Maturation functions of the HSV-1 tegument

HSV-1 外皮的成熟功能

• 批准号: 7105944
• 负责人: PRASHANT J DESAI
• 金额: $ 34.04万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105944
• 关键词: capsid; mutant; particle; protein protein interaction; proteins; virion

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(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，具有多种临床表现。单纯疱疹病毒(HSV)是普通人群中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很重要。我们的目标是了解病毒获得其感染外壳的机制以及病毒编码的功能在这一途径中的作用，主要是通过分析两种被膜蛋白-UL36和UL37基因产物的功能。我们的假设是，UL36和UL37基因产物为组装的颗粒成熟为具有感染性的病毒粒子指定了必要和独特的功能。这项建议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结构域，以及在这一过程中发生的相互作用。这可能会导致发现可以作为抗病毒干预靶点的新途径。实现这些目标的具体目标是：具体目标1：研究UL36和UL37在使用突变病毒的病毒颗粒成熟过程中的作用。UL36可以将衣壳转移到细胞质部位以获得最终包膜，而UL37在衣壳到达该部位之前是高尔基体稳定所必需的。将使用光学显微镜、电子显微镜和免疫-EM分析来确定UL36突变病毒颗粒与细胞骨架的关联，并分析初级神经元中的轴突运输。在UL37存在和不存在的情况下，将分析高尔基体的结构，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。特定目的2：UL36和UL37与病毒和细胞蛋白的蛋白质相互作用。蛋白质之间的相互作用对各种病毒和细胞过程至关重要。UL36和UL37编码的多肽将被用作探针，使用细胞生物学(细胞定位)、遗传学(酵母双杂交)、生化(蛋白质下拉)分析相结合的方法来确定蛋白质之间的相互作用。具体目标3：衣壳和成熟病毒粒子中UL36和UL37的结构研究。病毒粒子的衣壳结合分析和冷冻电子断层成像将阐明这些蛋白质与多蛋白组装(衣壳/病毒粒子)的联系。结构数据对于了解这些蛋白质如何在病毒出口中发挥作用很重要。具体目标4：确定UL36和UL37的功能结构域。转座子和定点突变将用于鉴定UL36和UL37基因的功能结构域。基因互补分析将被用来筛选功能活性的突变体。指定致命功能缺陷的突变将通过标记抢救/标记转移方法引入病毒。结果将是UL36和UL37的功能图谱，它确定了衣壳易位、高尔基体稳定、高尔基体运输、病毒粒子掺入和蛋白质-蛋白质相互作用的重要结构域。这一分析将确定有可能被用作抗病毒干预目标的蛋白质结构域