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，并且一些病原体基因组编码去泛素化酶（DUB）。卵巢肿瘤（OTU）结构域含有蛋白质最近被确定为港口DUB活性，并在整个进化过程中保守。克里米亚-刚果出血热病毒大蛋白（CCHFV-L）含有预测的OTU结构域，为进一步研究这种复杂病毒蛋白的功能提供了切入点。在这项研究中，CCHFV-L蛋白的OTU结构域和其他基序可能参与蛋白酶功能的亚基将被分析其潜在的切割泛素从不同的基板。在原核细胞中表达的来自合成开放阅读框的纯化蛋白质将在体外对其蛋白酶活性给出初步提示。DUB特异性的小活性位点修饰剂将用于鉴定真核细胞中的活性位点半胱氨酸和进一步的半胱氨酸蛋白酶活性和特异性。将泛素化的细胞或病毒底物蛋白捕获在活性位点内的非活性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这种病毒组分在感染期间的生物学作用，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.