Interactions of herpes simplex virus with nectin-1 at cell junctions

描述（由申请人提供）：单纯疱疹病毒（HSV）是一种非常常见的人类病原体，在传播到感觉神经元之前感染上皮和皮肤，并在那里建立终身感染。单纯疱疹病毒的进入是一个复杂的分子过程。一个重要的步骤是HSV糖蛋白D （gD）与细胞受体相互作用，诱导病毒包膜与质膜或内体膜融合。神经元和上皮细胞上的主要gd受体之一是细胞粘附分子nectin-1。本研究的长期目标是解释病毒如何利用细胞途径到达细胞连接处的连接蛋白-1，刺激内吞作用并与宿主膜融合。为实现这些目标，提出了两个具体目标。在目的1中，将定义在HSV进入时gD与连接蛋白-1结合时激活的细胞途径。表达Nectin-1的细胞将暴露于膜结合或可溶性形式的gD中，以确定参与受体内化的细胞反应。gD与nectin-1结合引发的信号将与自然配体nectin-3的信号进行比较，后者在细胞连接处和突触处与nectin-1相互作用。最后，参与受体内吞作用的途径将与HSV进入时激活的途径相关。在目标2中，将定义HSV进入对细胞连接完整性的影响。由于gD结合影响连接素-1介导的细胞粘附，gD对细胞连接结构的影响将被研究。病毒和细胞需要HSV颗粒在质膜上冲浪，并在细胞连接处接触连接蛋白-1。这一运动对于进入极化上皮细胞的重要性将被分析。该项目将扩展HSV附着和结合受体的研究，以探索由病毒诱导的直接细胞反应的新方面。最先进的技术和新试剂将用于探索由HSV结合到细胞表面引发的细胞机制。在这个项目中开发的创新方法将允许通过使用活细胞共聚焦显微镜实时直接观察病毒和细胞成分来研究HSV在细胞连接处的进入。这项研究将增加我们对HSV感染早期步骤的理解，并确定抗病毒治疗行动的新靶点。单纯疱疹病毒1型和2型（HSV-1和HSV-2）分别感染了80%和20%的美国成年人，目前的治疗方法并没有减少HSV再激活和传播的发生，因此需要开发新的治疗策略。这项研究将确定病毒与细胞结合的直接后果，并确定病毒进入的细胞机制。通过确定病毒进入宿主的新要求，本研究将为干扰这一过程的新治疗方法提供靶点。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a very common human pathogen that infects epithelia and skin before spreading to sensory neurons where it establishes a lifelong infection. Entry of HSV is a complex molecular process. One essential step is the interaction of HSV glycoprotein D (gD) with a cellular receptor to induce fusion of the viral envelope with the plasma membrane or an endosomal membrane. One of the main gD-receptor on neurons and epithelial cells is the cell adhesion molecule nectin-1. The long-term objectives of this study are to explain how the virus exploits cellular pathways to reach nectin-1 at cell junctions, stimulate endocytosis and fuse with the host membrane. Two specific aims are proposed to achieve these objectives. In aim 1 the cellular pathways that are activated when gD binds to nectin-1 during HSV entry will be defined. Nectin-1 expressing cells will be exposed to either membrane bound or soluble forms of gD to identify cellular responses involved in receptor internalization. The signals elicited by gD binding to nectin-1 will be compared to those of a natural ligand, nectin-3, which interacts with nectin-1 at cell junctions and at synapses. Finally, pathways involved in receptor endocytosis will be correlated with pathways activated during HSV entry. In aim 2 the effect of HSV entry on the integrity of cellular junctions will be defined. Since gD binding affects nectin-1-mediated cell adhesion, the effects of gD on the architecture of cell junctions will be studied. The viral and cellular needs for HSV particles to surf on the plasma membrane and access nectin-1 at cellular junctions will be defined. The importance of this movement for entry into polarized epithelial cells will be analyzed. This project will extend studies of HSV attachment and binding to receptors toward novel aspects of the immediate cellular responses induced by the virus. State of the art technology and new sets of reagents will be used to explore the cellular mechanisms triggered by HSV binding to the cell surface. Innovative approaches developed in this project will allow studies of HSV entry at cellular junctions by direct observation of viruses and cell components in real time using live cell confocal microscopy. This study will increase our understanding of the early steps of HSV infection and identify new targets for antiviral therapeutic actions. Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect 80% and 20% of all American adults respectively and current treatments do not reduce the occurrence of HSV reactivation and transmission, thus new therapeutic strategies need to be developed. This study will determine the immediate consequences of the virus binding to the cell and identify cellular mechanisms involved in virus entry. By identifying new requirements for virus entry in the host, this study will provide targets for novel therapeutic approaches to interfere with this process.