• YAKHAK HOEJI
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• v.45 no.6
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• pp.708-714
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• 2001

Many studies have provided evidences that hepatitis B surface antigen (HBsAg) including preS region could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. We established CHO cell lines, IY-CHO-2 and IY-CHO-11 expressing high levels of HBsAg containing preS2 and S protein by stable transfection method. These cell lines expressed the correct size (about 1 kb in length) of HBsAg mRNA as expected. The purified protein from the culture supernatants of the clones showed the same sizes as those expressed in native hepatitis B virus (24 kDa, 27 kDa, 34 kDa and 36 kDa). Antibody productivity of CHO-derived HBsAg protein at lower dose challenge was higher than the protein containing S region alone expressed in yeast system. These results indicate that CHO-derived HBsAg protein containing preS2 and S region can be effectively used for a better immune response as a HBV vaccine.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• BMB Reports
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• v.29 no.2
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• pp.180-182
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• 1996

One of the essential functions of virus surface proteins is the recognition of specific receptors on target cell membranes, and cellular receptors play an important role in viral pathogenesis. But the earliest steps of hepatitis B virus (HBV) infection, such as hepatocyte receptor interaction with the virus, are poorly understood. Previous work has suggested an important role of the preS1 region of HBV envelope protein in mediating viral binding to hepatocytes. Although hepatitis B virus (HBV) infection appears to be initiated by specific binding of virions to cell membrane structures via one or potentially several viral surface proteins, data showing the identification or isolation of the HBV receptor (s) are not yet available. The receptor-like proteins on the plasma membrane surface of HepG2 cells that bind to PreS1 were separated and identified using affinity chromatography, and the amino-terminal amino acid sequences of the receptor-like proteins were determined.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.11a
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• pp.39-40
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• 1993

Hepatitis B virus (HBV)is a member of the Hepadna virus family whose members share a characteristic virion structure and genome size, around 3.2kb in a paritially double-stranded form. The genome of HBV contains four overlapping open reading frames designated as P(polymerase). C(core), S(surface antigen)and X. The X gene has potential to encode 154 amino acids protein.

• Bulletin of the Korean Chemical Society
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• v.28 no.10
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• pp.1756-1762
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• 2007

Envelope proteins of virus contain a segment of hydrophobic amino acids, called as fusion peptide, which triggers membrane fusion by insertion into membrane and perturbation of lipid bilayer structure. Potential fusion peptide sequences have been identified in the middle of L or M proteins or at the N-terminus of S protein in the envelope of human hepatitis B virus (HBV). Two 16-mer peptides representing the N-terminal fusion peptide of the S protein and the internal fusion peptide in L protein were synthesized, and their membrane disrupting activities were characterized. The internal fusion peptide in L protein showed higher activity of liposome leakage and hemolysis of human red blood cells than the N-terminal fusion peptide of S protein. Also, the membrane disrupting activity of the extracellular domain of L protein significantly increased when the internal fusion peptide region was exposed to N-terminus by the treatment of V8 protease. These results indicate that the internal fusion peptide region of L protein could activate membrane fusion when it is exposed by proteolysis.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.844-850
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• 2000

The specific binding and internalization of viral particles is an essential step for the successful infection of viral pathogens. In the case of the hepatitis B virus (HBV), virions bind to the host cell via the preS domain of the viral surface antigen and are subsequently internalized by endocytosis. HBV-preS specific receptors are primarily expressed on hepatocytes, however, viral DNA and proteins have also been detected in extrahepatic sites, suggsting that celluar recepators for HBV may also exist on extrahepatic cells. Recently, an EBV-transformed B-cell line was identified onto which the preS region binds in a receptor-ligand specific manner. In this study, this specific interaction was further characterized, and the binding region within the preS protein was locaized. Also the internalization after host cell attachment was visualized and analyzed by fluorescence-labeled HBV-preS1 proteins using confocal microscopy. Energy depletion by sodium azide treatment effectively inhibited the internalization of the membrane-bound preS1 ligands, thereby indicating an energy-dependent receptor-mediated endocytotic pathway. Accordingly, the interaction of HBV-pres! with this specific B-cell line may serve as an effective model for an infection pathway in extrahepatic cells.

• BMB Reports
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• v.38 no.6
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• BMB Reports
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• v.29 no.2
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• BMB Reports
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• v.41 no.9
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• pp.640-644
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• 2008

The preS1 surface protein of the hepatitis B virus (HBV) is a key factor involved in initial viral entry into hepatocytes. It has been long postulated that an anti-HBV effect should be achievable using peptide fragments of the preS1. Recent reports demonstrated that several preS1-derived lipo-peptides in genotype D HBV exhibit nano to picomolar inhibitory activity against HBV infection. In this study, an acylated analog of a preS1 fragment, a 21-residue lipo-peptide (named 7524 BVS7) with a sequence of palmitoyl-GMGTNLSVPNPLGFFPDHQLDC-$NH_2$, from genotype C HBV was produced base upon a previous structural study and was shown potently inhibits HBV infection with an $IC_{50}$ of $\approx$ 20 nM.

• Molecules and Cells
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• v.28 no.1
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• pp.49-55
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• 2009

Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV viral proteins, the small hepatitis delta antigen (SHDAg) and the large hepatitis delta antigen (LHDAg), on $NF-{\kappa}B$ signaling pathway. In this study, we demonstrated that $TNF-{\alpha}-induced$ $NF-{\kappa}B$ transcriptional activation was increased by LHDAg but not by SHDAg in both HEK293 and Huh7 cells. Furthermore, LHDAg promoted TRAF2-induced $NF-{\kappa}B$ activation. Using coimmunoprecipitation assays, we demonstrated that both SHDAg and LHDAg interacted with TRAF2 protein. We showed that isoprenylation of LHDAg was not required for the increase of $NF-{\kappa}B$ activity. We further showed that only LHDAg but not SHDAg increased the $TNF-{\alpha}-mediated$ nuclear translocation of p65. This was accomplished by activation of $I{\kappa}B_{\alpha}$ degradation by LHDAg. Finally, we demonstrated that LHDAg augmented the COX-2 expression level in Huh7 cells. These data suggest that LHDAg modulates $NF-{\kappa}B$ signaling pathway and may contribute to HDV pathogenesis.