TY  - GEN
AV  - public
KW  - receptor 
KW  -  visualization 
KW  -  microscopy 
KW  -  pharmacodynamics
N2  - The human hepatitis B virus (HBV) is characterized by a pronounced liver tropism and a restricted host range. While the viral determinants essential for the host cell entry are well characterized, little is known about cellular factors involved in early steps of HBV infection. Proteoglycans have been described as primary attachment factors, but neither a receptor molecule nor the exact entry pathway is elucidated yet. Peptides comprising the first 47 amino acids (HBVpreS/2-48myr) of the preS1-domain of the HBV surface protein L have been shown to inhibit an infection in vitro and in vivo with high specificity. The full inhibitory potential of HBVpreS/2-48myr relies on the integrity of an essential region (amino acids 9 - 15) and the presence of an acylation. This correlates with viral requirements for infectivity: mutations in the essential region (amino acids 11, 12 and 13) or the removal of the myristoylation render HBV particles non-infectious. It therefore is assumed that HBVpreS/2-48myr and the virus address a common factor on the hepatocyte. This work aimed to visualize and characterize the interaction with this factor. Fluorescence microscopy and flow cytometry showed that fluorescently labeled peptides (HBVpreS/2-48myr-K-FITC) bind to the plasma membrane of differentiated hepatocytes in a sequence- and myristoylation-dependent manner. The binding was not restricted to HBV-susceptible cells like primary hepatocytes (PH) from human or tupaia, and HepaRG cells, but was also detected on PH from non-permissive species (mouse, rat, dog and woodchuck). This demonstrated that a binding-competent preS1-receptor is present also in non-susceptible species. The refractoriness of these cells towards HBV infection therefore must be independent from the receptor interaction. HBV infects only differentiated hepatocytes. Correlating to that, de-differentiation of PH was accompanied by a loss of the ability to bind HBVpreS/2-48myr-K-FITC. Vice versa, HepaRG cells gained binding competence during differentiation, demonstrating that the expression of a functional preS1-receptor depends on the differentiation status of a cell. Sustaining this assumption, hepatoma cell lines like HepG2 and HuH7 did neither bind HBVpreS/2-48myr-K-FITC. Their non-permissiveness therefore can inter alia be explained by a lack of a functional receptor. To determine the affinity of the peptide-receptor interaction, binding curves for HBVpreS/2-48myr-K-FITC-binding to the cell surface of PH were calculated with data from flow cytometry and mass spectrometry. This revealed a bimodal mechanism, consisting of (i) a sequence- and myristoylation-dependent binding to a receptor with a high affinity (KD ~ 60 nM), and (ii) a non-specific, low-affinity-interaction (KD > 2000 nM), that depended only on the myristoylation. Analysis of the binding kinetics on PH showed that equilibrium of the high-affinity interaction was established after 10 minutes. Thereby, the peptide-receptor complexes exhibited an extraordinary high stability over time (t1/2 ~ 11 hours), which indicated a low metabolic turn-over rate. These complexes were tightly associated with the actin cytoskeleton, as they did not show lateral movement within the membrane after photobleaching and co-localized with actin. In order to extend these findings and to investigate the interaction of HBV particles with cells, fluorescently labeled HBV-Alexa488 was produced and characterized on a single-particle level. Chemical labeling of cell-culture derived virus yielded infectious particles of a high purity and bright fluorescence. Detection of HBV-Alexa488 on HepaRG cells showed a binding behavior similar to that of unlabeled HBV. Binding could be enhanced with PEG and inhibited by heparin. The labeled virus produced here can be applied for future single-virus tracing experiments.
A1  - Meier, Anja
ID  - heidok11245
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/11245/
Y1  - 2010///
TI  - Visualization and Characterization of HBV-Receptor Interactions
ER  -