15, 16 (3) Besides preS1, another essential element of HBV/HDV infectivity has been assigned to the antigenic loop (AGL) of the S-domain.17 Replacement of cysteine residues in the AGL rendered HDV and HBV (Yi Ni, unpubl. results) noninfectious. Since some of these cysteines (e.g., Cys-124)

participate in intramolecular disulfide bonds, the sensitivity against reducing agents hints at the involvement of disulfide-bridge rearrangements during virus entry.18 Since only L-protein containing SVPs are able to bind hepatocytes from Tupaia belangeri the S-protein/domain is probably not essential for hepatocyte-specific binding.19 Consistent with the selleck screening library results of the mutational analyses, HBV preS1-derived lipopeptides,

mimicking the myristoylated N-terminal preS1-infectivity domain, efficiently inhibit HBV and HDV infection of HepaRG cells, PHH, and PTH.7, 20-23 The activity of the peptides requires myristoylation and the integrity of an internal sequence (9-NPLGFFP-15) which is highly conserved between primate hepadnaviruses.21 Since their inhibitory effect remains for several hours after preincubation they probably inactivate a cellular receptor.24 In the present study we used fluorescently labeled, myristoylated HBVpreS1-peptides to analyze the presence and turnover kinetics of this HBVpreS1-specific receptor on hepatocytes from different species. ACP-196 We investigated

whether receptor expression coincides with the species specificity of HBV and demonstrate highly specific binding of the preS1-lipopeptide to permissive cells (PHH, PTH, HepaRG). Unexpectedly, we detected specific binding to hepatocytes from non-susceptible species such as mouse, rat, rabbit, and dog, but not pig, cynomolgus, or rhesus monkey. Expression of a functional HBVpreS1-receptor was associated with the differentiation state of the hepatocyte: MCE No binding was observed in undifferentiated HepaRG cells or dedifferentiated PMH and PHH. HepG2 and HuH7 cells were unable to bind the peptide even after dimethyl sulfoxide (DMSO)-induced differentiation. Kinetic studies demonstrated a slow turnover and a constrained lateral movement of the receptor complex at the plasma membrane (PM), possibly due to a cytoskeleton interaction. DIPEA, N,N-Diisopropylethylamine; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; ge, genome equivalent; HBTU, O-(benzotriazol-1-yl)-N,N,N ′,N ′-tetramethyluronium hexafluorophosphate; HSPG, heparan sulfate proteoglycan; L-protein, hepatitis B virus large surface protein; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PHH, primary human hepatocytes; PMH, primary mouse hepatocytes; PTH, primary Tupaia belangeri hepatocytes.