Structure of cytomegalovirus nuclease, UL98

巨细胞病毒核酸酶 UL98 的结构

基本信息

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
项目类别：
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

项目摘要

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，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 CMV 感染。所有疱疹病毒都编码碱性核酸酶 (AN)，而 AN 是最高度保守的病毒之一。然而，AN 在病毒复制中发挥的作用尚不清楚，并且来自α疱疹病毒亚科的 AN 的 DNA 酶活性存在差异，建议通过去除新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 γ 疱疹病毒亚科的 AN 具有 RNase 活性，并且具有通过降解 mRNA 来关闭宿主蛋白翻译的功能，无论 β 疱疹病毒 AN 具有相似的作用还是具有与 β 疱疹病毒不同的功能。目前的应用是一个成熟的多研究者合作，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，并识别 UL98 的小分子抑制剂，以抑制疱疹病毒亚科的发生。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型来预测 UL98 活性位点和大肠杆菌表达的 UL98 的突变，以确认活性位点的重要性。构建了 UL98 无效病毒，发现其生长明显减弱，这表明 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有效的抗病毒活性。 blue PRL 是一种 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的：确定野生型 UL98 的晶体结构。和两个催化缺陷的 UL98 突变体，以及与 DNA 和抑制剂 atanyl blue PRL 复合的 UL98 结构与γ疱疹病毒 AN 的现有结构的比较将允许识别这些蛋白质中的每一种的独特结构特征。 UL98 结构还可能表明新的功能域，这将有助于指导突变的设计，以剖析 UL98 的生化活性和复制中的功能作用。 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，此类抑制剂将作为药理学探针，以补充和扩展 UL98 功能和生物化学研究。可能为抗病毒药物的开发提供重要的先导结构。这些进展将使 UL98 作为抗病毒靶标得到广泛应用，并最终可能产生用于治疗 CMV 感染的新型抗病毒药物。