HSV-Receptor Interaction in Entry and Pathogenesis

描述(申请人提供)：单纯疱疹病毒(HSV)可引起唇疱疹、眼部和生殖器感染、新生儿感染和脑炎。该病毒在感觉神经节建立了终生潜伏感染。包膜含有11种病毒编码的糖蛋白，其中4种是病毒进入所必需的。它们是受体结合蛋白Gd、Gb和Gh和Gl的复合体。有几个细胞分子充当进入受体，在每种情况下，受体都与GD结合。大多数菌株可以使用其中的两种，HveA(Hvem)和Nectin-1(HveC)。HveA(一种肿瘤坏死因子受体)在T淋巴细胞中含量最高，而Nectin-1(一种细胞黏附分子)在上皮细胞和神经细胞中含量最高。这项资助的具体目的是：(1)表征GD和Nectin-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；以及(3)开展HSV GB的结构和功能研究。在目标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。根据结晶学数据，当Gd与HveA结合时，它经历了两次构象变化。我们假设，当GD与Nectin-1结合时，至少其中之一也会发生，这种变化可能在以后的进入步骤中发挥作用。解决GD/Nectin-1复合体的结构将检验这一假设，这也是该项目的主要目标。这些研究结合定向突变将增强我们对这种蛋白质-蛋白质相互作用的理解。Nectin-1可以与自身以及在细胞贴壁连接处与其他Nextin相互作用。我们推测，在这些连接处，GD作为Nectin-1的配基，从而使受体可用于病毒传播到下一个细胞。虽然Nectin-1在上皮细胞和神经细胞上比HveA更丰富，但这两种受体在HSV发病中的作用尚不清楚。我们开发了一组GD突变体，它们使用一个或另一个受体的能力发生了变化。在目标2中，我们将测试携带这些突变的病毒感染小鼠并导致带状疱疹病毒病的能力。虽然gB、Gh和gl是病毒被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。在目标3中，我们将对gB的结构功能进行研究，以阐明其在病毒入侵中的作用。我们将跟踪观察到，在进入时，GB在细胞上分裂成脂筏。最终，我们希望定义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胞外结构域。这种蛋白质的晶体会发生衍射，因此我们建议用X射线结晶学来解决GB的结构问题。这项赠款中的研究可能导致基于HSV进入过程的靶点开发新的抗病毒治疗方法。

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.