Molecular Mechanism of HSV Entry and Spread

描述（由申请方提供）：本项目的长期目标是确定单纯疱疹病毒（HSV）侵入人体细胞和病毒扩散至神经元组织的分子基础。HSV感染是世界范围内的主要健康问题，感染了世界人口的60-90%，其中许多人一生都受到初始和复发性病变的困扰。除此之外，HSV疾病在新生儿和越来越多的免疫功能低下个体中的发病率和失明风险以及甚至致死性。3-O-硫酸化硫酸乙酰肝素（3-OS HS）通过为包膜糖蛋白D（gD 1）提供结合位点，作为HSV-1型的特异性进入受体。最近的一些数据表明，3-OS HS可以介导所有形式的原发性感染，从HSV-1进入细胞到病毒通过介导细胞融合的细胞间传播。细胞间融合以及病毒包膜与宿主细胞膜的融合似乎需要gD/3-OS HS复合物与三种额外的病毒糖蛋白gB、gH和gL协同作用。目前尚不清楚受体结合如何触发导致融合的事件序列。将进行涉及gD 1（野生型和突变型）、gD 2和3-OS HS变体的广泛研究，以更好地了解gD和3-OS HS之间产生相互作用的结构和生化要求。将使用多种试验，包括3-OS HS与gD的体外和体内结合、细胞融合和病毒进入。此外，为了确定3-OS HS在HSV-1进入中的意义，将在一些天然靶细胞中进行研究以检查3-OS HS在组织嗜性中的表达和潜在贡献。这些研究将通过使用原代细胞培养、蛋白质表达测定、通过噬菌体展示筛选产生的新肽和siRNA（小干扰RNA）技术来辅助。所产生的试剂也可用于评价3-OS HS的其他生物学功能。

DESCRIPTION (provided by the applicant): The long-term goal of this project is to determine the molecular basis of herpes simplex virus (HSV) invasion of human cells and spread of the virus to neuronal tissues. HSV infection is a major health problem worldwide infecting 60-90% of the world's population, many of which are plagued by initial and recurrent lesions for their entire lives. Adding to that is the morbidity and the risk of blindness from ocular lesions and even lethality of HSV disease in neonates and in increasing number of immunocompromised individuals. The 3-O-sulfated heparan sulfate (3-OS HS) serves as a specific entry receptor for HSV type-1 by offering binding sites for envelope glycoprotein D (gD1). Some recent data suggests that 3-OS HS can mediate all forms of primary infection, from HSV-1 entry into cells to cell-to-cell spread of the virus by mediating cell fusion. The cell-to-cell fusion as well as the fusion of the viral envelope with host cell membrane appear to require gD/3-OS HS complex in concert with three additional viral glycoproteins, gB, gH and gL. It is not clear how the receptor binding triggers the sequence of events that result in fusion. Extensive studies involving gD1 (wild-type and mutant forms), gD2, and 3-OS HS variants will be performed to develop a better understanding of the structural and biochemical requirements for a productive interaction between gD and 3-OS HS. Multiple assays that include in vitro and in vivo binding of 3-OS HS with gD, cell fusion, and viral entry will be used. Further, to determine the significance of 3-OS HS in HSV-1 entry, studies will be performed in some natural target cells to examine expression and the potential contribution of 3-OS HS in tissue tropism. These studies will be aided by the use of primary cell cultures, protein expression assays, novel peptides generated by phage display screening, and siRNA (small-interfering RNA) technology. The reagents generated would also be useful for evaluating other biological functions of 3-OS HS as well.