Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues

The N-terminal pre-S domain of the large hepatitis B virus (HBV) envelope protein plays a pivotal role at the initial step of the viral entry pathway. In the present study, the entire pre-S domain was mapped for infectivity determinants, following a reverse-genetics approach and using in vitro infection assays with hepatitis delta virus (HDV) or HBV particles. The results demonstrate that lesions created within the N-terminal 75 amino acids of the pre-S region abrogate infectivity, whereas mutations between amino acids 76 and 113, overlapping the matrix domain, had no effect. In contrast to the results of a recent study (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. 103:6730-6734, 2006), the deletion of a cell membrane translocation motif (TLM) located between amino acids 148 and 161 at the C terminus of pre-S2 did not interfere with the infectivity of the resulting HDV or HBV mutants. Furthermore, a series of large deletions overlapping the pre-S2 domain were compatible with infectivity, although the efficiency of infection was reduced when the deletions extended to the pre-S1 domain. Overall, the results demonstrate that the activity of the pre-S domain at viral entry solely depends on the integrity of its first 75 amino acids and thus excludes any function of the matrix domain or TLM.     

Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection

The presence of the two "large" surface proteins of hepatitis B virus (HBV), P39 and GP42 of pre-S1-hepatitis B surface antigen, was assayed in the serum of an experimentally infected chimpanzee by using antibodies to a pre-S1-specific fusion protein synthesized in Escherichia coli. The immune response to pre-S1-hepatitis B surface antigen was monitored by using the pre-S1 fusion protein as an antigen. pre-S1 proteins were detected in the serum early in the course of infection and prevailed as long as hepatitis B surface antigen did, together with hepatitis B e antigen and viral DNA. Thus, the pre-S1 antigen can be considered a novel diagnostic marker for acute HBV infection. Antibodies to pre-S1, both immunoglobulin M and G classes, were also detected early in infection, shortly after the appearance of the pre-S1 antigen, suggesting its strong immunogenicity in vivo. The anti-pre-S1 antibodies therefore also represent an early serological marker for acute HBV infection and, owing to their early appearance and persistence, may play a role in the neutralization of the virus.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor.     

Quantitative analysis of the interaction between the envelope protein domains and the core protein of human hepatitis B virus

Interaction between preformed nucleocapsids and viral envelope proteins is critical for the assembly of virus particles in infected cells. The pre-S1 and pre-S2 and cytosolic regions of the human hepatitis B virus envelope protein had been implicated in the interaction with the core protein of nucleocapsids. The binding affinities of specific subdomains of the envelope protein to the core protein were quantitatively measured by both ELISA and BIAcore assay. While a marginal binding was detected with the pre-S1 or pre-S2, the core protein showed high affinities to pre-S with apparent dissociation constants (K(D)(app)) of 7.3+/-0.9 and 8.2+/-0.4microM by ELISA and BIAcore assay, respectively. The circular dichroism analysis suggested that conformational change occurs in pre-S through interaction with core protein. These results substantiate the importance of specific envelope domains in virion assembly, and demonstrate that the interaction between viral proteins can be quantitatively measured in vitro.     

Topology of the large envelope protein of duck hepatitis B virus suggests a mechanism for membrane translocation during particle morphogenesis.

We have investigated the membrane topology of the large envelope protein of duck hepatitis B virus (DHBV) by protease protection and Western blot analysis, using monoclonal antibodies specific for the pre-S and S regions of the DHBV envelope to characterize protease-resistant polypeptides. These studies showed that DHBV L protein exhibits a mixed membrane topology similar to that of human hepatitis B virus L, with approximately half of the L molecules displaying pre-S on the surface of virus particles and the remainder with pre-S sequestered inside the virus envelope. The C-terminal region of DHBV pre-S was susceptible to protease digestion on all DHBV particle L protein, indicating that this region was externally disposed. DHBV L protein pre-S was entirely cytosolic immediately after synthesis. Our data, therefore, suggested that an intermediate form of the DHBV L molecule exists in mature envelope particles in which L is partially translocated or exists in a translocation-ready conformation. Incubation of virus particles at low pH and 37 degrees C triggered conversion of this intermediate into a fully translocated form. We have proposed a model for pre-S translocation based on our results that invokes the presence of an aqueous pore in the virus envelope, most likely created by oligomerization of transmembrane domains in the S region. The model predicts that pre-S is transported through this pore and that a loop structure is formed because the N terminus remains anchored to the inner face of the membrane. This translocation process occurs during particle morphogenesis and may also be a prerequisite to virus uncoating during infection.     

Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B

Hep 3B, a human hepatoma cell line was examined for its RNA hybridizable to the hepatitis B virus sequence. Using probes that covered different regions of the hepatitis B virus genome, five species of RNA were observed of sizes 4.0, 3.3, 2.9, 2.6 and 2.2 kilobases. The RNAs covered surface antigen gene, pre-S and X regions. None of them had a core antigen sequence. RNA with a 4.0 kilobase size was the most abundant. Using S1 nuclease analysis, its 5' end of hepatitis B virus sequence was mapped at pre-S region and its 3' end of viral sequence was mapped at DR region.     

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.     

Hepatitis B viral factors and clinical outcomes of chronic hepatitis B

Hepatitis B virus (HBV) infection is an important health problem and the major cause of chronic hepatitis, cirrhosis as well as hepatocellular carcinoma (HCC) worldwide. The natural history of chronic HBV infection can be divided into 4 dynamic phases in HBV carriers who acquire the virus early in life. In general, the frequency and severity of hepatitis flares in the immune clearance or reactivation phase predict disease progression in HBV carriers, and early HBeAg seroconversion typically confers a favorable outcome. In contrast, late or absent HBeAg seroconversion after multiple hepatitis flares accelerates the progression of chronic hepatitis to cirrhosis. Recently, several hepatitis B viral factors predictive of clinical outcomes have been identified. For example, serum HBV DNA level at enrollment is the best predictor of adverse outcomes (cirrhosis, HCC and death from liver disease) in adults with chronic HBV infection. In addition, HBV genotype C, basal core promoter (BCP) mutant and pre-S deletion mutant are associated with increased risk of HCC development. In conclusion, hepatitis B viral factors such as serum HBV DNA level, genotype and mutants have already been clarified to influence disease progression of chronic hepatitis B. Further studies are needed to investigate the pathogenic mechanism of each viral factor.     

Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity

Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.     

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.     

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.