In spite of decades of efforts made by many researchers the cellular binding proteins required for infection, the compartments in which HBV particles are taken up as well as the location in which fusion and release of core particles in the cytoplasma of target cells occur all remain unknown and are now to be identified. New methods of resolving these problems have become available by application of the infection system we have optimized using primary hepatocyte cultures of Tupaia belangeri (order Scandentia, south-east Asian tree shrew). This system of infection is homologous to scarcely available primary human hepatocytes. Because of the high degree of differentiation it is superior to extant hepatoma cell lines, both in efficiency and in significance of the results.
Binding and uptake of HBV are to be characterized both by confocal (B5) and by cryo-electron microscopy (Z4) using new instruments (B5) as well as by biochemical analysis of whole virus or of virus components after cell fractionation. In detail, the uptake pathway of the virus is to be characterized by expression of key proteins of the uptake pathway and their corresponding dominant negative mutants. Further questions relate to the dependence of infection upon pH value, redox potential and contingent proteolysis within characterized cell compartments as well as the intracellular localization of core particle release in the cytoplasm of the cells.
Additionally, receptor structures that are involved are to be identified by binding and crosslinking studies. In the last grant period we were able to determine the minimal viral binding sequence on the surface of the virus that is essential for infection. To this end we used chemically synthesized myristolated HBV pre-S1 peptides. These peptides or subviral particles will now be covalently bound to the surface of hepatocytes using specific binding molecules by application of heterologous or homo-bifunctional cross linkers. Binding can be performed on previously isolated hepatocytes in culture or after application of the viral binding partner in situ during perfusion of the tupaia liver or liver lobes. Determination of covalently bound protein complexes will then be undertaken biochemically following 2D-gel electrophoresis and tryptic in situ digestions by means of mass spectrometry and HPLC as well as N-terminal microsequencing in cooperation with Project Z1.
The cell culture system that we have established will facilitate measurement of HBV infectivity and of neutralizing antibodies. It will therefore also be useful in studying the infectivity of naturally occuring escape mutants and to determine the protection afforded by antibodies to mutated PreS or S domains