1. Volume 129, Issue 1, Pages 234-245 (July 2005)
Mapping of the Hepatitis B Virus Attachment Site by Use of Infection-Inhibiting preS1 Lipopeptides and Tupaia Hepatocytes 
Dieter Glebe, Stephan Urban, Eva V. Knoop, Nilgün Çaǧ, Peter Krass, Stefanie Grün, Aiste Bulavaite, Kestutis Sasnauskas, Wolfram H. Gerlich 
Gastroenterology 
Volume 129, Issue 1, Pages (July 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Schematic presentation of hepatitis B virus. (A) The viral DNA is drawn as a single or double line. The viral DNA polymerase is depicted with the priming domain (pr) and the catalytic domain (RT). The nucleocapsid (core or HBc) is shown in black. The surface proteins S-HBs, M-HBs, and L-HBs are shown with the S-domain, the preS2 domain, and the preS1 domain. (B) Postulated topology of the large hepatitis B surface protein (L-HBs) with the preS at the virus surface. In the nonassembled state, the bottom reflects the cytosolic side or, after budding, the interior of the virus. The molecules of the membrane bilayer are depicted in gray and white. Predicted membrane-associated helices are numbered I to V. The topology of helices III to V is not exactly known. N-glyc, binding site of complex N-linked glycan; myr, myristoylation site. The dotted line represents the postulated liver attachment site in the preS1 domain. The binding site of MAb MA18/7 is indicated.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Inhibitory activity of myristoylated HBV preS1 peptides on HBV infection. Primary tupaia hepatocytes were incubated with purified HBV particles containing 2000 genome equivalents per cell in the presence of the respective preS1 peptides for 15 hours at 37°C. HBeAg production of infected cultures was determined from day 9–12 after infection and is displayed as a percentage of control infection without competing preS1 peptides. A myristoylated preS peptide from an avian hepadnavirus (avi myr-preS 2–44) served as a negative control. The dotted line indicates the cutoff.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Inhibitory activity of HBV preS1 peptides with different N-terminal hydrophobic residues. Primary tupaia hepatocytes were treated as described in Figure 2. N-terminal acyl moieties of preS1 2–48 peptides are indicated: palm, palmitoyl; myr, myristoyl; oct, octanoyl; pent, pentanoyl; chol, cholesteryl; ∅, no peptide. The dotted line indicates the cutoff.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Mapping of preS1 amino acid sequences required for infection inhibition. (A) Primary tupaia hepatocytes were incubated with purified HBV particles in the presence of the respective myristoylated peptides as described in Figure 2. The dotted line indicates the cutoff. (B) Schematic illustration of HBV preS1 peptides (numbering for genotype D). Numbers within the arrows show the approximate 50% inhibitory concentration. The binding site of neutralizing MAb MA18/7 is indicated. (C) Sequence comparison of preS1 sequence 1–50 of consensus HBV genotypes A–H, woolly monkey HBV (WMHBV), and preS of heron hepatitis B virus (HHBV). Sequences contributing to infection inhibition are shaded more or less strongly depending on the inhibitory effect of the corresponding region. Region 9–18 is essential. X, naturally occurring HBV preS1 deletions or artificially introduced deletions in preS1 peptides (amino acids 20–27); Con, consensus sequence of all HBV genotypes; afr, African genosubtype; aus, Australian genosubtype.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Inhibitory activity of myristoylated preS1 peptides with deleted epitopes of neutralizing antibodies against HBV surface proteins. Primary tupaia hepatocytes were treated as described in Figure 2. Deletions within preS1 peptides are indicated (eg, Δ20–21). Myristoylated Woolly monkey HBV (WMHBV) preS1 peptide 2–48 served as a natural mutant because it contains 3 mutations in amino acid region 20–27 compared with the HBV preS1 consensus sequence (Figure 4C). The dotted line indicates the cutoff; wt, wild type.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Kinetics of infection inhibition by myristoylated preS1 peptide 2–48. (A) Primary tupaia hepatocytes were infected with 2000 genome equivalents in the presence of 100 or 1 nmol/L HBV myristoylated preS1 2–48 peptide at the indicated conditions. I, Cells were preincubated with peptide for 0.5 hours at 37°C before addition of the virus. Either virus was given directly to the cells without removal of the peptide, or peptide was removed first by washing the cells 3 times with medium and further incubating with virus after 0, 2, and 4 hours. II, Peptide and virus were simultaneously given to the cells. III, Cells were first incubated with virus, and peptide was given after 0.5, 1, 2, and 4 hours to the cells. All cells were washed after 15 hours at 37°C 3 times with medium. (B) Cells were incubated with purified virus as in (A) but in the absence of inhibitory peptides. Viral inoculum was removed after the indicated times, and cells were washed extensively and further cultivated. HBeAg production of infected cultures was determined as described in Figure 2. ∅, no peptide. The dotted line indicates the cutoff.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Binding of preS1 peptides to various cell types. Different primary cells or cell lines were incubated with 5 μmol/L of either nonmyristoylated (A) or myristoylated HBV preS1 peptide 2–48 (B and D–H) for 2 hours at 37°C. After incubation, cells were extensively washed with medium, fixed, and immune-stained for bound preS1 peptide (red) and counterstained with Meyer’s hemalaun. (A) Primary tupaia hepatocytes incubated with nonmyristoylated or (B) myristoylated HBV preS1 peptide 2–48. (C) Primary tupaia hepatocytes without peptide. (D) Myristoylated peptide with tupaia nonparenchymal liver cells, (E) mouse liver cell line AML-12, or (F) human cervix carcinoma cell line HeLa. (G) The human hepatoma cell line HepG2 usually showed no binding, but (H) occasionally very few cells on the same coverslip showed signs of bound peptide. (I) preS1 staining of the HBV-producing cell line HepG All panels have same magnification (bar = 50 μm).
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Myristoylated HBV preS1 peptide 2–48 inhibits binding of HBV subviral particles to primary tupaia hepatocytes. Tupaia hepatocytes were incubated for 1 hour with 1 μmol/L peptide and thereafter with purified preS1-containing subviral particles for 1 hour at 37°C. After fixation, immunostaining was performed with a MAb against the S-domain (red) and counterstained with Meyer’s hemalaun (blue). (A and B) Cells preincubated with myristoylated HBV preS1 peptide 2–48. (C and D) Preincubation with myristoylated avian preS peptide as a noninhibiting control (A and C magnification, 200×; B and D magnification, 400×; bar = 50 μm).
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
The preS1-dependent binding of HBV to primary tupaia hepatocytes. Immunostaining of tupaia hepatocytes after incubation with purified HBV subviral particles from human plasma or yeast (2 μg/mL each) for 1 hour at 37°C with a MAb against the S-domain (red) and counterstaining with Meyer’s hemalaun (blue) is shown. Binding of plasma-derived particles (containing preS1, preS2, and S-domain) is detected, as seen in (A) and (B), but not after incubation of hepatocytes with recombinant subviral particles containing only the S-domain (C and D). (E and F) Binding could be restored by using yeast-derived recombinant particles containing preS1 (2–48/79–108) and the S-domain. The black arrows indicate the specific preS1-dependent immunostaining of viable cells, whereas the gray arrows indicate preS1-independent binding to round apoptotic cells (A, C, and E magnification, 100×; B, D, and F magnification, 200×; bar = 50 μm).
Gastroenterology  , DOI: ( /j.gastro )
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