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

病毒感染的第一个基本步骤是病毒衣壳与宿主细胞表面的特定受体相互作用。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)样胞外区(D1和D2)组成，一个跨膜区和一个胞内区。CAR与CVB的相互作用是通过D1区与病毒衣壳表面结合而实现的。这种相互作用导致病毒衣壳的构象改变，使病毒衣壳蛋白VP4丢失，从而形成病毒改变的(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转的过程。病毒衣壳与宿主受体相互作用后形成的A颗粒在包括柯萨奇病毒在内的短小病毒科的家族中广泛存在。A-颗粒的形成也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关键步骤。CVB相互作用中CAR-D1结构域的功能已被很好地描述，但在此背景下，对D2结构域的功能知之甚少。腺病毒与其细胞受体CAR的相互作用也是通过与D1域结合而介导的。但进一步的研究表明，CAR D2结构域对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的信息。为了解决这个问题，应该消除CAR D2结构域，并分析得到的CAR突变体的受体特性。因此，受体功能的评估将从病毒与受体的结合、内化到细胞内、A-颗粒的形成和病毒的复制来进行。为了进一步分析D2结构域的功能，我打算用结构相似的蛋白质中的同源结构域取代D2结构域，并研究得到的突变CAR蛋白的CVB受体活性。CAR在每个细胞外Ig样结构域D1和D2上分别被两个独立的糖基化位点修饰。糖基化是蛋白质的一种可能的修饰，在蛋白质的折叠、定位和稳定性方面起着重要作用。在项目的第三部分，我想要研究CAR-D1和/或D2结构域的糖基化对CVB受体功能是否重要。因此，我打算构建单个和双个糖基化缺陷的CAR突变体，并测试它们的受体特性，如上所述。

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.