Molecular probes for a vOTU from CCHFV using a fluorogenic peptide

描述(申请人提供)：克里米亚-刚果出血热(CCHF)病毒是一种单链RNA(-)奈罗病毒，可导致人类发烧、虚弱和严重出血。根据病毒的系统发育变异、传播途径和不同的治疗设施，CCHF的病死率从5%到70%不等。CCHF最初发现于俄罗斯和刚果，现已迅速蔓延到欧洲、亚洲和非洲的大片地区。最近，前往受CCHF影响的地区的美国公民和军事交通大幅增加，特别是南亚。因此，CCHF和/或其滴虫媒介传播到美利坚合众国的风险很大。2009年，一名驻阿富汗的美国士兵在CCHF死亡，突显了这一风险。目前，还没有FDA批准的治疗CCHF的疫苗或治疗方法。最近的报道发现了一个卵巢肿瘤蛋白水解酶(Votu)的病毒同源物，并暗示它通过切割翻译后修饰蛋白泛素(Ub)和Ub样干扰素模拟基因15(ISG15)而下调干扰素1型免疫反应。此外，CCHFV Votu的同系物被认为在经济破坏性的单链RNA(+)动脉病毒、猪呼吸和生殖综合征和马动脉炎病毒中发挥类似的作用。甚至像破坏性的水稻条纹病毒这样的植物病毒也被证明具有病毒卵巢肿瘤结构域的蛋白水解酶同源物。这项建议将实施一种经济有效的基于荧光的酶分析方法来鉴定CCHFV Votu的分子探针，并评估该分子探针支架是否可以作为选择性抑制其他病毒和真核卵巢肿瘤结构域蛋白的基础。具体地说，将在MLPCN设施中进行使用荧光肽和MLPCN文库的高通量筛选活动。最初从CCHFV中确定的Votu分子探针随后将使用二次、正交和三次体外测试以及体内测试进行优化。抑制CCHFV的Votu功能的分子探针所提供的信息将使我们不仅能够深入了解CCHF的Votu，而且还可以了解VOTU在病毒逃避先天免疫系统方面的作用。此外，从Votu分子探针收集的信息也将有助于确定开发针对VOTU及其真核超家族亲属的预防药物的实用性，这些药物对人类的先天反应进行负面调节。

DESCRIPTION (provided by applicant): Crimean-Congo hemorrhagic fever (CCHF) virus is a ssRNA (-) Nairovirus that produces fever, prostration, and severe hemorrhages in humans. Fatality rates for CCHF range from 5-70% based on phylogenetic variation of the virus, transmission route, and different treatment facilities. Originally identified in Russia and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recently, U.S. citizen and military traffic has increased substantially to the regions affected by CCHF, specifically South Central Asia. As a result, there is a substantial risk for transmission of CCHF and/or its tick vector to the United States of America. This risk has been recently highlighted by a CCHF death of an American Soldier based in Afghanistan in 2009. Currently, there is no FDA approved vaccine or therapeutic for treatment of CCHF. Recent reports have identified a viral homologue of the ovarian tumor protease (vOTU) and implicated its involvement down-regulation of the Interferon type 1 immune response through cleavage of post- translational modifying proteins ubiquitin (Ub) and Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of CCHFV's vOTU have been suggested to perform similar roles in the economically devastating ssRNA (+) Arteriviruses, Porcine Respiratory and Reproduction Syndrome and Equine Arteritis viruses. Even plant viruses such as the damaging rice stripe virus have been shown to possess a viral ovarian tumor domain protease homologue. This proposal will implement a cost effective fluorescent-based enzymatic assay to identify molecule probes for a vOTU from CCHFV as well as assess whether the molecular probe scaffold can serve as a basis to selectively inhibit other viral and eukaryotic ovarian tumor domain proteases. Specifically a high-throughput screening campaign using a fluorogenic peptide and the MLPCN library will occur at a MLPCN facility. Molecular probes initially identified for the vOTU from CCHFV will be subsequently optimized using secondary, orthogonal, and tertiary in vitro assays as well as in vivo ones. The resulting information provided by molecular probes inhibiting the function of vOTU from CCHFV will allow the necessary insight into not only the role of CCHF's vOTU, but vOTUs in general, in viral evasion of the innate immune system. Additionally, information gathered from vOTU molecular probes would also assist in determining the practicality of developing prophylactics targeting vOTUs and their eukaryotic superfamily relatives that negatively regulate the human innate response.