In the course of the functional characterization of hepatitis B surface proteins, a motif with cell permeable properties was discovered within the PreS2 region termed translocation motif (TLM). In this context two aspects of TLM were investigated. First we established a TLM fusion protein with hepatitis B virus core to generate cell permeable capsid particles as a novel tool for gene and protein transfer. Moreover we characterized the significance of TLM in the life cycle of the hepatitis B virus. For mediating cell permeability of HBV capsid particles, it is essential for TLM to be exposed on the shell surface, so that it is easily accessible. For that purpose on the one hand the TLM was introduced at the N-terminus via a 14 amino acid linker, on the other hand the TLM was inserted into a region of the core protein designated as spike tip. The ability of the particles to penetrate biological membranes was shown by incubation of immortalized adherent cells and primary dendritic cells with the different constructs and subsequent immunofluorescence microscopy. Furthermore, we tried to package specific nucleic acids into the modified capsids in two different approaches, using a prokaryotic as well as a eukaryotic expression system. In the first step of the prokaryotic approach, capsids were denatured to obtain core protein dimers. Consecutively the denatured proteins were renatured in the presence of the DNA of interest, yielding capsids with integrated DNA. To mimic the viral mechanism for RNA integration into core particles, we triple-transfected eukaryotic cells with expression constructs coding for the core protein, the viral polymerase and a packaging transcript. Particle assembly was confirmed by sucrose density gradient analysis. Generation of hepatitis B viral particles mutated in the TLM Using a HBV subtype adr4 genome, we introduced mutations in the TLM coding region by site-directed mutagenesis, leading to an alteration of the hydropathy profile as well as disruption of the alpha-helix formation as suggested by computer aided structure prediction. Transfection experiments with these constructs showed, that the mutation of TLM does neither impair the expression of viral proteins nor the secretion and maturation of viral particles. By this means, we were able to establish a system for the generation of TLM deficient particles, to investigate role of this motif for the viral life cycle.
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In the course of the functional characterization of hepatitis B surface proteins, a motif with cell permeable properties was discovered within the PreS2 region termed translocation motif (TLM). In this context two aspects of TLM were investigated. First we established a TLM fusion protein with hepatitis B virus core to generate cell permeable capsid particles as a novel tool for gene and protein transfer. Moreover we characterized the significance of TLM in the life cycle of the hepatitis B viru...
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