CAR Function In Virus Tropism And Cell-Cell Contact

描述(申请人提供)：柯萨奇B组病毒(CBV)和腺病毒(ADS)是病毒性心肌炎的主要原因，CBV与胰腺炎和糖尿病有关。所有的CBV和许多ADS都通过附着在柯萨奇病毒和腺病毒受体(CAR)上而启动感染，CAR是一种细胞表面糖蛋白，其生理功能尚未确定。这一建议既涉及CAR在病毒趋向性中的作用，也涉及其生理功能。 我们最近发现，CAR是上皮细胞紧密连接的一个功能组件，是一种特殊的细胞间接触，是细胞旁溶质移动的屏障。在极化的上皮细胞上，CAR被隔离在紧密的连接中；因此，上皮单层抵抗CBV和ADS的感染。一些CBV还附着在第二个受体DAF上，DAF是一种分子，其在感染中的作用尚不清楚。初步数据表明，虽然DAF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粒子的构象变化。然而，DAF是针对极化细胞的顶端表面的，并且DAF结合的CBV变体有效地感染上皮单层；我们认为与DAF的相互作用为病毒在上皮和粘膜表面的感染提供了一种机制。在第一系列拟议的实验中，我们将确定CAR和DAF在感染过程中的功能，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DAF允许CBV穿过上皮表面的假设 在第二系列实验中，我们将在活体模型中确定CAR在CBV发病机制中的作用。在人体组织中，CAR mRNA在心脏和胰腺中表达最高，这两个器官是人类和动物感染模型中CBV的主要靶器官。我们将使用有条件的基因打靶策略来确定组织特异性CAR的表达是否对CBV感染以及病毒诱导的心脏和胰腺病理是必不可少的。 在最后一组实验中，我们将探索CAR的生理功能。我们发现CAR既是紧密连接的结构成分，也是紧密连接的功能成分，CAR与主要的连接蛋白ZO-1相互作用，CAR胞外区之间的同型相互作用介导细胞黏附，有助于连接的完整性。我们将确定参与CAR介导的同型黏附的结构，并定义CAR‘S与其他对连接形成和功能至关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.