Structure of cytomegalovirus nuclease, UL98

 描述(申请人提供)：人巨细胞病毒(CMV)是一种疱疹病毒，是贝塔疱疹病毒亚家族的原型。巨细胞病毒会导致移植患者和艾滋病患者的肺炎、失明和死亡，以及新生儿的智力低下和听力损失。迫切需要开发新的抗病毒药物来治疗CMV感染。所有疱疹病毒都编码一种碱性核酸酶(AN)，ANS是最保守的疱疹病毒蛋白之一。然而，ANS在病毒复制中扮演的角色还没有被很好地理解，并且在不同的亚家族之间存在差异。来自α疱疹病毒亚家族的ANS的DNA酶活性被认为是通过从新复制的病毒DNA中移除分支或不寻常的结构来促进重组或促进DNA包装。相反，来自伽玛疱疹病毒亚家族的ANS具有RNase活性，并通过降解mRNAs来关闭宿主蛋白翻译。贝塔疱疹病毒ANS是否具有贝塔疱疹病毒亚家族所特有的作用或功能尚不清楚。目前的应用是一个成熟的多研究人员合作，重点是定义CMV AN，UL98的结构和生化活性，了解其在CMV复制中的机制作用，并识别UL98的小分子抑制剂，以探索其作为抗病毒靶标的潜力。我们的初步研究使用同源建模来预测UL98的活性位点和大肠杆菌表达的UL98的突变，以确认活性位点残基对DNA酶活性的重要性。构建了一个UL98缺失的病毒，发现UL98对CMV的生长有显著的减毒作用，这表明UL98对CMV的复制至关重要，并提示UL98的小分子抑制剂可能具有强大的抗病毒活性。为了支持后者，UL98核酸酶活性的抑制剂阿坦尼蓝PRL已被证明能抑制CMV的复制。目前的应用只有一个目的：确定野生型UL98和两个催化缺陷型UL98突变体的晶体结构，以及UL98与DNA和抑制剂atanyl Blue PRL的络合。将UL98的结构与伽马疱疹病毒ANS的现有结构进行比较，将有助于识别每种蛋白质独有的结构特征。UL98的结构还可能提示新的功能结构域，这将有助于指导突变的设计，以剖析UL98‘S的生化活性和复制中的功能角色。重要的是，UL98与抑制剂atanyl Blue PRL的复合结构将提供有价值的机制和结构见解，并将提供信息并使基于结构的其他UL98抑制剂的识别成为可能。这些抑制剂将作为药理探针，补充和扩展对UL98‘S功能的遗传和生化研究，并可能为抗病毒开发提供重要的先导结构。这些进展将扩大对UL98作为抗病毒靶标的追求，并最终可能导致治疗CMV感染的新型抗病毒药物。

 DESCRIPTION (provided by applicant): Human cytomegalovirus (CMV) is a herpes virus and prototype of the beta herpes virus subfamily. CMV causes pneumonitis, blindness, and death among transplant and AIDS patients, and mental retardation and hearing loss among newborns. There is a pressing need for development of new antiviral drugs to treat CMV infections. All herpes viruses encode an alkaline nuclease (AN) and ANs are among the most highly conserved herpes virus proteins. However, the roles that ANs play in virus replication are not well understood and differ between subfamilies. DNase activities of ANs from the alpha herpes virus subfamily are proposed to promote recombination or facilitate DNA packaging by removing branches or unusual structures from newly replicated viral DNA. In contrast, ANs from the gamma herpes virus subfamily have RNase activity and function to shut off host protein translation by degrading mRNAs. Whether beta herpes virus ANs serve similar roles or have functions unique to the beta herpes virus subfamily is not known. The current application is a well-established multi-investigator collaboration focused on defining the structure and biochemical activities of the CMV AN, UL98, understanding its mechanistic roles in CMV replication, and identifying small molecule inhibitors of UL98 to explore its potential as an antiviral target. Our initial studies used homology modeling to predict the UL98 active site and mutagenesis of E. coli-expressed UL98 to confirm the importance of active site residues for DNase activity. A UL98-null virus was constructed and found to be profoundly growth-attenuated, demonstrating that UL98 is critically important for CMV replication and suggesting that small molecule inhibitors of UL98 may have potent antiviral activity. In support of the latter atanyl blue PRL, an inhibitor of UL98 nuclease activity, has been shown to inhibit CMV replication. The current application has one aim: to determine crystal structures of wild type UL98 and two catalytically-deficient UL98 mutants, as well as UL98 complexed with DNA and with the inhibitor atanyl blue PRL. Comparison of the UL98 structure with existing structures of gamma herpes virus ANs will allow identification of structural features that are unique to each of these proteins. The UL98 structure may also suggest novel functional domains that will help guide the design of mutations to dissect UL98's biochemical activities and functional roles in replication. Importantly, the structure of UL98 complexed with the inhibitor atanyl blue PRL will provide valuable mechanistic and structural insights and will inform and enable structure-based identification of additional UL98 inhibitors. Such inhibitors will serve as pharmacological probes to complement and extend genetic and biochemical studies of UL98's functions and may provide important lead structures for antiviral development. These advances will enable expanded pursuit of UL98 as an antiviral target and may ultimately lead to novel antivirals for treating CMV infections.