Structure of cytomegalovirus nuclease, UL98

巨细胞病毒核酸酶 UL98 的结构

• 批准号: 8969474
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8969474
• 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; 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; public health relevance; repaired; small molecule; 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（由申请人提供）：人类巨细胞病毒（CMV）是一种疱疹病毒，是β疱疹病毒亚科的原型。 CMV 会导致移植患者和艾滋病患者出现肺炎、失明和死亡，以及新生儿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巨细胞病毒感染。所有疱疹病毒都编码碱性核酸酶 (AN)，AN 是最高度保守的疱疹病毒蛋白之一。然而，AN 在病毒复制中发挥的作用尚不清楚，并且不同亚科之间的作用也不同。来自α疱疹病毒亚科的AN的DNA酶活性被认为可以通过从新复制的病毒DNA中去除分支或异常结构来促进重组或促进DNA包装。相比之下，来自 γ 疱疹病毒亚科的 AN 具有 RNase 活性，并具有通过降解 mRNA 来关闭宿主蛋白翻译的功能。 β 疱疹病毒 AN 是否具有相似的作用或具有 β 疱疹病毒亚科特有的功能尚不清楚。目前的申请是一项完善的多研究者合作，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，并鉴定 UL98 的小分子抑制剂以探索其作为抗病毒靶点的潜力。我们最初的研究使用同源模型来预测 UL98 活性位点和大肠杆菌表达的 UL98 的诱变，以确认活性位点残基对 DNase 活性的重要性。构建了 UL98 无效病毒，发现其生长明显减弱，这证明 UL98 对于 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 blue PRL（一种 UL98 核酸酶活性抑制剂）已被证明可以抑制 CMV 复制。目前的应用有一个目标：确定野生型 UL98 和两种催化缺陷型 UL98 突变体，以及与 DNA 和抑制剂 atanyl blue PRL 复合的 UL98 的晶体结构。将 UL98 结构与伽玛疱疹病毒 AN 的现有结构进行比较，将能够识别这些蛋白质中每种蛋白质独特的结构特征。 UL98 结构还可能表明新的功能域，这将有助于指导突变设计，以剖析 UL98 的生化活性和复制中的功能作用。重要的是，UL98 与抑制剂 atanyl blue PRL 复合的结构将提供有价值的机制和结构见解，并将为其他 UL98 抑制剂的基于结构的鉴定提供信息和实现。此类抑制剂将作为药理学探针来补充和扩展 UL98 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。这些进展将有助于扩大 UL98 作为抗病毒靶点的研究范围，并可能最终开发出用于治疗 CMV 感染的新型抗病毒药物。