Kartierung von umhüllungsrelevanten Aminosäureresten auf dem Hepatitis B Virus Kapsid

During morphogenesis of the hepatitits B virus (HBV) the cytosolic build nucleocapsid containing the viral genome interacts with transmembrane viral surface proteins to get enveloped into the ER lumen and secreted out of an infected liver cell. Therefore a direct interaction between the capsid and surface proteins are predicted. The crystal structure of recombinant HBV capsids has recently been published. To mapped sites on the capsid surface important for envelopment by viral surface proteins 52 single amino acids were mutated to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core-negative HBV genome in transiently transfected liver cells, immunoprecipitation of capsids and virions and radioactive labeling of the viral genome by the endogenous polymerase reactions (epr). 13 point mutations impeded nucleocapsid detection by epr, 28 were wild type (wt) and 11 mutations allowed nucleocapsid formation but blocked their envelopment and virion formation to undetectable levels. These mutations cluster in two small areas on the capsid surface. These areas are proposed to interact with envelope proteins during virion morphogenesis.Cell lines susceptible to HBV infection are still not available and functional HBV receptors has not yet been identified. However, transfection of the viral genome in human liver cell lines allows the formation of infectious virus indicating that the early steps in the viral life cycle are blocked. It is hypothesized that the block might be due to the lack of a viral receptor and might be overcome by expression of an artificial receptor for binding and uptake of virions. To generate such an receptor the cDNA for the heavy and light chains of the monoclonal anti-preS1 antibody MA18/7 binding to the large HBV envelope protein was cloned from mouse hybridoma cells via RT-PCR. The 3`-end of the heavy chain was fused to a transmembrane domain of IgD. It could be shown that both chains were stably expressed in hepatoma cells, that they were located at the cell membrane and directed to the outside of the cell. Incubation of MA18/7 transfected cells with HBV virions showed a specific binding of virions to the cells but no infection could be observed.