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: 8516432
• 负责人: DONALD M COEN
• 金额: $ 59.68万
• 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/8516432
• 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)：这项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)的中心步骤，并利用这一理解进行药物发现。该项目特别与健康有关，因为治疗巨细胞病毒需要新药。在这个应用中，我们研究了与核出口有关的巨细胞病毒蛋白。其中一种蛋白质，UL97蛋白激酶，已经是一个既定的药物靶点。另外两种蛋白质UL50和UL53相互作用形成核出口复合体(NEC)，可以作为新的药物靶点。具体目的1是研究UL97在未分裂细胞和分裂细胞中对产生传染性病毒的重要作用；特别是UL97在核出口中的唯一作用是否是层蛋白A/C的磷酸化以破坏核层。一种主要的方法是分析表达Lamin A/C显性负性突变体的重组HCMV，以确定该病毒是否可以在分裂细胞中复制和破坏核膜以及野生型病毒，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是研究NEC的功能(S)。将构建不表达UL50或UL53或具有UL50-UL53相互作用缺陷等更特异变化的巨细胞病毒突变体，并借助电子显微镜等技术确定其在核出口的阻断(S)。为什么在没有UL97的情况下，HCMV NEC不足以破坏核层，将进行调查。在HCMV感染细胞中与NEC相互作用的候选蛋白将被研究，以探索这样的蛋白被招募来通过内核膜影响核衣壳萌发的假设。这些蛋白质将被测试与NEC在细胞中的共同定位，它们是否直接与NEC相互作用，如果是，则绘制相互作用的决定因素图。将使用包括RNA干扰在内的技术来研究这些蛋白质对巨细胞病毒复制的重要性。如果这些研究未能发现相互作用的蛋白质的作用，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NEC的结构。保留疱疹病毒间保守序列的NEC亚基的截短版本的结构正在由核磁共振确定，亚基界面的位置也是如此，这可能导致复合体的核磁共振结构。改进该复合体晶体的努力将继续下去，目标是获得高分辨率的晶体结构。具体目标4是使用放大的发光均相分析或其他分析建立抑制NEC亚单位相互作用的小分子的高通量分析。这项检测将用于筛选小分子和天然产物的文库。然后将检测“HITS”的生化活性和特异性，以及抗HCMV活性和细胞毒性，长期目标是将它们开发成抗病毒药物。 公共卫生相关性：巨细胞病毒会导致免疫受损人群的严重疾病，并与免疫能力人群中的许多疾病有关。由于目前的药物有很大的局限性，因此需要相当多的新药来对抗巨细胞病毒。建议的研究不仅应该提供有助于了解药物靶点和机制的信息，而且还应该直接旨在发现新的抗HCMV药物。