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（由适用提供）：人类巨细胞病毒（CMV）是β疱疹病毒亚科的疱疹病毒和原型。 CMV导致移植和AIDS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。所有疱疹病毒均编码酒精核酸酶（AN）和ANS是最保守的疱疹病毒蛋白之一。但是，ANS在病毒复制中扮演的角色在亚家族之间尚未充分理解和不同。提出了来自Alpha疱疹病毒亚家族的ANS的DNase活性，以通过去除新复制的病毒DNA的分支或异常结构来促进重组或促进DNA包装。相比之下，来自伽马疱疹病毒亚家族的ANS具有RNase活性和功能，可以通过降解mRNA来关闭宿主蛋白的翻译。尚不清楚β疱疹病毒ANS ANS是否发挥相似的作用，还是具有β疱疹病毒特有的功能。当前的应用程序是一项良好的多入侵者协作，旨在定义CMV AN，UL98的结构和生化活动，了解其在CMV复制中的机械作用，并确定UL98的小分子抑制剂，以探索其潜在的潜在抗病毒目标。我们的初步研究使用同源性建模来预测UL98活性位点和大肠杆菌表达的UL98的诱变，以确认活性位点保留在DNase活性中的重要性。构建了UL98-NULL病毒并发现其深刻的生长，表明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潜在的抗病毒活性。为了支持后者的亚烷基蓝PRL，这是一种UL98核酸酶活性的抑制剂，已被证明可抑制CMV复制。当前的应用有一个目的：确定野生型UL98和两个催化性UL98突变体的晶体结构，以及与DNA和抑制剂视ananyl蓝色PRL复合的UL98。将UL98结构与伽马疱疹病毒ANS的现有结构进行比较将允许识别这些蛋白质中独有的结构特征。 UL98结构还可能提出新的功能领域，这些域将有助于指导突变的设计，以剖析UL98的生化活性和复制中的功能作用。重要的是，与抑制剂亚烷基蓝色PRL复合的UL98的结构将提供有价值的机械和结构见解，并将为基于结构的额外UL98抑制剂提供信息并实现结构的识别。这种抑制剂将作为药理问题，以补充和扩展UL98功能的遗传和生化研究，并可能为抗病毒发育提供重要的铅结构。这些进步将使对UL98作为抗病毒药靶的扩大追求能够扩大，并最终可能导致用于治疗CMV感染的新型抗病毒药。