Structure of cytomegalovirus nuclease, UL98

巨细胞病毒核酸酶 UL98 的结构

• 批准号: 9086246
• 负责人: Martin K Safo
• 金额: $ 7.63万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9086246
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Antiviral Agents; Attenuated; Biochemical; Biological Assay; Blindness; Cessation of life; Cidofovir; Collaborations; Complement; Complex; Computer Simulation; Congenital Abnormality; Crystallization; Cytomegalovirus; Cytomegalovirus Infections; DNA; DNA Packaging; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; Deoxyribonucleases; Development; Disease; Dissection; Dose-Limiting; Escherichia coli; Evaluation; Fetus; Foscarnet; Future; Ganciclovir; Genetic; Genetic Recombination; Goals; Growth; HIV; Health; Herpesviridae; Herpesvirus 1; Homologous Protein; Homology Modeling; Human; Human Herpesvirus 4; Human Herpesvirus 8; Immune; In Vitro; Laboratories; Lead; Ligands; Mental Retardation; Messenger RNA; Molecular; Mutagenesis; Mutation; Newborn Infant; Nucleotides; Outcome; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Play; Pregnancy; Proteins; Pulmonary Inflammation; Recombinant Proteins; Recombinants; Research Personnel; Retinitis; Ribonucleases; Roentgen Rays; Role; Structure; T-Lymphocyte; Techniques; Therapeutic; Toxic effect; Translations; Transplant Recipients; Transplantation; Viral; Viral Proteins; Virus; Virus Replication; X ray diffraction analysis; X-Ray Diffraction; base; deafness; design; drug development; drug discovery; endoexonuclease; experience; hearing impairment; in utero; inhibitor/antagonist; insight; member; mutant; novel; nuclease; pathogen; prevent; protein complex; protein expression; prototype; repaired; small molecule; small molecule inhibitor; viral DNA; viral alkaline nuclease; viral resistance

 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.

 描述（由申请方提供）：人巨细胞病毒（CMV）是一种疱疹病毒，是β疱疹病毒亚科的原型。巨细胞病毒可导致移植和艾滋病患者的肺炎、失明和死亡，以及新生儿的智力迟钝和听力损失。迫切需要开发新的抗病毒药物来治疗CMV感染。所有疱疹病毒编码碱性核酸酶（AN），并且AN是最高度保守的疱疹病毒蛋白。然而，AN在病毒复制中所起的作用还不清楚，并且在亚家族之间存在差异。来自α疱疹病毒亚家族的AN的DNA酶活性被提出通过从新复制的病毒DNA中去除分支或不寻常的结构来促进重组或促进DNA包装。相比之下，来自γ疱疹病毒亚家族的AN具有RNA酶活性和通过降解mRNA来关闭宿主蛋白质翻译的功能。β疱疹病毒AN是否具有类似的作用或具有β疱疹病毒亚家族特有的功能尚不清楚。目前的申请是一个完善的多研究者合作，专注于定义CMV AN，UL98的结构和生化活性，了解其在CMV复制中的机制作用，并确定UL98的小分子抑制剂，以探索其作为抗病毒靶点的潜力。我们的初步研究使用同源模建来预测E.大肠杆菌表达的UL98，以确认活性位点残基对DNA酶活性的重要性。构建了一种UL98无效病毒，发现其具有显著的生长减毒作用，表明UL98对CMV复制至关重要，并表明UL98的小分子抑制剂可能具有有效的抗病毒活性。为了支持后者，Atanyl蓝PRL，一种UL98核酸酶活性的抑制剂，已显示抑制CMV复制。本申请具有一个目的：确定野生型UL98和两种催化缺陷型UL98突变体以及与DNA和与抑制剂阿塔尼蓝PRL复合的UL98的晶体结构。UL98结构与γ疱疹病毒AN的现有结构的比较将允许鉴定对这些蛋白质中的每一个都是独特的结构特征。UL98的结构也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在复制中的生化活性和功能作用。重要的是，与抑制剂阿塔尼蓝PRL复合的UL98的结构将提供有价值的机制和结构见解，并将为其他UL98抑制剂提供信息和实现基于结构的鉴定。这种抑制剂将作为药理学探针，以补充和扩展UL98功能的遗传和生化研究，并可能为抗病毒药物的开发提供重要的先导结构。这些进展将扩大UL98作为抗病毒靶点的研究，并可能最终导致治疗CMV感染的新型抗病毒药。