Importance of the D2-domain of coxsackievirus-adenovirus-receptor (CAR) for virus-receptor-interaction of coxsackievriuses

病毒感染的第一步是病毒衣壳与宿主细胞表面特定受体的相互作用。柯萨奇病毒-腺病毒受体（CAR）是柯萨奇- b病毒（CVB）的细胞受体，可引起多种疾病，尤其是心肌炎和胰腺炎。CAR定位于细胞表面，由两个免疫球蛋白（Ig）样胞外结构域（D1和D2）、一个跨膜结构域和一个细胞质结构域组成。CAR和CVB的相互作用是通过D1结构域与病毒衣壳表面的结合介导的。这种相互作用引起病毒衣壳的构象变化，病毒衣壳蛋白VP4丢失，从而形成病毒改变(a)颗粒。这些颗粒已经失去了感染细胞的能力，它们的形成是一个不可逆的过程。病毒衣壳与宿主受体相互作用后形成a颗粒在小核糖核酸科中广泛存在，包括CVB。a粒子的形成还导致病毒基因组进入细胞，这是病毒感染和病毒复制开始的重要步骤。CAR-D1结构域在CVB相互作用中的功能已被很好地表征，但在这种情况下，对D2结构域的功能一无所知。腺病毒与其细胞受体CAR的相互作用也通过与D1结构域的结合介导。但进一步的研究表明，CAR - D2结构域对于腺病毒进入靶细胞也是必不可少的。对CVB病毒-受体相互作用过程中CAR - D2结构域功能的研究为病毒摄取机制或抗病毒治疗方案提供了新的有趣信息。为了解决这个问题，应该消除CAR - D2结构域，并分析由此产生的CAR突变体的受体特性。因此，对受体功能的评估将从病毒与受体的附着、内化到细胞、a粒子的形成和病毒复制等方面进行。为了进一步分析D2结构域的功能，我打算用具有相似结构特征的蛋白的同源结构域代替D2结构域，并研究由此产生的突变CAR蛋白的CVB受体活性。CAR分别被细胞外ig样结构域D1和D2中的两个独立糖基化位点修饰。糖基化是一种可能的蛋白质修饰，在蛋白质的折叠、定位和稳定性中起着重要作用。在项目的第三部分，我想研究CAR-D1和/或D2结构域的糖基化对CVB受体功能是否重要。因此，我打算构建单和双糖基化缺陷的CAR突变体，并测试它们的受体特性。

The first essential step in the viral infection is the interaction of the viral capsid with the specific receptor on the surface of the host cell. The coxsackievirus-adenovirus-receptor (CAR) is the cellular receptor of the Coxsackie-B-viruses (CVB), which cause different diseases especially myocarditis and pancreatitis. CAR is localized on the cell surface and consists of two immunoglobulin (Ig)-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmatic domain. Interaction of CAR and CVB is mediated by binding of the D1 domain with the surface of the viral capsid. This interaction induces a conformational change of the viral capsid with loss of the viral capsid protein VP4 and thereby formation of viral altered (A)-particles. These particles have lost their capacity to infect cells and their formation is an irreversible process. The formation of A-particles after interaction of the viral capsid with the host receptor is widespread in the family of the picornaviridae, including CVB. A-particle formation results also in delivery of the viral genome into the cell, an essential step during virus infection and beginning of the viral replication.The function of the CAR-D1 domain during CVB interaction is well characterized but in this context nothing is known about function of the D2 domain. The interaction of adenoviruses with their cellular receptor CAR is also mediated by binding to the D1 domain. But further investigation showed that the CAR D2 domain is also essential for adenovirus uptake into the target cells. Investigation about function of the CAR D2 domain during virus-receptor-interaction of CVB provides new interesting information, regarding viral uptake mechanisms or antiviral therapy options. Addressing this issue the CAR D2 domain should be eliminated and the receptor properties of the resulting CAR mutant will be analyzed. Thereby, evaluation of the receptor function will be sectioned in attachment of the virus to the receptor, internalization into the cell, formation of A-particles and viral replication.To further analyze the function of the D2 domain I intent to substitute the D2 domain with homologous domains from proteins with similar structural features and investigate the CVB receptor activity of the resulted mutated CAR proteins.CAR is modified by two independent glycosylation sites in each extracellular Ig-like domain D1 and D2, respectively. Glycosylation is a possible modification of proteins and can play an important role in folding, localization and stability of proteins. In the third part of the project I want to investigate whether the glycosylation of the CAR-D1 and/or D2 domain is important for the CVB receptor function. Therefore I intend to construct single and double glycosylation-deficient CAR mutants and test their receptor properties as described above.