Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion-and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) H epadna-or hepatitis B viruses (HBV) are small enveloped DNA viruses that cause acute and chronic liver infections in mammals and birds. In the case of HBV, progression to the chronic state significantly increases the risk of developing liver cirrhosis and hepatocellular carcinoma. Because 400 million people worldwide suffer from chronic HBV infection, HBV is one of the most important human pathogens. Hepadnaviruses possess remarkable host specificities and preferentially replicate in hepatocytes of their respective hosts. 1 Until recently, in vitro HBV infections were only successful in primary human hepatocytes (PHH) or hepatocytes of related primates. 2,3 Systematic investigations of HBV early infection events were thus difficult and varied with the quality of the hepatocyte preparation. The observation that hepatocytes of Tupaia belangeri are susceptible for HBV 4,5 and the recent establishment of the HepaRG cell line, which also supports the full HBV replication cycle, 6 resolved this limitation and facilitated detailed studies on the contribution of HBV envelope protein domains in virus attachment and entry.The HBV envelope consists of three membrane proteins termed large (L-), middle (M-), and small (S-) proteins. They are encoded in one open reading frame with three in-phase start codons. 1 L-and M-share the S-domain, which serves as a membrane anchor but accom-

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