An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.     

Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus

The susceptibility of the tree shrew Tupaia belangeri to human hepatitis B virus (HBV) has been demonstrated both in vivo and in vitro. In this study, we show that purified HBV infects primary T. belangeri hepatocyte cultures in a very specific manner, as detected by HBV covalently closed circular DNA, mRNA, HBV e antigen, and HBsAg production. A monoclonal antibody (MAb), MA18/7, directed against the pre-S1 domain of the large HBs protein, which has been shown to neutralize infectivity of HBV for primary human hepatocytes, also blocked infection of primary Tupaia hepatocytes. MAbs against the pre-S2 domain of HBs inhibited infection only partially, whereas an S MAb and polyvalent anti-HBs antibodies neutralized infection completely. Thus, both pre-S1 and S antigens are necessary for infection in the tupaia. Using subviral particles, >70% of primary Tupaia hepatocytes are capable of specific binding of pre-S1-rich HBsAg, showing localization in distinct membrane areas. The data show that the early steps of HBV infection in Tupaia hepatocyte cultures are comparable to those in the human system.     

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.     

Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain.

Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes.     

Emergence of and takeover by hepatitis B virus (HBV) with rearrangements in the pre-S/S and pre-C/C genes during chronic HBV infection.

We have shown, by analyzing serial serum samples from a chronic hepatitis B virus (HBV) carrier, the emergence of HBV DNA molecules with nucleotide rearrangements in the pre-S/S and pre-C/C genes. Serum samples were obtained at four different times (1983, 1985, 1988, and 1989) from an HBsAg- and HBeAg-positive carrier with chronic hepatitis. The polymerase chain reaction was used to amplify the pre-S/S and pre-C/C genes. The amplified products were cloned, and 8 to 10 independent clones were sequenced. In 1983 and 1985 only one type of HBV DNA molecule was observed. Nucleotide divergence relative to the adw2 subtype was 4.7, 7.2, and 1.6%, for the pre-S1, pre-S2, and S regions, respectively, and 2.2 and 3.9% for the pre-C and C regions, respectively. In 1988 and 1989, HBV DNA forms with marked rearrangements of both the pre-S/S and pre-C/C regions were evidenced. In the pre-S/S region, they comprised two distinct HBV DNA molecules. The first showed nucleotide divergence of 20.4, 14.8, and 3.3% for the pre-S1, pre-S2, and S regions when compared with the adw2 sequence. In addition, nucleotide deletions in the pre-S1 region led to the appearance of a stop codon. The second was created by recombination between the original and mutated HBV DNA. In the pre-C/C region, the mutated viral DNA showed 11.7% divergence when compared with the adw2 sequence. A point mutation led to the creation of a stop codon in the pre-C region, together with an insertion of 36 nucleic acids in the core gene. Most of this DNA insertion was identical to that reported in an independent HBV isolate but showed no significant homology with known sequences. Semiquantitative estimation of the proportion of wild-type and mutated HBV DNA molecules showed a marked increase in the mutated forms during the period of follow-up. Sucrose gradient analysis indicated that the defective HBV DNA molecules were present in circulating virions. Western immunoblot analysis showed the appearance of modified translation products. Our findings thus indicate the emergence of and gradual takeover by mutated HBV DNA forms during the HBV chronic carrier state. The rearrangements we observed in the pre-S/S and pre-C/C genes might lead to changes in the immunogenicity of the viral particles and thus affect the clearance of the virus by the immune system.     

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.     

Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis

The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.     

Protection of tree shrews by pVAX-PS DNA vaccine against HBV infection

The immunological protection of pVAX-PS, a DNA vaccine, was assessed in the tree shrews model. pVAX-PS was constructed by inserting the gene encoding the middle (pre-S2 plus S) envelope protein of HBV into a plasmid vector pVAX1. Tree shrews (Tupaia belangeri chinenesis) were experimentally infected with human HBV by inoculation with human serum positive for HBV markers. DNA vaccination-induced seroconversion and antibody to HBV surface antigen (anti-HBs) were analyzed by ELISA, and protective effects elicited by pVAX-PS vaccination against subsequent HBV challenge were evaluated by detection of HBV seromarkers and observation of hepatic lesions in HBV-infected tree shrews. The results shown that anti-HBs were detectable in serum at week 2 after pVAX-PS vaccination and peaked at week 4, and immunization with pVAX-PS decreased the positive conversion rate of HBV seromarkers and relieved hepatic lesions in tree shrews challenged with HBV. These results indicated that pVAX-PS immunization could induce remarkable humoral immune response and prevent the experimental tree shrews from infection of HBV.     

The influence of glycosylation on secretion,stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.     

应用系统发育树分析DDBJ基因库中HBV基因序列的基因型

为了充分利用核酸库中的HBV序列信息,探讨DDBJ核酸库中HBV基因序列的基因分型,采用Clustal X（1.8）软件比较HBV基因序列前S区序列差异并产生系统发育树。通过对下载的1471条HBV基因序列进行系统分析。获得了228条前S/S区完整的HBV基因序列,其中有66条序列的基因型已被各种方法所证实。利用软件分析绘制了基于228条HBV前S区基因序列的系统发育树。66条已知基因型HBV基因序列在系统发育树上的分型与其原有基因型完全吻合。在228条HBV基因序列中,有207条序列分别属于A、B、C、D、E、F和G等7个基因型,但另外21条序列不能归属于上述7个基因型的任何一种,而且它们又分为彼此相互独立存在的两群,暂分别称之为未分型I和未分型Ⅱ,经比较未分型I、Ⅱ和其他7个基因型前S区核苷酸序列,发现未分型I、Ⅱ和D型前S区都有33个核苷酸缺失,但三者基因缺失片段的位置和形式各不相同,但其它六型前S区无大片段基因缺失。结果说明采用基于前S区的系统发育树基因分型分析方法正确可靠,除了现已证实的7个基因型外,尚可能存在另外两个新的HBV基因型。     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein

The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Beta.     

Association of Hepatitis B Virus Pre-S Deletions with the Development of Hepatocellular Carcinoma in Qidong,China

Background/Aim

To investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China.

Methods

We conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC.

Results

After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5′ end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063–5.573) and 3.065 (1.099–8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease.

Conclusion

Pre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.     

Immunoadhesins containing pre-S domains of hepatitis B virus large envelope protein are secreted and inhibit virus infection

Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S. The pre-S1 region has important functions in the HBV life cycle. Thus, as an approach to studying these roles, the pre-S1 and/or pre-S2 sequences of HBV (serotype adw2, genotype A) were expressed as N-terminal fusions to the Fc domain of a rabbit immunoglobulin G chain. Such proteins, known as immunoadhesins (IA), were highly expressed following transfection of cultured cells and, when the pre-S1 region was present, >80% were secreted. The IA were myristoylated at a glycine penultimate to the N terminus, although mutation studies showed that this modification was not needed for secretion. As few as 30 amino acids from the N terminus of pre-S1 were both necessary and sufficient to drive secretion of IA. Even expression of pre-S1 plus pre-S2, in the absence of an immunoglobulin chain, led to efficient secretion. Overall, these studies demonstrate an unexpected ability of the N terminus of pre-S1 to promote protein secretion. In addition, some of these secreted IA, at nanomolar concentrations, inhibited infection of primary human hepatocytes either by hepatitis delta virus (HDV), a subviral agent that uses HBV envelope proteins, or HBV. These IA have potential to be part of antiviral therapies against chronic HDV and HBV, and may help understand the attachment and entry mechanisms used by these important human pathogens.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression,Telomere Length,Senescence, Inflammation and Fibrosis

BackgroundChronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase.MethodsLiver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro.Results13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to
in vitro induction of cellular senescence, which had no effect.ConclusionChronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.