Human Cytomegalovirus Nuclear Egress: Molecular Mechanisms and Drug Targeting

人类巨细胞病毒核出口：分子机制和药物靶向

• 批准号: 8150329
• 负责人: DONALD M COEN
• 金额: $ 62.05万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8150329
• 关键词: Antiviral Agents; Applications Grants; Biochemical; Biochemistry; Biological; Biological Assay; Biological Factors; Cell Nucleus; Cell physiology; Cells; Complex; Conserved Sequence; Cyclin-Dependent Kinases; Cytomegalovirus; Cytoplasm; Defect; Disease; Dominant-Negative Mutation; Drug Delivery Systems; Drug effect disorder; Drug resistance; Electron Microscopy; Enzymes; Exhibits; Ganciclovir; Goals; Grant; Health; Herpesviridae; Human; Human Papillomavirus; Immunity; Immunocompetent; In Vitro; Infection; Interphase Cell; Lamin Type A; Lead; Location; Maps; Membrane; Membrane Proteins; Molecular; Molecular Genetics; Murid herpesvirus 1; Nuclear; Nuclear Inner Membrane; Nuclear Lamina; Nuclear Protein; Nuclear Structure; Nucleocapsid; Pharmaceutical Preparations; Phosphorylation; Population; Process; Production; Protein Kinase; Proteins; RNA Interference; Reagent; Recombinants; Recruitment Activity; Research; Resistance; Resolution; Retinoblastoma; Retinoblastoma Protein; Role; Simplexvirus DNA polymerase; Specificity; Structure; Techniques; Testing; Ursidae Family; Viral; Virus; Virus Replication; Work; X-Ray Crystallography; base; combat; cytotoxicity; design; drug discovery; drug mechanism; fascinate; high throughput screening; improved; inhibitor/antagonist; insight; interest; maribavir; mutant; pathogen; public health relevance; small molecule; small molecule libraries; tumor

DESCRIPTION (provided by applicant): This application's long-term objectives are to understand the central steps by which human cytomegalovirus (HCMV) nucleocapsids transit from the nucleus to the cytoplasm (nuclear egress), and to exploit that understanding for drug discovery. This project is especially health-related, as new drugs are needed for treatment of HCMV. In this application, HCMV proteins that are involved in nuclear egress are investigated. One of these proteins, the UL97 protein kinase, is already an established drug target. Two other proteins, UL50 and UL53, interact to form a nuclear egress complex (NEC) that can serve as a new drug target. Specific aim 1 is to investigate the roles of UL97 that are important for production of infectious virus in both non-dividing and dividing cells; in particular, whether the only role of UL97 in nuclear egress is phosphorylation of lamin A/C to disrupt the nuclear lamina. A principal approach will be to analyze a recombinant HCMV expressing a dominant negative mutant of lamin A/C in place of UL97 to determine if that virus can replicate and disrupt nuclear lamina as well as wild type virus in dividing cells or as well as a virus expressing human papillomavirus E7 in non-dividing cells. Specific aim 2 is to investigate the function(s) of the NEC. HCMV mutants that fail to express UL50 or UL53 or that have more specific alterations such as defects in UL50-UL53 interactions will be constructed and their block(s) in nuclear egress will be determined with the aid of techniques including electron microscopy. Why the HCMV NEC is not sufficient to disrupt nuclear lamina in the absence of UL97 will be investigated. Candidate proteins that interact with the NEC in HCMV-infected cells will be investigated to explore the hypothesis that such proteins are recruited to effect budding of nucleocapsids through the inner nuclear membrane. These proteins will be tested for co-localization with the NEC in cells, whether they interact directly with the NEC, and, if so, to map determinants of the interaction. The importance of these proteins for HCMV replication will be investigated using techniques including RNA interference. Should these studies fail to uncover a role for interacting proteins, in vitro studies to test whether the NEC can promote membrane curvature and vesiculation will be undertaken. Specific aim 3 is to determine the structure of the NEC. The structures of truncated versions of NEC subunits that retain sequences that are conserved among herpesviruses are being determined by NMR, as are the locations of the subunit interfaces, which can lead to an NMR structure of the complex. Efforts to improve crystals of the complex will continue, with the goal of obtaining a high resolution crystal structure. Specific aim 4 is to establish a high throughput assay for small molecules that inhibit subunit interactions of the NEC using an amplified luminescent proximity homogeneous assay or other assay. This assay will be used to screen libraries of small molecules and natural products. "Hits" will be then assayed for biochemical activity and specificity, and for anti-HCMV activity and cytotoxicity, with the long range goal of developing them into antiviral drugs. PUBLIC HEALTH RELEVANCE: HCMV causes severe disease in people with impaired immunity, and is associated with a number of diseases in the immunocompetent population. There is considerable need for new drugs to combat HCMV, as current drugs have major limitations. The research proposed should not only provide information that could aid in understanding drug targets and mechanisms, but aims directly to discover new anti-HCMV drugs.

描述（由申请人提供）：本申请的长期目标是了解人巨细胞病毒（HCMV）核衣壳从细胞核转运至细胞质（核出口）的中心步骤，并利用该理解进行药物发现。该项目尤其与健康相关，因为治疗HCMV需要新药。在本申请中，研究了参与核出口的HCMV蛋白。其中一种蛋白质，UL 97蛋白激酶，已经是一个既定的药物靶点。另外两种蛋白质UL 50和UL 53相互作用形成核出口复合物（NEC），可以作为新的药物靶点。具体目标1是研究UL 97在非分裂和分裂细胞中对感染性病毒产生的重要作用;特别是，UL 97在核出口中的唯一作用是否是核纤层蛋白A/C的磷酸化以破坏核纤层。主要方法是分析表达核纤层蛋白A/C的显性负突变体代替UL 97的重组HCMV，以确定该病毒是否可以在分裂细胞中复制和破坏核纤层以及野生型病毒，或者在非分裂细胞中复制和破坏表达人乳头瘤病毒E7的病毒。具体目标2是调查NEC的功能。将构建不能表达UL 50或UL 53或具有更特异性改变（例如UL 50-UL 53相互作用中的缺陷）的HCMV突变体，并将借助于包括电子显微镜在内的技术来确定它们在核出口中的阻断。将研究为什么HCMV NEC不足以在不存在UL 97的情况下破坏核纤层。将研究与HCMV感染的细胞中的NEC相互作用的候选蛋白质，以探索这样的蛋白质被募集以影响核衣壳通过内核膜出芽的假设。将测试这些蛋白质与NEC在细胞中的共定位，它们是否与NEC直接相互作用，如果是，则绘制相互作用的决定因素。这些蛋白质对HCMV复制的重要性将使用包括RNA干扰在内的技术进行研究。如果这些研究未能揭示相互作用蛋白质的作用，将进行体外研究以测试NEC是否可以促进膜弯曲和囊泡形成。具体目标3是确定国家选举委员会的结构。保留疱疹病毒中保守序列的NEC亚基的截短形式的结构正在通过NMR确定，亚基界面的位置也是如此，这可以导致复合物的NMR结构。将继续努力改进复合物的晶体，目标是获得高分辨率的晶体结构。具体目标4是使用扩增发光邻近均相测定或其他测定建立抑制NEC亚基相互作用的小分子的高通量测定。该测定将用于筛选小分子和天然产物的文库。然后将对“点击”进行生化活性和特异性以及抗HCMV活性和细胞毒性分析，长期目标是将其开发成抗病毒药物。 公共卫生关系：HCMV在免疫力受损的人群中引起严重疾病，并且与免疫活性人群中的许多疾病相关。由于目前的药物有很大的局限性，因此相当需要新的药物来对抗HCMV。这项研究不仅应该提供有助于理解药物靶点和机制的信息，而且应该直接旨在发现新的抗HCMV药物。