Efficient Inhibition of Hepatitis B Virus Infection by a preS1-binding Peptide

Abstract

Entry inhibitors are promising novel antivirals against hepatitis B virus (HBV) infection. The existing potential entry inhibitors have targeted the cellular receptor(s). In this study, we aim to develop the first entry inhibitor that inhibits HBV infection via targeting viral particles. The preS1 segment of the large envelope glycoprotein of HBV is essential for virion attachment and infection. Previously, we obtained a preS1-binding short peptide B10 by screening a phage display peptide library using the N-terminal half of preS1 (residues 1 to 60, genotype C). We report here that by means of concatenation of B10, we identified a quadruple concatemer 4B10 that displayed a markedly increased preS1-binding activity. The main binding site of 4B10 in preS1 was mapped to the receptor binding enhancing region. 4B10 blocked HBV attachment to hepatic cells and inhibited HBV infection of primary human and tupaia hepatocytes at low nanomolar concentrations. The 4B10-mediated inhibition of HBV infection is specific as it did not inhibit the infection of vesicular stomatitis virus glycoprotein pseudotyped lentivirus or human immunodeficiency virus type 1. Moreover, 4B10 showed no binding activity to hepatic cells. In conclusion, we have identified 4B10 as a promising candidate for a novel class of HBV entry inhibitors.

Figure 1. B10 concatemers exhibited increased preS1- and HBV-binding activities.

(A) Schematic representation of B10 concatemers. B10 monomers were depicted as dark gray boxes (left) and in boldface letters (right). Terminal protective residues and internal tri-peptide spacers (SP) were italicized. (B) B10 concatemers exhibit an increased activity in binding to preS1/2-48^myr. FITC-labelled preS1/2-48^myr captured by the immobilized N-biotinylated B10 monomer or concatemer was quantified via fluorescence measurment. (C) B10 concatemers exhibit an increased activity in binding to HBV. Captured HBV particles were detected with HRP-conjugated polyclonal anti-HBsAg antibody. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 2. Mapping of the main binding site of 4B10 in preS1.

(A) Schematic presentation of mutant preS1/2-48^myr peptides. IC₅₀, concentration for achieving 50% inhibition of the interaction between preS1/2-48^myr-FITC and 4B10, calculated based on the data plotted in (B). The five-amino acid deletions were depicted as lines and the myristoyl groups as open circles. Places where alanine was substituted were indicated. (B) Peptide competition assays. Each mutated preS1/2-48^myr peptide was used as a competitor of preS1/2-48^myr-FITC to bind to immobilized N-biotinylated 4B10. The fluorescence intensity was determined and normalized to that from the control competitorless wells. The numbers on the x axis represent the molar ratios of competitor versus preS1/2-48^myr-FITC. BSA, bovine serum albumin. (C) 4B10-binding activities of alanine substituted preS1/2-48^myr peptides. Wild type or alanine substituted preS1/2-48^myr-FITC was incubated with immobilized N-biotinylated 4B10. The fluorescence intensity was determined and relative binding activity was calculated, with the activity of the wild type peptide taken as 1.0. *p < 0.05; ***p < 0.001; ns, not significant.

Figure 3. 4B10 inhibited HBV attachment to hepatic cells.

(A) 4B10-FITC and LA-20-FITC did not bind to PMH. Fluorescence-activated cell sorting detected the binding of preS1/2-48^myr-FITC but not 4B10-FITC and LA-20-FITC to PMH. (B) 4B10 inhibited the binding of preS1/2-48^myr-FITC to PMH. PMH-bound preS1/2-48^myr-FITC peptides were visualized via confocal fluorescent microscopy. (C,D) 4B10 inhibited HBV attachment to PMH. (C) HBV virions bound to PMH were quantified via real-time PCR. (D) Immunofluorescent staining of cell-bound HBsAg. (E) 4B10 and LA-20 had no cytotoxic effect on PMH. Cell viability was measured using CCK-8 test.

Figure 4. 4B10 inhibited HBV infection of primary tupaia hepatocytes

. HBsAg (A,C) and HBeAg (B,D) levels in the culture supernatant of PTH infected with HBV preincubated with varying concentrations of 4B10: (A,B) time course; (C,D) relative HBsAg and HBeAg levels at day 12 post infection. The HBsAg and HBeAg levels derived from the culture supernatant without 4B10 preincubation were taken as 100% respectively. (E) 4B10 and LA-20 had no cytotoxic effect on PTH.

Figure 5. 4B10 inhibited HBV infection of primary human hepatocytes.

Relative HBsAg (A) and HBeAg (B) levels at day 14 post infection in the culture supernatant of PHH infected with HBV preincubated with varying concentrations of 4B10. The HBsAg and HBeAg levels derived from the culture supernatant without 4B10 preincubation were taken as 100% respectively. (C) 4B10 and LA-20 had no cytotoxic effect on PHH. The proliferation of PHH was measured using CellTiter-Glo Luminescent Cell Viability Assay.

Figure 6. Intracellular HBcAg expression was diminished in PTH and PHH infected with HBV in the presence of 4B10.

Intracellular HBcAg in PTH (A) and PHH (B) at day 12 and day 14 post-infection was detected via immunofluorescence using an anti-HBcAg antibody. Concentrations of peptides pre-incubated with HBV are indicated.

Figure 7. 4B10 had no inhibitory effect on the infections of VSV-G pseudotyped lentivirus and HIV-1.

(A) Huh7 cells were infected with VSV-G pseudotype lentivirus expressing EGFP in the presence of increasing concentrations of 4B10. Percentages of EGFP positive cells were calculated using fluorescence-activated cell sorting and plotted in (B). (C) TZM-bl cells harboring the HIV-1 LTR-luciferase reporter were infected with replication-deficient HIV-1/b16 (left) and HIV-1/sh1.81 (right) in the presence of increasing concentrations of 4B10. Intracellular luciferase activities were determined and plotted.