Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes. Author summary Viruses utilize a variety of mechanisms to manipulate and suppress host responses to infection. One specific mechanism used by nairoviruses is the production of a deubiquitinating enzyme, termed the vOTU, that disrupts the innate immune response. This enzyme has been shown to play a key role in efficient replication of Crimean-Congo hemorrhagic fever virus (CCHFV), a severe human pathogen causing outbreaks with high case fatality rates. Recent genomic studies have revealed a high degree of sequence variation for the vOTU among nairoviruses, but knowledge relating to the functional impact of this diversity is lacking. Here we investigated the effects of this diversity on the structure and function of vOTUs from a wide range of nairoviruses. This revealed that vOTUs from different nairoviruses possess distinct preferences for certain host proteins. In addition, we found that different vOTUs possess distinguishing structural features, including a unique motif present in one that was previously undescribed. Utilizing this information, we were able to provide a rational basis for the observed differences in the vOTUs. This work provides a foundation to understand nairovirus evolution by providing insight into a mechanism that influences virus host adaptation and pathogenesis.