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. 2007 Apr;81(8):4244-54.
doi: 10.1128/JVI.01270-06. Epub 2006 Dec 27.

Identification and characterization of peptides that interact with hepatitis B virus via the putative receptor binding site

Qiang Deng  1 , Jian-wei Zhai, Marie-Louise Michel, Jun Zhang, Jun Qin, Yu-ying Kong, Xin-xin Zhang, Agata Budkowska, Pierre Tiollais, Yuan Wang, You-hua Xie
Affiliations

Identification and characterization of peptides that interact with hepatitis B virus via the putative receptor binding site

Qiang Deng et al. J Virol. 2007 Apr.

Abstract

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.

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Figures

FIG. 1.
FIG. 1.
Library construction, screening, and verification of PreS-binding phages. (A) A phage library was constructed for the surface display of linear 12-mer peptides (X12). The pIII secretion signal facilitates the transport of the fusion peptide to the phage surface. The arrows indicate the cleavage site for the signal peptide by pelB and the glycine tether for enhanced flexibility for the peptide. (B) Recombinant Thio-PreS (left) and CBD-PreS (right) were analyzed by SDS-PAGE. Lanes 1 and 4, E. coli lysate before IPTG (isopropyl-β-d-thiogalactopyranoside) induction; lanes 2 and 5, E. coli lysate after IPTG induction; lanes 3 and 6, the affinity-purified products. PreS fusions are indicated by arrowheads. (C) The PreS-binding property of the enriched phage pool after the final round of screening was assessed by phage-ELISA. Closed circles, assay with Thio-PreS; open circles, assay with thioredoxin; triangles, phages from the original library assayed with Thio-PreS. OD450, optical density at 450 nm. Error bars indicate standard deviations.
FIG. 2.
FIG. 2.
GST pull-down assays for the verification of PreS-binding properties of the peptides. (A) Western blot with anti-PreS1 MAb 125E11. An equal amount of MBP-PreS was applied to beads conjugated with GST-p2 (lane 2), GST-p5 (lane 3), GST-p18 (lane 4), and GST (lane 5). Lane 1 shows the input of MBP-PreS. (B) Western blot with anti-MBP MAb. Comparable amount of MBP-PreS (lanes 4, 6, and 8) and MBP (lanes 3, 5, and 7) were applied to beads conjugated with GST-p2 (lanes 3 and 4), GST-p5 (lanes 5 and 6), and GST-p18 (lanes 7, 8). Lanes 1 and 2 show the MBP and MBP-PreS inputs, respectively. (C) Mapping of the peptide binding site within PreS. Comparable amount of indicated MBP-PreS fragments were applied to beads conjugated with GST-p18, GST-p2, or GST-p5. PreS fragments as MBP fusion proteins are denoted above each lane.
FIG. 3.
FIG. 3.
Identification and mutational analysis of a consensus PreS-binding motif. (A) Peptide alignment. Highly conserved residues are shaded and in bold. The deduced consensus sequence (pC) is W1T2X3W4W5. (B) GST pull-down assays. MBP-PreS11-65 was used for the binding assays (lanes 2, 4, and 6). Parallel controls were performed with MBP-PreS11-20 (lanes 1, 3, and 5). Lanes 1 and 2, GST-p18; lanes 3 and 4, GST-pC, with the sequence W1T2N3W4W5; lanes 5 and 6, W4W5→AA mutant of GST-pC. (C) GST pull-down assays with A, F, or S residue substitutions. The substitutions are denoted above each lane.
FIG. 4.
FIG. 4.
Capture of HBV particles by the synthetic consensus peptide. (A) Sequences of the biotin-conjugated synthetic consensus peptide (pC) and mutant peptide (pM). Pentapeptide sequences are in bold. Mutated residues in pM are indicated in bold italics. The spacers in both peptides are depicted by arrows. (B) Medium of HepG2 culture transfected with p3.6II (left) or pcDNA3-SHBs (right) was concentrated and applied to biotin-peptide-immobilized streptavidin-coated wells. Bound particles were revealed by detection of HBs by ELISA. Comparable HBs loads (normalized by HBs) were assayed. (C) Serially 1:2 diluted HBV sera with an initial 106 viral DNA copies were applied, and bound viral particles were detected by anti-HBs antibody. OD450, optical density at 450 nm. Error bars indicate standard deviations.
FIG. 5.
FIG. 5.
LPL is a novel HBV binding protein. (A) Schematic view of the three-dimensional model of human LPL (25). The PreS-binding motif is exposed in a surface loop, residues 382 to 396 between the stacked sheets in the C-terminal domain. (B) GST-pC (lanes 1 and 2), GST-LPLc (lanes 3 and 4), and GST-LPLcm (W393W394→AA) (lanes 5 and 6) were assessed for binding to PreS11-65 (lanes 1, 3, and 5) and PreS11-20 (lanes 2, 4, and 6). (C) Virus capture assay with FLAG-tagged LPLc. HBV particles from serum samples were applied to FLAG-LPLc-immobilized streptavidin-coated wells. OD450, optical density at 450 nm. Error bars indicate standard deviations. (D) Virus capture by recombinant LPL produced in COS cells. Upper panel, recombinant LPL protein with a C-terminal FLAG tag (lane 1) and the corresponding mutant (lane 2) from lysates of transfected COS cells were verified by Western blotting with anti-FLAG MAb. Lane 3, COS cell lysate without transfection. Lower panel, LPL proteins from cell lysates were applied to anti-FLAG-coated wells and used to capture virus particles.
FIG. 6.
FIG. 6.
Conservation of the PreS-binding motif in non-human lipoprotein lipases. (A) Sequence alignment around the PreS-binding motif. The active sites are in bold and underlined. hLPL, human LPL; mLPL, mouse LPL; rLPL, rat LPL; bLPL, bovine LPL. (B) HBV capture by bLPL. Purified bLPL was directly applied to microwells. BSA, bovine serum albumin as a negative control. OD450, optical density at 450 nm.
FIG. 7.
FIG. 7.
HBV is bridged to the surface of THP-1 cells by LPL. Differentiated THP-1 cells were incubated with 10 nM dexamethasone to stimulate the production of LPL (16). (A and B) Immunostaining of LPL expressed on the cells with anti-LPL MAb 5D2 without (A) or with (B) heparin pretreatment. (C and D) Immunostaining of HBV bound to the cells by anti-HBs MAb without (C) or with (D) heparin pretreatment. (E and F) Competition assay using FITC-labeled anti-HBs polyclonal antibody for the detection of HBV binding. Cells were coincubated without (E) or with (F) the anti-LPL MAb 5D2. DAPI (4′,6′-diamidino-2-phenylindole)-stained nuclei are in blue. Signals of FITC- or Alexa 488-labeled antibody are in green.
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