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

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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.