HSV-Receptor Interaction in Entry and Pathogenesis

描述（由申请人提供）：单纯疱疹病毒（hsv）引起唇疱疹，眼睛和生殖器感染，新生儿感染和脑炎。这种病毒在感觉神经节中形成终身潜伏感染。包膜包含11种病毒编码的糖蛋白，其中4种是病毒进入所必需的。它们是gD，受体结合蛋白，gB和gH和gL的复合物。几个细胞分子充当进入受体，每种情况下受体都与gD结合。大多数菌株可以使用其中的两种，HveA （HVEM）和连接素- 1 （HveC）。HveA（一种肿瘤坏死因子受体）在T淋巴细胞上最丰富，而连接素- 1（一种细胞粘附分子）在上皮细胞和神经元细胞上丰富。该资助的具体目的是：(1)表征gD和连接蛋白- 1之间的相互作用；(2)确定HSV进入受体在病毒感染小鼠模型中的作用；(3)开展HSV gB的结构-功能研究。在Aim 1中，我们的目标是增加我们对gD和nectin-1在体外和细胞上相互作用的理解。晶体学数据显示，gD与HveA结合时，会发生两种构象变化。我们假设，当gD结合连接蛋白- 1时，这些变化中至少有一个也会发生，这种变化可能在随后的进入步骤中发挥作用。gD/nectin-1复合物结构的解决方案将检验这一假设，是本项目的主要目标。这些研究结合靶向诱变将增强我们对这种蛋白质-蛋白质相互作用的理解。Nectin-1可以反式与自身以及细胞粘附连接处的其他连接蛋白相互作用。我们推测gD在这些连接处作为连接素-1的配体，从而使受体可用于病毒传播到下一个细胞。虽然上皮细胞和神经元细胞上的连接蛋白-1似乎比HveA更丰富，但这两种受体在HSV发病机制中的作用尚不清楚。我们开发了一组gD突变体它们使用一种或另一种受体的能力发生了改变。在目标2中，我们将测试携带这些突变的病毒感染小鼠并引起带状虫状疾病的能力。虽然gB、gH和gL是病毒包膜与宿主细胞质膜融合所必需的，但它们在这一过程中的作用尚不清楚。在Aim 3中，我们将开展gB的结构功能研究，以阐明其在病毒侵入中的作用。我们将继续观察在进入时gB在细胞上分裂成脂筏的情况。最终，我们想要确定这种相互作用的细胞目标。最近，我们克隆、表达和纯化了大量的gB外结构域。这种蛋白质的晶体可以衍射，因此我们建议用x射线晶体学来解决gB的结构。这项资助的研究可能会导致基于HSV进入过程靶点的抗病毒治疗新方法的发展。

DESCRIPTION (provided by applicant): Herpes simplex viruses (HSVs) cause cold sores, eye and genital infections, neonatal infections and encephalitis. The virus establishes lifelong latent infections in sensory ganglia. The envelope contains eleven virus-encoded glycoproteins of which four are essential for virus entry. These are gD, the receptor binding protein, gB and a complex of gH and gL. Several cell molecules serve as entry receptors and in each case the receptor binds gD. Most strains can use two of these, HveA (HVEM) and nectin- 1 (HveC). Whereas HveA (a TNF receptor) is found most abundantly on T lymphocytes, nectin- 1 (a cell adhesion molecule) is abundant on epithelial and neuronal cells. The specific aims of this grant are: (1) to characterize the interaction between gD and nectin- 1; (2) to determine the role HSV entry receptors in a mouse model of virus infection; and (3) to carry out structure-function studies of HSV gB. In Aim 1, our goal is to increase our understanding of the interaction between gD and nectin-1 in vitro, and on cells. When gD binds HveA, it undergoes two conformational changes as revealed by crystallographic data. We hypothesize that at least one of these also occurs when gD binds nectin- 1 and this change may play a role in later steps of entry. Solution of the structure of the gD/nectin-1 complex will test this hypothesis and is a major goal of this project. These studies combined with targeted mutagenesis will enhance our understanding of this protein-protein interaction. Nectin-1 can interact in trans with itself as well as with other nectins at cellular adherens junctions. We speculate that gD acts as a ligand for nectin-1 at these junctions so that receptor is available for virus spread to the next cell. Although nectin-1 appears to be more abundant than HveA on epithelial and neuronal cells, the role of these two receptors in HSV pathogenesis is not known. We developed a panel of gD mutants with altered ability to use one or the other receptor. In Aim 2, we will test the ability of viruses carrying these mutations to infect and cause zosteriform disease in mice. Although gB, gH and gL are required for fusion of the viral envelope with the plasma membrane of the host cell, their role in this process is not understood. In Aim 3, we will carry out structure function studies of gB in order to clarify its role in virus entry. We will follow up on the observation that gB partitions into lipid rafts on cells at the time of entry. Ultimately, we want to define the cellular targets of this interaction. Recently, we cloned, expressed and purified large quantities of the gB ectodomain. Crystals of this protein diffract and we therefore propose to solve the structure of gB by X-ray crystallography. The studies in this grant may lead to development of new approaches for antiviral therapeutics based on targets of the HSV entry process.