Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases

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.

蜱传内罗病毒（Tick-borne nairoviruses）隶属于布尼亚病毒目（Bunyavirales），其编码一种卵巢肿瘤结构域蛋白酶（ovarian tumor domain protease, OTU），该蛋白酶可通过逆转泛素（ubiquitin, Ub）与干扰素刺激基因15产物（interferon-stimulated gene product 15, ISG15）介导的蛋白质翻译后修饰，从而抑制宿主先天免疫应答。Ub在真核生物中高度保守，而ISG15仅存在于脊椎动物中，且序列多样性显著。既往针对ISG15多样性对病毒蛋白-ISG15相互作用影响的研究，仅聚焦于单一物种的ISG15或有限种类的内罗病毒OTU。为更全面地解析OTU-ISG15相互作用的全貌，本研究通过生化实验评估了14种不同内罗病毒的OTU对12个物种ISG15的相对活性，结果发现ISG15的活性主要局限于特定的内罗病毒谱系，这大体上与已知的病毒-宿主关联相符。为揭示驱动OTU对ISG15亲和力的潜在分子机制，本研究解析了库普病毒（Kupe virus）与甘贾姆病毒（Ganjam virus）的OTU结合绵羊ISG15的X射线晶体结构，并分别与克里米亚-刚果出血热病毒（Crimean-Congo hemorrhagic fever virus）、埃尔韦病毒（Erve virus）的OTU结合人源与鼠源ISG15的复合物结构进行了比对分析。通过突变与结构分析，本研究鉴定出ISG15上的7个残基，这些残基主要影响内罗病毒OTU对ISG15的物种特异性识别。此外，本研究还鉴定出OTU上影响ISG15偏好性的残基，提示病毒OTU存在适配不同宿主ISG15的潜力。本研究结果为进一步开发溯源内罗病毒-宿主关联的研究方法，以及阐明ISG15多样性对内罗病毒感染的完整影响奠定了基础。