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: 8704099
• 负责人: DONALD M COEN
• 金额: $ 64.72万
• 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/8704099
• 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 Targeting; 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类巨细胞病毒（HCMV）核衣壳从细胞核转移到细胞质（核出口）的核心步骤，并利用这一理解进行药物发现。这个项目尤其与健康有关，因为治疗HCMV需要新的药物。在本应用中，研究了参与核输出的HCMV蛋白。其中一种名为UL97蛋白激酶的蛋白质已经成为一种确定的药物靶点。另外两种蛋白UL50和UL53相互作用形成核出口复合物（NEC），可以作为新的药物靶点。具体目的1是研究UL97在非分裂细胞和分裂细胞中对感染性病毒产生的重要作用；特别是UL97在核输出中的唯一作用是否只是磷酸化核纤层蛋白A/C从而破坏核纤层。一种主要的方法是分析一种表达层状蛋白A/C的显性阴性突变体代替UL97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层以及野生型病毒，或者在非分裂细胞中表达人乳头瘤病毒E7的病毒。具体目标2是调查NEC的功能。HCMV突变体不能表达UL50或UL53，或有更具体的改变，如UL50-UL53相互作用的缺陷，将被构建，它们在核出口的阻滞将通过包括电子显微镜在内的技术来确定。为什么在没有UL97的情况下，HCMV NEC不足以破坏核层将被研究。我们将研究在hcmv感染细胞中与NEC相互作用的候选蛋白，以探索这些蛋白通过核膜影响核衣壳出芽的假设。这些蛋白质将在细胞中测试与NEC的共定位，是否与NEC直接相互作用，如果是，绘制相互作用的决定因素。这些蛋白对HCMV复制的重要性将通过包括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的作用，则将进行体外研究，以测试NEC是否可以促进膜曲率和囊泡。具体目标3是确定选举管理委员会的结构。保留疱疹病毒中保守序列的NEC亚基的截断版本的结构正在通过核磁共振确定，亚基界面的位置也可以确定，这可以导致复合物的核磁共振结构。将继续努力改进该复合物的晶体，以获得高分辨率的晶体结构。具体目标4是使用放大发光接近均质分析或其他分析方法，建立抑制NEC亚基相互作用的小分子高通量分析。该试验将用于筛选小分子和天然产物的文库。“命中”将被检测生化活性和特异性，以及抗hcmv活性和细胞毒性，其长期目标是将其开发成抗病毒药物。