Maturation functions of the HSV-1 tegument

HSV-1 外皮的成熟功能

• 批准号: 7846535
• 负责人: PRASHANT J DESAI
• 金额: $ 8.36万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846535
• 关键词: Affinity; Antiviral Agents; Axon; Axonal Transport; Binding; Biochemical; Biogenesis; Biological Assay; Biology; Capsid; Cell Line; Cell Nucleus; Cell physiology; Cell surface; Cells; Cellular biology; Clinical; Complex; Cytoplasm; Cytoskeleton; Data; Defect; Development; Dimensions; Electron Microscopy; Epitopes; General Population; Genes; Genetic; Goals; Golgi Apparatus; Growth; Herpesviridae; Herpesvirus 1; Human; Image; Immunoelectron Microscopy; In Vitro; Infection; Intervention; Lead; Life; Location; Maps; Mediating; Methods; Microtubules; Morphogenesis; Mutation; Neurons; Nuclear; Outcome; Pathway interactions; Principal Investigator; Process; Protein Footprinting; Protein Region; Proteins; Recruitment Activity; Role; Signal Transduction; Simplexvirus; Site; Site-Directed Mutagenesis; Specific qualifier value; Structure; Tertiary Protein Structure; Testing; Time; Translating; Vertebral column; Viral; Viral Genome; Virion; Virus; Virus Replication; base; electron tomography; imaging modality; light microscopy; mutant; neuronal cell body; novel; particle; pathogen; polypeptide; protein function; protein protein interaction; trafficking; yeast genetics; yeast two hybrid system

DESCRIPTION (provided by applicant): Herpesviruses are important human pathogens that result in life-long persistent infections with numerous clinical manifestations. Herpes simplex virus (HSV) is among the most frequently encountered pathogen by the general population. Thus understanding the biology of this virus is important in the development of efficacious treatments of these infections. Our goal is to understand the mechanism by which the virus acquires its infectious coat and the role of the virus encoded functions in this pathway, primarily by analyzing the functions of two tegument proteins, the UL36 and UL37 gene products. Our hypothesis is that the UL36 and UL37 gene products specify essential and unique functions for the maturation of the assembled particle into an infectious virion. The goals of this proposal are to understand how these two proteins function in the morphogenesis of the infectious particle, identification of the functional domains required for these activities and the interactions that occur during this process. This could potentially lead to the discovery of novel pathways that can be targeted by antiviral intervention. The specific aims to achieve these goals are: Specific Aim 1: Investigating the role of UL36 and UL37 in the maturation of the virus particle using mutant viruses. UL36 may act to translocate capsids to the cytoplasmic site for final envelopment, whereas, UL37 is required for Golgi stabilization prior to the arrival of capsids at this site. Light microscopy, electron microscopy and immuno-EM assays will be used to determine the association of UL36 mutant virus particles with the cellular cytoskeleton and analyze axonal transport in primary neurons. The Golgi structure will be analyzed in the presence and absence of UL37 and the contribution of UL36, capsid assembly and cis-acting signals for facilitating Golgi stabilization will be determined. Specific Aim 2: Protein-protein interactions of UL36 and UL37 with virus and cellular proteins. Protein-protein interactions are critical for a variety of viral and cellular processes. The UL36 and UL37 encoded polypeptides will be used as probes to identify protein-protein interactions using a combination of cell biology (cellular localization), genetic (yeast two-hybrid), biochemical (protein pull-down) assays. Specific Aim 3: Structural studies of UL36 and UL37 in capsids and the mature virion. A capsid binding assay and cryo-electron tomography imaging of virions will illuminate the association of these proteins with a multi-protein assembly (capsid/virion). Structural data is important for understanding how these proteins function in virus egress. Specific Aim 4: Identification of functional domains of UL36 and UL37. Transposon and site-directed mutagenesis will be used to identify functional domains of the UL36 and UL37 genes. Genetic complementation assays will be used to screen the mutants for functional activity. Mutations that specify lethal defects in function will be introduced into the virus using a marker-rescue/marker-transfer method. The outcome will be a functional map of UL36 and UL37 that identifies domains important for capsid translocation, Golgi stabilization, Golgi trafficking, virion incorporation and protein-protein interactions This analysis will identify domains of the proteins that have the potential to be used as targets for antiviral intervention

描述（由申请人提供）：疱疹病毒是重要的人类病原体，可导致终身持续感染，并具有多种临床表现。单纯疱疹病毒（HSV）是普通人群最常遇到的病原体之一。因此，了解该病毒的生物学对于这些感染有效治疗的发展至关重要。我们的目标是了解病毒获得其感染性外套的机制以及该病毒在该途径中的作用的作用，主要是通过分析两个Tegument蛋白的功能，即UL36和UL37基因产物。我们的假设是，UL36和UL37基因产物为组装粒子成熟到传染性病毒体中指定了必不可少的独特功能。该提案的目标是了解这两种蛋白质如何在传染性粒子的形态发生，识别这些活动所需的功能域以及在此过程中发生的相互作用。这可能会导致发现可以通过抗病毒干预靶向的新型途径。实现这些目标的具体目的是：特定目标1：使用突变病毒研究UL36和UL37在病毒颗粒成熟中的作用。 UL36可能作用于capsids转移到细胞质部位以进行最终包膜，而在capsids到达该部位之前，Golgi稳定需要UL37。光显微镜，电子显微镜和免疫EM分析将用于确定UL36突变病毒颗粒与细胞骨骼的关联，并分析原代神经元中的轴突转运。将在存在和不存在UL37的情况下分析高尔基体结构，并确定UL36，Capsid组装和顺式作用信号的贡献，以促进高尔基体稳定。特定目标2：UL36和UL37与病毒和细胞蛋白的蛋白质 - 蛋白质相互作用。蛋白质 - 蛋白质相互作用对于多种病毒和细胞过程至关重要。 UL36和UL37编码的多肽将用作探针，以结合细胞生物学（细胞定位），遗传学（酵母两杂化），生化（蛋白质下拉）测定法，以鉴定蛋白质蛋白质相互作用。特定目的3：在衣壳和成熟的病毒体中UL36和UL37的结构研究。 CAPSID结合测定和病毒体的冷冻电子层析成像将阐明这些蛋白质与多蛋白质组装（CAPSID/VIRION）的关联。结构数据对于理解这些蛋白质在病毒出口中的功能很重要。特定目标4：识别UL36和UL37的功能域。转座子和定点诱变将用于鉴定UL36和UL37基因的功能结构域。遗传互补测定将用于筛选突变体以进行功能活性。指定功能中指定致命缺陷的突变将使用标记词/标记转移方法引入病毒。结果将是UL36和UL37的功能图，它可以确定对衣壳易位，高尔基体稳定，高尔基贩运，GOLGI贩运，Virion掺入和蛋白质 - 蛋白质相互作用重要的结构域。该分析将确定蛋白质的结构域，这些蛋白质具有可作为抗病毒干预措施靶标的靶标