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

描述(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，它先感染上皮和皮肤，然后传播到感觉神经元，在那里它会终身感染。单纯疱疹病毒的进入是一个复杂的分子过程。一个基本的步骤是单纯疱疹病毒糖蛋白D(GD)与细胞受体的相互作用，以诱导病毒被膜与质膜或内膜融合。神经元和上皮细胞上的主要GD受体之一是细胞黏附分子Nectin-1。这项研究的长期目标是解释病毒如何利用细胞途径在细胞连接处到达Nectin-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。在目标1中，将定义当gD在HSV进入过程中与Nectin-1结合时被激活的细胞通路。表达Nectin-1的细胞将暴露于膜结合或可溶性的GD，以确定参与受体内化的细胞反应。Gd与Nectin-1结合所引发的信号将与天然配体Nectin-3的信号进行比较，Nectin-3在细胞连接和突触与Nectin-1相互作用。最后，参与受体内吞作用的通路将与HSV进入时激活的通路相关联。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Nectin-1介导的细胞黏附，因此将研究Gd对细胞连接结构的影响。病毒和细胞对单纯疱疹病毒颗粒在质膜上冲浪并在细胞连接处接触Nectin-1的需求将被定义。这一运动对于进入极化上皮细胞的重要性将被分析。该项目将把单纯疱疹病毒与受体的附着和结合的研究扩展到病毒诱导的即时细胞反应的新方面。最先进的技术和新的试剂将被用来探索HSV结合到细胞表面引发的细胞机制。该项目开发的创新方法将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感染早期步骤的了解，并为抗病毒治疗寻找新的靶点。单纯疱疹病毒1型和2型(HSV-1和HSV-2)分别感染80%和20%的美国成年人，目前的治疗方法并不能减少HSV重新激活和传播的发生，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，这项研究将为干扰这一过程的新的治疗方法提供靶点。

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.