Hepatitis B virus (HBV) can infect only a few cell types including primary Tupaiahepatocytes (PTH) which were used in this work. The aim of the study was to characterizethe early steps of HBV infection using pharmacological inhibitors and confocal microscopy.The distinction between endocytosis in general and direct fusion with the plasmamembrane was made by using hypertonic sucrose concentrations. The fusion of Sendaivirus (SeV) with the plasma membrane and its infectivity was not affected, whereas theinfection of control viruses known to be taken up via endocytosis, e.g. Semliki Forest virus(SFV) and Simian virus 40 (SV40), was impeded. The HBV infection was inhibited in adose-dependent manner, suggesting an endocytic uptake. The dynamin inhibitor Dynasorecompletely inhibited the entry of SFV (clathrin-mediated endocytosis) and SV40 (caveolinmediatedendocytosis), but had no effect on HBV suggesting a dynamin-independentinternalization. Furthermore, the inhibitor of macropinocytosis EIPA did not impair HBVinfection. HBV was also not able to stimulate the macropinocytic activity of PTH. Alsophagocytosis could be ruled out as an entry mechanism. The actin inhibitors Jasplakinolide(actin-stabilisation) or Cytochalasin D (actin-depolymerisation) resulted in a partialinhibition of the early steps of HBV infection. The intracellular transport of HBV could beconstrained by utilization of Nocodazole, an inhibitor of microtubule-polymerisation. HBVdoes not require an acidic pH within the endosomal compartments for the initiation of aproductive infection cycle and therefore differs from many other viruses that areinternalized via endocytosis (e.g. SFV, which was tested as control virus) and also from therelated hepatitis B virus of the duck. No parallels to the retrograde pathway of SV40 wereobserved, since the HBV infection showed no sensitivity towards inhibitors of specificintracellular transport mechanisms (Brefeldin A), ER protein folding and quality factors(MG-132) and the translocation process to the cytosol (Thapsigargin). Together withprevious data it can be concluded that HBV is internalized via a not yet known, possiblyvirus-induced, endocytic uptake mechanism, which is clathrin-, caveolin-, lipid raft- anddynamin-independent and differs from the process of phagocytosis and macropinocytosis.
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