly global geographical distribution. Several are known causative agents of human disease, including Crimean–Congo hemorrhagic fever virus (CCHFV), which has a case fatality rate that can exceed 30%. Nairoviruses encode an ovarian tumour domain protease (OTU) that can suppress the innate immune response by reversing post-translational modifications by ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15). As a result, the OTU has been identified as a potential target for the development of CCHFV therapeutics. Despite sharing the same general fold, nairoviral OTUs show structural and enzymatic diversity. The CCHFV OTU, for example, possesses activity towards both Ub and ISG15, while the Hazara virus (HAZV) OTU interacts exclusively with Ub. Virology studies focused on the OTU have mostly been restricted to CCHFV, which requires BSL-4 containment facilities. Although HAZV has been proposed as a BSL-2 alternative, differences in the engagement of substrates by CCHFV and HAZV OTUs may present complicating factors when trying to model one using the other. To understand the molecular underpinnings of the differences in activity, a 2.78 Å resolution crystal structure of HAZV OTU bound to Ub was solved. Using structure-guided site-directed mutagenesis, HAZV OTUs were engineered with altered or eliminated deubiquitinase activity, including one with an exclusive activity for ISG15. Additionally, analysis of the structure yielded insights into the difference in inhibition observed between CCHFV and HAZV OTUs with a Ub-based inhibitor. These new insights present opportunities to utilize HAZV as a model system to better understand the role of the OTU in the context of infection.