CAR Function In Virus Tropism And Cell-Cell Contact

描述（由申请方提供）：科萨基B病毒（CBV）和腺病毒（Ad）是病毒性心肌炎的主要病因，CBV与胰腺炎和糖尿病有关。所有CBV和许多Ad通过附着于柯萨奇病毒和腺病毒受体（CAR）（一种其生理功能尚未确定的细胞表面糖蛋白）来启动感染。该建议涉及CAR在病毒嗜性中的作用及其生理功能。 我们最近发现CAR是上皮细胞紧密连接的功能组分，紧密连接是一种专门的细胞间接触，作为细胞旁溶质运动的屏障。在极化的上皮细胞上，CAR被隔离在紧密连接中;因此，上皮单层抵抗CBV和Ad的感染。一些CBV还附着在第二种受体上，这种分子在感染中的作用仍然知之甚少。初步数据表明，虽然病毒感染可能会改变病毒进入细胞的途径，但它不会引发感染所必需的病毒体构象变化。然而，ESTA是针对极化细胞的顶端表面，和DAF结合CBV的变体有效地感染上皮细胞单层，我们建议与ESTA的相互作用提供了一种机制，在上皮和粘膜表面的病毒感染。在第一系列拟议的实验中，我们将确定CAR和BAF在感染期间的功能，定义CAR结合病毒和DAF结合变体的病毒进入途径，并测试附着到BAF允许CBV穿过上皮表面的假设。 在第二系列实验中，我们将在体内模型中定义CAR在CBV发病机制中的功能。在人类组织中，CAR mRNA在心脏和胰腺中表达最高，这是人类和动物感染模型中的主要CBV靶器官。我们将使用条件性基因靶向策略来确定组织特异性CAR表达是否对CBV感染以及心脏和胰腺中病毒诱导的病理学至关重要。 在最后一组实验中，我们将探索CAR的生理功能。我们已经发现CAR是紧密连接的结构和功能组分，CAR与主要连接蛋白ZO-1相互作用，CAR胞外结构域之间的同型相互作用介导细胞粘附并有助于连接完整性。我们将确定参与CAR介导的同型粘附的结构，并定义CAR与其他对连接形成和功能重要的蛋白质的相互作用。

DESCRIPTION (provided by applicant): Coxsackie B viruses (CBV) and Adenoviruses (Ads) are the major causes of viral myocarditis, and CBV are implicated in the pancreatitis and diabetes. All CBV and many Ads initiate infection by attachment to the coxsackievirus and adenovirus receptor (CAR), a cell surface glycoprotein whose physiologic function has not been defined. This proposal concerns both CAR's role in virus tropism and its physiologic function. We recently found that CAR is a functional component of the epithelial cell tight junction, a specialized intercellular contact that serves as a barrier to the paracellular solute movement. On polarized epithelial cells, CAR is sequestered in tight junctions; consequently, epithelial monolayers resist infection by both CBV and Ads. Some CBV also attach to a second receptor, DAF, a molecule whose role in infection has remained poorly understood. Preliminary data indicate that while DAF may alter the route by which virus enters a cell, it does not trigger conformational changes in the virion essential that are essential for infection. However, DAF is targeted to the apical surface of polarized cells, and a DAF-binding CBV variant efficiently infects epithelial monolayers; we propose that interaction with DAF provides a mechanism for virus infection at epithelial and mucosal surfaces. In the first series of proposed experiments, we will determine the functions of CAR and DAF during infection, define the route of virus entry for CAR-binding viruses and DAF-binding variants, and test the hypothesis that attachment to DAF permits CBV to cross epithelial surfaces In a second series of experiments we will define CAR's function in CBV pathogenesis in an in vivo model. In human tissues, CAR mRNA is most highly expressed in the heart and pancreas, the major CBV target organs both in humans and in animal models of infection. We will use a conditional gene targeting strategy to determine whether tissue-specific CAR expression is essential for CBV infection, and for virus-induced pathology in the heart and pancreas. In a final group of experiments we will explore CAR's physiologic functions. We have found that CAR is both a structural and functional component of the tight junction, that CAR interacts with the major junctional protein ZO-1, and that homotypic interactions between CAR extracellular domains mediate cell adhesion and contribute to junctional integrity. We will determine the structures involved in CAR-mediated homotypic adhesion, and define CAR' s interactions with other proteins important for junction formation and function.