Charakterisierung der Proteaseaktivität und Spezifität der OTU-Domäne (ovarian tumour domain) des L-Proteins des Krim-Kongo-Fieber-Virus

泛素加工酶在宿主-病原体相互作用中起重要作用，一些病原体基因组编码去泛素化酶（DUBs）。卵巢肿瘤（OTU）结构域的蛋白质最近被鉴定为具有DUB活性，并且在整个进化过程中是保守的。克里米亚-刚果出血热病毒大蛋白（CCHFV-L）含有一个预测的OTU结构域，为进一步研究这种复杂病毒蛋白的功能提供了一个切入点。在本研究中，将分析含有OTU结构域的CCHFV-L蛋白亚基和可能参与蛋白酶功能的其他基序，以分析它们从不同底物中切割泛素的潜力。从合成的开放阅读框中纯化的蛋白在原核细胞中表达，将首次提示其在体外的蛋白酶活性。dub特异性的小活性位点修饰剂将用于鉴定活性位点半胱氨酸，并进一步表征真核细胞中的蛋白酶活性和特异性。在活性位点捕获泛素化细胞或病毒底物蛋白的失活蛋白酶突变体将允许鉴定CCHFV-L靶蛋白，并提供对病毒感染受损的细胞机制的深入了解。CCHFV-L蛋白利用细胞功能的新机制的发现将揭示该病毒成分在感染过程中的生物学作用，并为潜在的抗病毒药物干预提供一个窗口。

Ubiquitin processing enzymes play an important role in host-pathogen interactions and some pathogen genomes encode for deubiquitinating enzymes (DUBs). Ovarian tumour (OTU) domain containing proteins were recently identified to harbour DUB activity and are conserved throughout evolution. Crimean-Congo hemorrhagic fever virus large protein (CCHFV-L) contains a predicted OTU domain, providing an entry point to further examine the functions of this complex viral protein. In this study, subunits of CCHFV-L protein containing the OTU domain and additional motifs possibly participating in protease function will be analysed for their potential to cleave ubiquitin from different substrates. Purified proteins expressed in prokaryotic cells from synthetic open reading frames will give first hints on their protease activity in vitro. Small active site modifiers specific for DUBs will be used to identify the active site cysteine and further characterise protease activity and specificity in eukaryotic cells. Inactive protease mutants trapping ubiquitinated cellular or viral substrate proteins within the active site will allow identification of CCHFV-L target proteins and provide insight into cellular mechanisms impaired by viral infection. The discovery of novel mechanisms of the CCHFV-L protein to exploit cellular functions will shed light on the biological role of this viral component during infection, and provide a window for potential antiviral pharmaceutical intervention.