Molecular probes for a vOTU from CCHFV using a fluorogenic peptide

摘要/摘要 克里米亚-刚果出血热(CCHF)病毒是一种引起发热的单链RNA(-)奈罗病毒， 虚弱和严重的人类大出血。CCHF的死亡率从5%到70%不等，基于 病毒的系统发育变异、传播途径和不同的治疗设施。原来是这样的 CCHF发现于俄罗斯和刚果，现已迅速蔓延到欧洲、亚洲、 还有非洲。最近，前往该地区的美国公民和军事交通大幅增加 受到CCHF的影响，特别是中南亚。因此，有很大的风险 将CCHF和/或其扁虱媒介传播到美利坚合众国。这一风险一直是 最近突出的是CCHF在209年一名驻扎在阿富汗的美国士兵的死亡。 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 已经确定了卵巢肿瘤蛋白水解酶(Votu)的病毒同源物，并与其有关 干扰素-1通过切割POST-1参与下调免疫应答 翻译修饰蛋白泛素(Ub)和类Ub干扰素模拟基因15(ISG15)。 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，猪呼吸和繁殖综合征 和马动脉炎病毒。甚至像破坏性的水稻条纹病毒这样的植物病毒也受到了 显示具有病毒卵巢肿瘤结构域蛋白水解酶同源物。该提案将实施一项 经济高效的基于荧光的酶标法鉴定Votu的分子探针 以及评估分子探针支架是否可以作为选择性地 抑制其他病毒和真核卵巢肿瘤结构域蛋白。具体地说，高吞吐量 使用荧光肽和MLPCN文库的筛选活动将在MLPCN进行 设施。最初从CCHFV中确定的Votu分子探针将随后进行优化 使用二次、正交和三次体外试验以及体内试验。由此产生的 抑制CCHFV Votu功能的分子探针提供的信息将允许 不仅对CCHF的VOTU，而且对VOTU在病毒逃避中的作用有必要的了解 先天免疫系统。此外，从Votu分子探测器收集的信息还将 协助确定开发针对vOTU及其真核细胞的预防性药物的可行性 对人类先天反应进行负面调控的超级家族亲戚。

Summary/Abstract 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 209. 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.