pre-S Protein Research Articles

Pre-S Deletion and Complex Mutations of Hepatitis B Virus Related to Advanced Liver Disease in HBeAg-Negative Patients

This longitudinal study investigated the interactions and roles of hepatitis B virus (HBV) genotypes, pre-S deletions, and core promoter and precore mutations on the progression of liver disease in hepatitis B e antigen (HBeAg)-negative patients. A total of 141 HBeAg-negative patients without liver cirrhosis or hepatocellular carcinoma at study entry were recruited for this study, including 45 inactive HBV carriers and 96 patients with HBeAg-negative chronic hepatitis B. The HBV genotypes and the sequences of pre-S, core promoter, and precore regions were determined. Compared with patients without developing liver cirrhosis, patients with the development of liver cirrhosis had higher rates of genotype C; pre-S deletions; C or G1753, T1762/A1764, T1766, and/or A1768 mutants; and G1799 variant. Cox regression analysis showed that older age, higher total bilirubin and HBV DNA levels, pre-S deletions, and T1766 and/or A1768 mutants were significantly associated with the development of liver cirrhosis. HBV with a complex mutation pattern (pre-S deletion, T1762/A1764, and T1766 and/or A1768 mutants) rather than a single mutation was associated with the development of liver cirrhosis, and the patterns of mutation combinations differed between HBV genotype B and C. Moreover, pre-S deletion was a significant risk factor for hepatocellular carcinoma. This study indicated that pre-S deletion and combined mutations of HBV are useful molecular markers for predicting the clinical outcomes of HBeAg-negative patients.

Preferential Gene Transfer of Lentiviral Vectors to Liver-Derived Cells, Using a Hepatitis B Peptide Displayed on GP64

One of the problems that limit the efficiency of viral gene therapy is the lack of specificity of viral particle binding. The development of techniques to target viral particles to specific cell types is therefore important. Because GP64 can efficiently pseudotype lentiviral vectors, we investigated the possibility of using GP64 for lentiviral vector particle targeting. A peptide derived from the hepatitis B virus (HBV) PreS1 protein, with known affinity for an unidentified receptor expressed on hepatocytes, was inserted at amino acid position 278 of the GP64 protein (PreS1-GP64). The GP64 and PreS1-GP64 proteins were expressed and incorporated into lentiviral particles at comparable levels. Flow cytometry measurements confirmed surface display of the PreS1 peptide. The highest titers of PreS1-GP64-pseudotyped lentiviral vectors were observed on liver-derived cell lines. Gene transfer of PreS1-GP64 lentiviral vectors was inhibited by coincubation with an antibody directed against the PreS1 peptide. These data suggest that the PreS1 peptide is involved in viral attachment to the cell surface. The insertion of targeting peptides into the GP64 envelope protein represents a potential approach for the targeting of lentiviral vectors to specific cell types.

Screening and cloning for proteins transactivated by the PS1TP5 protein of hepatitis B virus: A suppression subtractive hybridization study

To clone and identify human genes transactivated by PS1TP5 by constructing a cDNA subtractive library with suppression subtractive hybridization (SSH) technique. SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-myc-his(A)-PS1TP5 and pcDNA3.1(-)-myc-his(A) empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa I, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification. The subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown. One novel gene with unknown functions was found and named as PS1TP5TP1 after being electronically spliced, and deposited in GenBank (accession number: DQ487761). PS1TP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PS1TP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.

Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327). Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

Optimisation of secretion of recombinant HBsAg virus-like particles: Impact on the development of HIV-1/HBV bivalent vaccines

The hepatitis B surface antigen (HBsAg) assembles into virus-like particles (VLPs) that can be used as carrier of immunogenic peptides for the development of bivalent vaccine candidates. It is shown here that by respecting certain qualitative features of mammalian preS1 and preS2 protein domains upstream of HBsAg, foreign sequences can be inserted in their place while maintaining efficient secretion of VLPs. A polyepitope bearing HIV-1 epitopes restricted to the HLA-A*0201 class I allele was optimised for secretion as an HBsAg fusion protein by counterbalancing the generally hydrophobic class I epitopes with hydrophilic spacers, eliminating epitopes bearing cysteine residues, limiting the number of internal methionine residues to a minimum and adopting Homo sapiens codon usage. The optimised HIV-1 polyepitope-HBsAg recombinant protein with up to 138 residues assembled into efficiently secreted recombinant VLPs. DNA immunisation in HLA-A*0201 and HLA-A*0201/HLA-DR1 transgenic mice resulted in the recovery of humoral response against the carrier and enhanced levels of HIV-1 specific CD8 + T lymphocyte activation. Efficient self-assembly of recombinant HBsAg VLPs opens up the possibility of making efficient bivalent HBV/HIV vaccine candidates, which is particularly apposite given that the two viruses are frequently associated.

Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon

Presentation of class I major histocompatibility complex (MHC) is severely down-regulated on hepatocytes in chronic hepatitis caused by woodchuck hepatitis virus (WHV). To determine which of the viral proteins mediates class I MHC antigen suppression, cultured normal woodchuck hepatocytes were transfected with the complete WHV genome, sequences encoding individual virus proteins, or whole virus genomes in which transcription of selected proteins was disabled by site-specific mutagenesis. It was found that hepatocyte presentation of class I MHC antigen was significantly inhibited following transfection with complete WHV genome or with viral subgenomic fragments encoding envelope pre-S2 protein or pre-S1 protein, which naturally encompasses pre-S2 amino acid sequence. In contrast, hepatocytes transfected with WHV X gene alone demonstrated a profound enhancement in the class I antigen display, whereas those expressing virus major S protein or nucleocapsid (core) protein were not different from control hepatocytes. Analysis of the mutated WHV sequences confirmed that the envelope pre-S2 protein was responsible for inhibition of the class I MHC antigen display. Interestingly, treatment with recombinant woodchuck gamma interferon (rwIFN-gamma) restored the inhibited presentation of the class I antigen. Moreover, the class I antigen suppression was not associated with down-regulation of hepatocyte genes for class I MHC heavy chain, beta(2)-microglobulin, transporters associated with antigen processing, and proteasome subunits. These findings indicate that the defective presentation of class I MHC antigen on hepatocytes transcribing WHV is a consequence of posttranscriptional suppression exerted by virus pre-S2 protein and that this hindrance can be fully reversed by IFN-gamma.

P.093 Demonstration of a firm association between detection of IgG antibody reactivity towards the C-terminal part of the pres1 protein of HBV and the capacity to respond to interferon therapy in chronic hepatitis B

P.093 Demonstration of a firm association between detection of IgG antibody reactivity towards the C-terminal part of the pres1 protein of HBV and the capacity to respond to interferon therapy in chronic hepatitis B

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1–119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli ( E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni–NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.

Expression of HBV S and preS1 fusion gene in Pichia pastoris expression system

To clone and express the ss1 recombinant gene containing S gene and preS1 (10-50 AA) gene in P. pastoris expression system. The fusion gene ss1 containing the S (1-222 AA) gene and preS1 (10-50 AA) gene was constructed with PCR method. The fusion ss1 gene was cloned into the expression vector of pPIC3.5k. The linear vector DNA was transformed into the host cell of GS115 with electroporation method. After screening with G418, the product was induced to express with methanol and its antigenicity was analyzed. The molecular weight of expressed ss1 protein was about 30,000 dalton. The product was reactive to anti-HBs and anti-preS1 mAb. The fusion gene was efficiently expressed in P. pastoris expression system.The expressed products have the antigenicity of both S and preS1 protein.

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.

NACA as a Potential Cellular Target of Hepatitis B Virus PreS1 Protein

The mechanisms of the attachment and penetration of hepatitis B virus remain obscure. It has been demonstrated that the preS1 region is essential for viral assembly and infectivity, however, as its cellular receptor has still not been identified unequivocally, we used a yeast two-hybrid system to screen the cellular proteins that can interact with preS1 protein. The protein recovered from a human liver cDNA library was nascent polypeptide-associated complex alpha polypeptide. The interaction between preS1 and nascent polypeptide-associated complex alpha polypeptide was verified by mating experiment and coimmunoprecipitation of COS7 cell lysates expressing both proteins. Based on these results, we speculate that nascent polypeptide-associated complex alpha polypeptide is a functional target of hepatitis B virus preS1 protein in cells.

Further characterization of glycine decarboxylase (P120), a pre-S envelope-interacting protein in duck hepatitis B virus life cycle: Ji S Li, Shuping Tong, Shigenobu Kawai, Hong B Lee, Jack R Wands, Brown Medical Sch, Providence, RI

Further characterization of glycine decarboxylase (P120), a pre-S envelope-interacting protein in duck hepatitis B virus life cycle: Ji S Li, Shuping Tong, Shigenobu Kawai, Hong B Lee, Jack R Wands, Brown Medical Sch, Providence, RI

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection. pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of beta-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis. This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

Occult hepatitis B virus infection in chronic liver disease: Full-length genome and analysis of mutant surface promoter

Genome sequence of hepatitis B virus (HBV) from occult chronic infection is scarce. Fifty-six (9.4%) of 591 patients seronegative for hepatitis B surface antigen (HBsAg) with chronic liver disease were positive for HBV DNA. The complete HBV genome from 9 of these patients (S1-S9) and 5 controls positive for HBsAg (SWT.1-SWT.5) were analyzed. Overlapping genome fragment amplification, cloning, and sequencing was performed on these cases. Functional analysis of surface promoter was conducted using fusion construct. All patients with occult infection except one (S8) had a low viral titer. Eight patients had infection with genotype A (S1-S5, SWT.1-2, SWT.5) and 6 had infection with genotype D (S6-S9, SWT.3-4). S4 and S5.1 of genotype A had the characteristic nucleotide deletions in core and pre-S1 region seen in genotype D. The major observations in patients with occult HBV infection were as follows: frequent quasispecies variation, deletions in pre-S2/S region affecting the surface promoters (nt 3025-54) and pre-S protein (S3, S5, S6, S8), truncated precore (S6, S8, S7.1) and core (S9) owing to stop signal, alternate start codon for the Polymerase gene (S3, S9), and YMDD mutation (S1, S4, S9) in patients not on antiviral therapy. HBsAg and core proteins could be shown immunohistochemically in 3 of 5 liver biopsy specimens available. The mutant surface promoters (pre-S2 and S) on functional analysis showed alterations in HBsAg expression. These changes in the regulatory region with possible alterations in the ratio of large and small surface proteins along with other mutations in the genome may decrease the circulating HBsAg level synergistically, making the immunodetection in serum negative.

A subtractive fluorescence-activated cell-sorting strategy to identify mimotopes of HBV–preS protein from bacterially displayed peptide library

A novel subtractive fluorescence-activated cell-sorting (FACS) strategy using a model system is described here to identify disease-specific (DS) epitopes from a bacterially displayed random peptide library. In this process, preimmune serum was used as “Driver ” to block any common binding sites on the bacterial surface and the labeled anti-preS IgG polyclonal antibodies from immunized serum were used as “Tester” to enrich preS-specific mimotopes. Bacterial clones were identified out of this pool through an “antigen-independent” procedure only using both different sera samples. After four rounds of sub-FACS screening, 41 out of 50 bacterial clones were identified as reacting with the immunized serum but not reacting with the pre-immune one. Two motif sequences HQLD and DPAF were obtained from 13 clones. Immunization of mice with two representative bacterial clones elicited a strong specific response against native preS antigen in comparison with the control. This technique may provide a useful technology platform for high-throughput screening of disease-related epitope which is of importance to develop vaccine against some infectious diseases whose pathogen or immunodominant antigen is still unknown.

Identification of a glycosylation site in the woodchuck hepatitis virus preS2 protein and its role in protein trafficking.

The middle surface antigen (M-sAg) of hepadnaviruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of woodchuck hepatitis virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3-5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with virus assembly.

Surface expression of squamous cell carcinoma antigen (SCCA) can be increased by the preS1(21-47) sequence of hepatitis B virus.

A variant of the serpin squamous cell carcinoma antigen (SCCA) has been identified as a hepatitis B virus binding protein and high expression of SCCA has recently been found in hepatocarcinoma. Since HBV is involved in liver carcinogenesis, experiments were carried out to examine the effect of HBV preS1 envelope protein on SCCA expression. Surface and intracellular staining for SCCA was assessed by FACS analysis. Preincubation of HepG2 cells and primary human hepatocytes with preS1 protein or with preS1(21-47) tetrameric peptide significantly increased the surface expression of SCCA, without modification of its overall cellular burden, suggesting a surface redistribution of the serpin. An increase in HBV binding and internalization was observed after pre-incubation of the cells with preS1 preparations, compared to cells preincubated with medium alone. Pretreatment of cells with DMSO, while not influencing SCCA basal expression, was responsible for an increase in the efficiency of HBV internalization and this effect was additive to that obtained after incubation with preS1 preparations. In conclusion, the HBV preS1(21-47) sequence is able to induce overexpression of SCCA at the cell surface facilitating virus internalization, while the increased efficiency of HBV entry following DMSO addition is not mediated by SCCA.

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection.

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.

Identification of mimotopes by screening of a bacterially displayed random peptide library and its use in eliciting an immune response to native HBV-preS

Bacterially-displayed peptide libraries have been widely used as an alternative to phage-displayed peptide libraries in screening epitopes or mimotopes of antibodies. Using a protective monoclonal antibody (mAb) 3B9 against hepatitis B virus (HBV) preS protein as target, mimotopes were successfully screened from a FliTrx™ random peptide library. To monitor the enrichment ratios of each round and to isolate higher affinity clones from the library, a modified procedure was performed in which the titer of eluted bacteria from an antibody-coated well ( P value) was compared with that from a non-coated well ( N value). After sufficient enrichment of the library, bacterial colonies were randomly picked and identified further by the monoclonal bacterial P/ N value assay and Western blotting analysis. Immunization of mice with the selected bacterially-displayed mimotopes, including the enriched populations without clone identification, elicited strong specific immune responses against the recombinant preS protein. The present study provides a potentially rapid and effective strategy for the development of engineered live bacterial vaccines without the need for information about the aetiological agents or their antigens.