Mapping of the Hepatitis B Virus Attachment Site by Use of Infection-Inhibiting preS1 Lipopeptides and Tupaia Hepatocytes
2005; Elsevier BV; Volume: 129; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2005.03.090
ISSN1528-0012
AutoresDieter Glebe, Stephan Urban, E. V. Knoop, Nilgün Çaǧ, Peter Krass, Stefanie Grün, Aistė Bulavaitė, Kęstutis Sasnauskas, Wolfram H. Gerlich,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoBackground & aims: Studies on the early steps in the life cycle of hepatitis B virus have been hampered by the lack of readily available target cells. In this study, we mapped a defined virus attachment site to primary hepatocytes that is essential for infection. Methods: We used purified virus particles from human carrier plasma as an inoculum and primary cultures of tupaia hepatocytes as susceptible target cells and studied the inhibitory effect of amino-terminally acylated preS1-derived lipopeptides on infection interference. Results: Infectivity of virus could be blocked efficiently in this system by amino-terminally acylated peptides containing amino acids 2–18 from the preS1 domain. The addition of amino acids 28–48 enhanced the inhibitory capacity, whereas amino acids 49–78 did not contribute to inhibition. Myristoylated preS1 peptides 2–48 bound strongly to tupaia hepatocytes but not to nonhepatic cells or rodent hepatocytes and thereby inhibited infection even at concentrations of 1 nmol/L completely. Particles consisting only of the small hepatitis B surface protein—the active component of current hepatitis B vaccines—did not bind at all to tupaia hepatocytes, but the addition of the preS1 domain to the particles allowed binding. Conclusions: The preS1 sequence 2–48 mediates attachment of the virus to its target cells, whereas the small surface protein seems to be involved in other steps. These findings indicate that the current subunit hepatitis B vaccines may be improved by the addition of distinct preS1 epitopes. Moreover, preS1 lipopeptides are promising candidates for specific antiviral therapy against hepatitis B infections. Background & aims: Studies on the early steps in the life cycle of hepatitis B virus have been hampered by the lack of readily available target cells. In this study, we mapped a defined virus attachment site to primary hepatocytes that is essential for infection. Methods: We used purified virus particles from human carrier plasma as an inoculum and primary cultures of tupaia hepatocytes as susceptible target cells and studied the inhibitory effect of amino-terminally acylated preS1-derived lipopeptides on infection interference. Results: Infectivity of virus could be blocked efficiently in this system by amino-terminally acylated peptides containing amino acids 2–18 from the preS1 domain. The addition of amino acids 28–48 enhanced the inhibitory capacity, whereas amino acids 49–78 did not contribute to inhibition. Myristoylated preS1 peptides 2–48 bound strongly to tupaia hepatocytes but not to nonhepatic cells or rodent hepatocytes and thereby inhibited infection even at concentrations of 1 nmol/L completely. Particles consisting only of the small hepatitis B surface protein—the active component of current hepatitis B vaccines—did not bind at all to tupaia hepatocytes, but the addition of the preS1 domain to the particles allowed binding. Conclusions: The preS1 sequence 2–48 mediates attachment of the virus to its target cells, whereas the small surface protein seems to be involved in other steps. These findings indicate that the current subunit hepatitis B vaccines may be improved by the addition of distinct preS1 epitopes. Moreover, preS1 lipopeptides are promising candidates for specific antiviral therapy against hepatitis B infections. Hepatitis B virus (HBV) is the prototype of the Hepadnaviridae, a family of hepatotropic and highly species-specific viruses that are able to cause persistent infection of the liver with a high level of viremia.1van Regenmortel M.H.V. Fauquet C.M. Bishop D.H.L. Carstens E.B. Estes M.K. Lemon S.M. Maniloff J. Mayo M.A. McGeoch D.J. Pringle C.R. Wickner R.B. Virus taxonomy the classification and nomenclature of viruses. The seventh report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego2000Google Scholar An estimated 370 million people are chronically infected with HBV, and approximately 1.2 million people die annually from the sequelae of this infection. Various subunit types of HBV vaccines have been developed,2Shouval D. Hepatitis B vaccines.J Hepatol. 2003; 39: S70-S76Abstract Full Text Full Text PDF PubMed Google Scholar but the most commonly used vaccine type consists of only the smallest of the 3 coterminal HBV surface proteins (SHBs). Although this vaccine has been very successful in reducing the transmission of HBV, its mechanism of protection is not well understood. Classically, nondividing virus vaccines protect via neutralizing antibodies and mainly target the attachment sites of the virus to their cellular receptor(s). Numerous studies have described various HBV-binding molecules, but the proof that these binding partners are true receptors is still lacking.3De Meyer S. Gong Z.J. Suwandhi W. van Pelt J. Soumillion A. Yap S.H. Organ and species specificity of hepatitis B virus (HBV) infection a review of literature with a special reference to preferential attachment of HBV to human hepatocytes.J Viral Hepat. 1997; 4: 145-153Crossref PubMed Scopus (85) Google Scholar The difficulty in the study of attachment and uptake of HBV by its target cells is that only the liver in the living host is fully susceptible. Here 1 in 10 viral particles are sufficient to initiate infection.4Ulrich P.P. Bhat R.A. Seto B. Mack D. Sninsky J. Vyas G.N. 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In vitro experimental infection of primary human hepatocytes with hepatitis B virus.Gastroenterology. 1994; 106: 664-673Abstract PubMed Scopus (0) Google Scholar Recently, 2 more available in vitro systems for HBV infection studies have been established: a redifferentiatable hepatoma cell line, HepaRG,7Gripon P. Rumin S. Urban S. Le Seyec J. Glaise D. Cannie I. Guyomard C. Lucas J. Trepo C. Guguen-Guillouzo C. Infection of a human hepatoma cell line by hepatitis B virus.Proc Natl Acad Sci U S A. 2002; 99: 15655-15660Crossref PubMed Scopus (998) Google Scholar and primary hepatocyte cultures from Tupaia belangeri livers.8Köck J. Nassal M. MacNelly S. Baumert T.F. Blum H.E. von Weizsäcker F. Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus.J Virol. 2001; 75: 5084-5089Crossref PubMed Scopus (128) Google Scholar The susceptibility of tupaias for human HBV is surprising, because these animals are not primates but form their own order (Scandentia) within mammals, phylogenetically placed between Rodentia, Lagomorpha, and primates.9Nishihara H. Terai Y. Okada N. Characterization of novel Alu- and tRNA-related SINEs from the tree shrew and evolutionary implications of their origins.Mol Biol Evol. 2002; 19: 1964-1972Crossref PubMed Scopus (64) Google Scholar Despite the narrow species specificity of hepadnaviruses, HBV infection of tupaia hepatocytes is specific because we could show that monoclonal antibodies (MAbs) against the surface proteins of HBV (HBs) specifically neutralized the infectivity of HBV for tupaia hepatocyte cultures.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar The 3 co–carboxy-terminal HBs proteins (L-, M-, and S-HBs) are distinguished by 3 domains: preS1 only in L-HBs, preS2 in L-HBs and M-HBs, and S in all 3 HBs proteins11Heermann K.H. Goldmann U. Schwartz W. Seyffarth T. Baumgarten H. Gerlich W.H. Large surface proteins of hepatitis B virus containing the pre-s sequence.J Virol. 1984; 52: 396-402Crossref PubMed Google Scholar (Figure 1). We10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar and another group12Köck J. Baumert T.F. Delaney W.E. Blum H.E. von Weizsäcker F. Inhibitory effect of adefovir and lamivudine on the initiation of hepatitis B virus infection in primary tupaia hepatocytes.Hepatology. 2003; 38: 1410-1418PubMed Google Scholar showed that some, but not all, MAbs against the S-domain neutralized HBV infection of tupaia hepatocyte cultures. A MAb against preS1 inhibited HBV infectivity completely, whereas MAbs against the preS2 domain neutralized only partially or not at all.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar In this study, we wanted to distinguish the contribution of the preS1 and S-domain to attachment and infectivity and to narrow down the attachment site(s) to defined amino acids (AAs). It is known that the preS1 domain is acylated at glycine 2 with myristic acid13Persing D.H. Varmus H.E. Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.J Virol. 1987; 61: 1672-1677Crossref PubMed Google Scholar and that this modification is necessary for efficient hepadnaviral infectivity.14Macrae D.R. Bruss V. Ganem D. Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly.Virology. 1991; 181: 359-363Crossref PubMed Scopus (55) Google Scholar, 15Bruss V. Hagelstein J. Gerhardt E. Galle P.R. Myristylation of the large surface protein is required for hepatitis B virus in vitro infectivity.Virology. 1996; 218: 396-399Crossref PubMed Scopus (95) Google Scholar, 16Gripon P. Le Seyec J. Rumin S. Guguen-Guillouzo C. Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity.Virology. 1995; 213: 292-299Crossref PubMed Scopus (154) Google Scholar PreS-derived peptides of the duck hepadnavirus have been found to inhibit infection of duck hepatocytes, but their inhibitory power could be dramatically increased by amino-terminal myristoylation.17Urban S. Gripon P. Inhibition of duck hepatitis B virus infection by a myristoylated pre-S peptide of the large viral surface protein.J Virol. 2002; 76: 1986-1990Crossref PubMed Scopus (44) Google Scholar Recent studies have confirmed this effect also in the human system.18Gripon P. Carrie I. Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein.J Virol. 2005; 79: 1613-1622Crossref PubMed Scopus (284) Google Scholar Thus, we used HBV preS1 lipopeptides with an amino-terminal fatty acid and mapped a defined HBV attachment site to primary hepatocytes that is essential for infection. HBV and natural HBs antigen (HBsAg; genotype D; HBsAg subtype ayw2) were isolated from hepatitis B early antigen (HBeAg)-positive plasma of 2 asymptomatic chronic HBV carriers with normal transaminase levels in serum. One carrier (ID304) had 1.6 × 109 HBV genomes per milliliter and 55 μg/mL HBsAg, and the second (ID259) had 2 × 109 HBV genomes per milliliter and 10 μg/mL HBsAg. The purification was performed as previously described,19Glebe D. Gerlich W.H. Study of the endocytosis and intracellular localization of subviral particles of hepatitis B virus in primary hepatocytes.Methods Mol Med. 2004; 96: 143-151PubMed Google Scholar with some modifications: HBV and subviral particles from 350 mL of human plasma were pelleted through 10% and 15% sucrose for 15 hours at 25,000 rpm in a SW28.38 rotor (Beckman, Munich, Germany). The resuspended pellets were pooled and ultracentrifuged into a discontinuous sucrose density gradient (15%, 25%, 35%, 45%, and 60%) as described previously. HBV-containing fractions were identified by quantitative real-time polymerase chain reaction as previously described.20Jursch C.A. Gerlich W.H. Glebe D. Schaefer S. Marie O. Thraenhart O. Molecular approaches to validate disinfectants against human hepatitis B virus.Med Microbiol Immunol (Berl). 2002; 190: 189-197Crossref PubMed Scopus (43) Google Scholar The fractions containing HBsAg were identified by sodium dodecyl sulfate gel electrophoreses and silver staining, pooled, and further purified by CsCl density gradient flotation and discontinuous sucrose density gradient as previously described.19Glebe D. Gerlich W.H. Study of the endocytosis and intracellular localization of subviral particles of hepatitis B virus in primary hepatocytes.Methods Mol Med. 2004; 96: 143-151PubMed Google Scholar The SHBs gene and the hybrid gene, containing the 2–48/79–108 preS1 sequence fused to SHBs, was constructed by polymerase chain reaction techniques from plasmid HBV320 (a kind gift from Paul Pumpens, Department of Protein Engineering, Biomedical Research and Study Centre, University of Latvia, Riga, Latvia) containing HBV genotype D by using specific oligonucleotides.21Bichko V. Pushko P. Dreilina D. Pumpen P. Gren E. Subtype ayw variant of hepatitis B virus. DNA primary structure analysis.FEBS Lett. 1985; 185: 208-212Crossref PubMed Scopus (126) Google Scholar Both genes were separately inserted into the XbaI site of the yeast expression vector pFX7 under control of the hybrid GAL10-PYK1 promoter. The resulting plasmids pFX7-S and pFX7-preS1-S were used for transformation of yeast Saccharomyces cerevisiae strain FH4C. Selected transformants were used for the purification of recombinant proteins as previously described.22Sasnauskas K. Bulavaite A. Hale A. Jin L. Knowles W.A. Gedvilaite A. Dargeviciute A. Bartkeviciute D. Zvirbliene A. Staniulis J. Brown D.W. Ulrich R. Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.Intervirology. 2002; 45: 308-317Crossref PubMed Scopus (77) Google Scholar Peptides were synthesized and purified in the Department of Biomolecular Chemistry at the Zentrum für Molekulare Biologie (Heidelberg, Germany) and by Peptide Specialty Laboratories, GmbH (Heidelberg, Germany). Primary hepatocytes of T belangeri (Asian tree shrews) were isolated as previously described.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar In brief, the livers were perfused via the portal vein with Hanks solution (Invitrogen, Karlsruhe, Germany) containing 5 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, followed by perfusion with Dulbecco's modified Eagle medium (Invitrogen) containing 0.05% collagenase (Sigma, Deisenhofen, Germany). Hepatocytes were selectively pelleted 3 times at 40g for 6 minutes at 4°C. The first supernatant of this centrifugation was used to isolate nonparenchymal liver cells as previously described.23Michalopoulos G.K. Bowen W.C. Mule K. Stolz D.B. Histological organization in hepatocyte organoid cultures.Am J Pathol. 2001; 159: 1877-1887Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Hepatocytes were resuspended in tupaia hepatocyte medium (THM) and poured on collagen-coated 24-well plates (5 × 104 hepatocytes per well) as previously described.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar Plating efficiency was measured before infection either by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or by measuring released cytosolic lactate dehydrogenase (LDH) by freeze-thawing of randomly selected wells. The MTT assay involves the metabolic conversion of the water-soluble compound MTT to an insoluble formazan only in intact living cells. Variability as determined by MTT and LDH assay for each preparation was ≤10% (data not shown). The organ harvest from tupaias was approved by the local animal protection committee. Cell viability was determined by the MTT assay24Mosmann T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays.J Immunol Methods. 1983; 65: 55-63Crossref PubMed Scopus (48661) Google Scholar and confirmed by measuring released cytosolic LDH into the supernatant and the propidium iodide exclusion test.25Tanke H.J. van der Linden P.W. Langerak J. Alternative fluorochromes to ethidium bromide for automated read out of cytotoxicity tests.J Immunol Methods. 1982; 52: 91-96Crossref PubMed Scopus (34) Google Scholar The human cervix carcinoma cell line HeLa and the human hepatoma cell line HepG2 were obtained from the American Type Culture Collection (Manassas, VA). The HBV-producing cell line HepG2.2.15 was a kind gift from George Acs and Mary Ann Sells (Department of Biochemistry, Mont Sinai School of Medicine, New York, NY).26Sells M.A. Chen M.L. Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc Natl Acad Sci U S A. 1987; 84: 1005-1009Crossref PubMed Scopus (1048) Google Scholar AML-12 (an immortalized hepatocyte line from mice transgenic for transforming growth factor α) cells were provided by Nelson Fausto (Department of Pathology, University of Washington School of Medicine, Seattle, WA).27Wu J.C. Merlino G. Fausto N. Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.Proc Natl Acad Sci U S A. 1994; 91: 674-678Crossref PubMed Scopus (258) Google Scholar Cells were cultivated in Dulbecco's modified Eagle medium (Invitrogen) with the addition of 10% fetal calf serum at 37°C in a humidified incubator. For binding experiments, cells were trypsinized and cultivated on collagen-coated coverslips until use. Primary tupaia hepatocyte cultures were preincubated 3 days after plating with different lipopeptides at the indicated conditions and then incubated with purified HBV at concentrations of 2000 genome equivalents per cell in THM for 15 hours at 37°C. Cells were extensively washed and further cultivated in THM. Medium was changed every 3 days, and supernatant from days 9–12 was measured for the appearance of secreted HBeAg. All experiments were performed at least in 3 independent series, and the results of 1 representative experiment are shown in each case. HBeAg was determined quantitatively by a commercially available enzyme-linked immunosorbent assay (AxSym; Abbott Laboratories, Delkenheim, Germany) that reacts specifically with HBeAg but not with HBV core particles. Results were obtained as a signal-cutoff ratio and were converted to a percentage of the noninhibited control. Primary tupaia hepatocytes (1–5 × 103) were plated on collagen-coated coverslips in THM as described previously. Purified subviral particles from plasma containing all 3 HBs proteins or yeast-derived particles containing either only SHBs or the preS1 domains 2–48/79–108 fused to the SHBs protein were diluted in THM to yield a final concentration of 2 μg/mL. Primary tupaia hepatocytes were incubated for the times and temperatures indicated and washed several times with ice-cold THM. Cells were fixed with 3% paraformaldehyde for 0.5 hours at 4°C and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 0.5 hours at room temperature. HBsAg staining was performed with the APAAP staining kit (Dako, Hamburg, Germany) as previously described10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar by using monoclonal anti-HBs (Novocastra, Newcastle, UK). For inhibition of binding, cells were preincubated with 1 μmol/L myristoylated HBV preS1 peptides 2–48 for 1 hour at 37°C. Peptides were removed, and plasma-derived HBV subviral particles were incubated with the cells for the indicated times and concentrations and washed several times thereafter with ice-cold THM. Fixation, permeabilization, and HBsAg staining of the cells was performed as previously described. Primary tupaia hepatocytes and indicated cell lines cultivated on collagen-coated coverslips were incubated with 5 μmol/L of myristoylated HBV preS1 peptides 2–48 for 2 hours at 37°C in THM. Peptides were removed, and cells were washed several times thereafter with ice-cold THM. Cells were immediately fixed and permeabilized as previously described. Detection of HBV preS1 peptides was performed with an APAAP staining kit (Dako) as previously described by using an antiserum from rabbits (H863) that recognizes the preS1 and preS2 domain. For HBV infection of tupaia hepatocytes, we used highly purified plasma-derived HBV particles that contained less than 10% subviral HBsAg particles. We determined HBeAg secretion of infected cells as a marker of an established infection, because HBeAg is a nonstructural viral protein and therefore not present in the purified viral input. The amount of secreted HBeAg is approximately proportional to the amount of infectious HBV in the inoculum.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar We have shown previously that infection of primary tupaia hepatocytes can be inhibited by certain MAbs against the preS1 and S-domain.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar These findings lead to the question of whether preincubation of the cells with preS1 peptides alone could block infection of the cells by HBV. Preincubation of tupaia hepatocytes with a synthetic peptide comprising AA 2–48 of preS1 (genotype D) was sufficient to reduce HBV infection significantly (Figure 2). However, for complete inhibition of infection, large amounts of peptides were necessary (>10 μmol/L; data not shown). Linking a myristic acid to the peptides at the N-terminus (Gly-2) increased the inhibiting power of the peptide more than 1000-fold, because complete inhibition of infection could now be achieved by using 10 nmol/L of myristoylated preS1 peptide AA 2–48. Increasing the peptide length to AA 2–68 or 2–78 did not further increase the inhibitory potential of the peptide, but it resulted in a slightly but significantly reduced activity (Figure 2). Preincubation of the cells with an analogous myristoylated peptide from an avihepadnavirus (avi myr-preS 2–44 of heron HBV) did not inhibit HBV infection at all. This negative control showed the sequence specificity of HBV-derived preS1 peptide inhibitors and ruled out nonspecific effects of the myristic side chain on the susceptibility of tupaia hepatocytes for HBV. No cytotoxicity was induced by HBV infection or incubation with specific peptides even at the highest concentrations used, as determined by MTT assay, the propidium iodide exclusion test, and measurement of released LDH in the supernatant (data not shown). To analyze the role of the acyl side chain of the myristoylated HBV preS1 peptides during HBV infection interference, we substituted the myristoyl side chain with other hydrophobic moieties. First, we synthesized HBV preS1 peptides 2–48 with variable acyl chain lengths (Figure 3), resulting in palmitoyl (C16), myristoyl (C14), octanoyl (C8), and pentanoyl (C5) preS1 peptides 2–48. As shown by HBeAg secretion 12 days after infection, infection interference by the preS1 peptides decreased while the acyl side chain length of peptides 2–48 was shortened. Exchanging the myristoyl moiety of the peptide with a cholesteryl moiety resulted in strongly increased inhibition of HBV infection (Figure 3). To narrow down the HBV preS1 sequences required for inhibition, we used stepwise carboxy-terminally truncated myristoylated peptides containing AA 2–8, 2–18, 2–28, 2–39, and 2–48. Whereas peptide 2–48 was the most effective inhibitor (50% inhibitory concentration [IC50] 1000 nmol/L), whereas the addition of 10 AAs (AA 2–18) increased the inhibitory activity drastically (IC50 of approximately 30 nmol/L). It is interesting to note that the further addition of 10 AAs did not improve the inhibitory activity significantly but resulted in a slightly reduced activity (IC50 of approximately 40 nmol/L). However, increasing the peptide length further (peptides 2–39) resulted in strongly increased inhibition (IC50 of approximately 3 nmol/L). Peptides 2–48 showed the strongest inhibitory potential (IC50 >1000 nmol/L). In summary, HBV preS1 sequence 2–18 (genotype D) seems to contain the essential residues for inhibition, and sequence 29–48 is only an accessory. It is interesting to note that MAb MA18/7, which binds to HBV preS1 sequence 20–23,28Sominskaya I. Pushko P. Dreilina D. Kozlovskaya T. Pumpen P. Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes.Med Microbiol Immunol. 1992; 181: 215-226Crossref PubMed Scopus (55) Google Scholar neutralizes HBV infection of tupaia hepatocytes.10Glebe D. Aliakbari M. Krass P. Knoop E.V. Valerius K.P. Gerlich W.H. Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (158) Google Scholar However, introducing internal deletions at AA 20–21, 20–23, and 23–27 of myristoylated preS1-peptides 2–48 showed no significantly reduced inhibitory potential compared with the wild-type peptide (Figure 5). The inhibitory potential of HBV preS1 deletion mutant 20–23 is comparable to that of wild-type Woolly monkey preS1 peptide 2–48 (Figure 5, right), which has additional AA changes in its homologous sequence 23–27 compared with HBV preS1 (Figure 4C). We wanted to distinguish whether the inhibition by the peptides was due more to the blocking of receptors by preincubation of peptide or to competition caused by the simultaneous presence of peptide and virus. Therefore, we preincubated the cells with peptide, washed it away after 30 minutes, and thereafter added virus at 0, 2, and 4 hours (Figure 6A, panel I). At a peptide concentration of 100 nmol/L, inhibition of infection was still complete even when the virus was added after 4 hours. With a concentration of 1 nmol/L, susceptibility was almost completely reconstituted within 2 hours after removal of the peptide, whereas this concentration was sufficient to block infection completely when left in the culture medium (panel I). In fact, preincubation is not necessary, as shown in Figure 6A (panel II). Surprisingly, myristoylated preS1 peptides still showed an inhibitory effect on HBV infection when added after the viral inoculum. Increasing the time between the addition of virus and peptide reduced the inhibitory potential of the peptide step by step (panel III). Nevertheless, even 4 hours after the addition of the peptide, 50%–60% inhibition was achieved by the preS1 peptides (Figure 6A, panel III). As a control, we determined the effect of removal of the viral inoculum without peptides after different time points (Figure 6B). Maximum infectivity was achieved when virus was incubated for 15 hours at 37°C with the cells, whereas 30% or 50% infectivity was reached after 2 and 4 hours, respectively. Adding the peptides without removing the viral inoculum at these time points had the same effect (Figure 6A, panel III). These experiments showed that the myristoylated peptide 2–48 interacts with a cell-associated factor of hepatocytes that is necessary for infection, most probably a cell-surface receptor for HBV. For direct visualization of the binding activity of preS1 peptides to cellular membranes, we incubated various primary cells or cell lines with either nonmyristoylated or myristoylated HBV preS1 peptide 2–48 for 2 hours at 37°C and showed binding and uptake by immune cytochemistry (Figure 7). When primary tupaia hepatocytes were incubated with the nonmyristoylated peptide, only a few cells showed signs of bound peptide (Figure 7A). When the myristoylated peptide was used at the same concentrations, up to 25% of the cells were able to bind the peptide (Figure 7B). No signal was detected without peptide (Figure 7C) or primary rat hepatocytes (data not shown). Incubation of the myristoylated peptide with tupaia nonparenchymal liver cells showed no staining, thus indicating that the peptide binds specifically to primar
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