Characterization of a hepatitis B and hepatitis delta virus receptor binding site.
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) a myristoylated HBVpreS/2-48-GST fusion protein efficiently interferes with HBV infection of HepaRG cells; (2) deletions and point mutations in the highly conserved preS1 sequence between amino acids 11 through 21 result in the loss of infection inhibition activity; (3) hepatitis B viruses carrying single amino acid exchanges within this region lose infectivity; and (4) HDV infection of HepaRG cells can be inhibited by myristoylated HBVpreS peptides with the same specificity. In conclusion, HBV and HDV use at least one common step to enter hepatocytes and require a highly conserved preS1-sequence within the L-protein. This step is exceptionally sensitive toward inactivation by acylated HBVpreS1 peptides, which therefore represent a novel group of entry inhibitors that could be used for the treatment of hepatitis B and D.
Department of Molecular Virology, Otto-Meyerhof-Zentrum (OMZ), University of Heidelberg, Heidelberg, Germany.
Matthias
M
Engelke
Kerry
K
Mills
Stefan
S
Seitz
Petra
P
Simon
Philippe
P
Gripon
Martina
M
Schnölzer
Stephan
S
Urban
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006509:Hepatitis B / Q000517:prevention & control; D015611:Hepatitis B Virus, Duck / Q000235:genetics; D006515:Hepatitis B virus / Q000378:metabolism*; D003699:Hepatitis D / Q000517:prevention & control; D003698:Hepatitis Delta Virus / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D010449:Peptide Mapping; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D008074:Lipoproteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D019814:Myristic Acid; D005982:Glutathione Transferase