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，可导致终生持续感染并具有多种临床表现。单纯疱疹病毒（HSV）是普通人群最常见的病原体之一。因此，了解这种病毒的生物学对于开发这些感染的有效治疗方法非常重要。我们的目标是了解病毒获得感染外壳的机制以及病毒编码功能在此途径中的作用，主要通过分析两种外皮蛋白（UL36 和 UL37 基因产物）的功能。我们的假设是，UL36 和 UL37 基因产物指定了组装颗粒成熟为感染性病毒粒子的基本且独特的功能。该提案的目标是了解这两种蛋白质如何在感染性颗粒的形态发生中发挥作用，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。这可能会导致发现抗病毒干预的新途径。实现这些目标的具体目标是： 具体目标1：使用突变病毒研究UL36和UL37在病毒颗粒成熟中的作用。 UL36 可能会将衣壳转移到细胞质位点以进行最终包封，而 UL37 是衣壳到达该位点之前高尔基体稳定所必需的。光学显微镜、电子显微镜和免疫电镜测定将用于确定 UL36 突变病毒颗粒与细胞骨架的关联，并分析原代神经元中的轴突运输。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、衣壳组装和顺式作用信号对促进高尔基体稳定的贡献。具体目标 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，结合细胞生物学（细胞定位）、遗传（酵母双杂交）、生物化学（蛋白质下拉）测定来鉴定蛋白质-蛋白质相互作用。具体目标 3：衣壳和成熟病毒体中 UL36 和 UL37 的结构研究。病毒粒子的衣壳结合测定和冷冻电子断层扫描成像将阐明这些蛋白质与多蛋白质组装体（衣壳/病毒粒子）的关联。结构数据对于了解这些蛋白质在病毒出口中如何发挥作用非常重要。具体目标 4：鉴定 UL36 和 UL37 的功能域。转座子和定点诱变将用于鉴定 UL36 和 UL37 基因的功能域。遗传互补测定将用于筛选突变体的功能活性。使用标记拯救/标记转移方法将指定致命功能缺陷的突变引入病毒中。结果将是 UL36 和 UL37 的功能图谱，该图谱识别对衣壳易位、高尔基体稳定、高尔基体运输、病毒体掺入和蛋白质-蛋白质相互作用重要的结构域。该分析将识别有可能用作目标的蛋白质结构域。抗病毒干预