Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor.

UNLABELLED: Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes. IMPORTANCE: Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.

Antihepatitis B virus activity of a protein-enriched fraction from housefly (Musca domestica) in a stable HBV-producing cell line.

Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

Suppression of furin by interferon-γ and the impact on hepatitis B virus antigen biosynthesis in human hepatocytes.

The roles of furin and intrahepatic cytokines in chronic heptatitis B virus (HBV) infection remain largely unknown. Here, we examined the relations between furin, IL-10, IL-12ß, interferon (IFN)-γ, programed death (PD)-1, programed death ligand (PD-L)1, and the suppression of hepatitis B e antigen (HBeAg) and surface antigen (HBsAg) biosynthesis. Liver biopsies were performed on 20 chronically HBV-infected (15 HBeAg-positive and 5 HBeAg-negative) patients to assess liver inflammation/fibrosis, and mRNA levels of furin, IL-10, IL-12ß, IFN-γ, PD-1, and PD-L1 were assessed by quantitative real-time PCR. IFN-γ mRNA abundance was associated with lower furin mRNA levels and higher PD-1 and PD-L1 mRNA levels in liver tissue from HBeAg-positive patients. IL-10 and IL-12ß mRNA levels positively correlated with IFN-γ expression levels (P < 0.05). PD-L1 and furin mRNA levels were further assessed in IFN-γ-stimulated hepatoma cell lines with (HepG2.2.15 cells) and without (HepG2 and Huh7 cells) HBV replication. IFN-γ enhanced PD-L1 expression in hepatoma cells. In HepG2.2.15 cells, IFN-γ further suppressed furin and HBeAg expression. Furin inhibition and knockdown in HepG2.2.15 cells also down-regulated HBeAg and HBsAg biosynthesis. These data suggest that IFN-γ modulates the inflammatory response to avoid excessive hepatocyte damage through the enhancement of PD-1/PD-L1 expression, whereas furin suppression may contribute to a reduction in HBeAg/HBsAg biosynthesis.

Effects of hepatitis B virus precore and basal core promoter mutations on the expression of viral antigens: genotype B vs C.

Hepatitis B virus (HBV) genotypes/mutants are known to affect natural outcomes. The virologic differences among HBV genotype, precore and basal core promoter (BCP) mutations were investigated. HBV strains were isolated from 18 hepatitis B e antigen (HBeAg)-positive patients (nine genotype B and nine genotype C). All had precore and BCP wild-type sequences. After cloning of full-length HBV genome, the effects of viral genotype, precore and BCP mutations singly or additively on the expression of viral DNA and antigens were investigated by mutagenesis and transfection assays in Huh7 cells. Significant findings included the following: (i) expression of intracellular core protein increased when precore or BCP mutation was introduced in genotype C strains; (ii) expression of intracellular surface protein was lower in genotype C precore wild-type strain compared with genotype B; (iii) precore mutation was associated with a lower extracellular expression level of HBV DNA; (iv) secretion of hepatitis B surface antigen in genotype C was lower than that in genotype B; and (v) secretion of HBeAg in genotype B was lower than that in genotype C. No additive effect was observed by combining precore and BCP mutations. Hence, HBV genotype and precore/BCP mutations correlate with intrahepatic expression of viral antigens in vitro.

Increased in vivo immunological potency of HB-110, a novel therapeutic HBV DNA vaccine, by electroporation.

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.

The hepatitis B virus X gene potentiates c-myc-induced liver oncogenesis in transgenic mice.

The hepatitis B virus X protein (HBx) is thought to be implicated in the development of hepatocellular carcinoma, but its exact function remains controversial. Transgenic mice from PEX7 and AX16 lineages that express HBx in the liver under control of different viral regulatory elements develop no liver pathology (Billet et al., 1995). We have crossed these two mouse lineages with WHV/c-myc oncomice in which liver-specific expression of c-myc driven by woodchuck hepatitis virus (WHV) regulatory sequences causes liver cancer in all animals. The average tumor latency was shortened by 2 to 3 months in bitransgenic animals from all populations compared with simple c-myc transgenic littermates. At preneoplastic stages, adult bitransgenic mice showed four to fivefold enhanced expression of the c-myc transgene, increased hepatocyte proliferation and more extensive liver lesions compared with simple WHV/c-myc transgenics. Thus in this model, HBx alone has no direct pathological effect but it is shown to accelerate tumor development induced by c-myc. The data presented here firmly establish the oncogenic potential of HBx, apparently acting as a tumor promoter. This model offers unique opportunities to investigate the mechanisms by which HBx trans-activates the expression of target genes and deregulates the hepatocyte growth control in vivo.

A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein.

A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.

Rapid isolation of highly productive recombinant Chinese hamster ovary cell lines.

An expression system combining a unit for the expression of the gene of interest reinforced by the hepatitis B virus X transactivator and a selectable gene weakened by the insertion of an A+T-rich sequence derived from the 3'-untranslated region of the granulocyte-macrophage colony-stimulating factor mRNA is described. This vector allows rapid one-step isolation of highly productive Chinese hamster ovary clones.

Changing of hepatitis B virus markers in patients with bone marrow transplantation.

The hepatitis B virus (HBV) infection and its resulting hepatic abnormalities are very high in prevalence among the Taiwan population. They also seem to compose a major problem to patients subjected to bone marrow transplantation (BMT) due to intensive chemoradiotherapy. In this study, the sera of 42 patients were investigated before and after BMT to detect the presence of HBV markers and to test their liver function (LF). Being followed-up for 3-12 months after BMT, 12 out of 27 were found to have altered HBV markers according to the classification of the following: seroconversion of HBsAg, clearance of HBsAb, appearance of HBeAg, clearance of HBeAb, and acute hepatitis. Thirty-seven out of 42 patients (88.1%) were found in routine LF test to develop one or more abnormality; however, 90% of them turned normal within one year after BMT. Only one patient died of complications associated with fulminant hepatitis. In conclusion, the previous hepatic damage from HBV infection appears unlikely to increase the risk of posttransplant morbidity and mortality.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.

Antisense oligonucleotides are effective inhibitors of hepatitis B virus replication in vitro.

Antisense oligonucleotides are currently being used in numerous laboratories as potential anticancer and antiviral agents. The unique replication cycle of hepatitis B virus (HBV) contains several different steps which are potentially amenable to modulation by these molecules. We have examined the ability of 56 different single-stranded, oligodeoxyribonucleotides (14-23 nucleotides in length), which target several HBV-specific functions, to inhibit HBV replication in the human hepatoblastoma cell line, 2.2.15. None of the oligonucleotides examined were toxic at concentrations up to 500 microM. Oligonucleotides directed against the HBV surface antigen (HBsAg) gene (S gene), the preS1 open reading frame, and the HBV core antigen (HBcAg) gene (C gene) were effective at depressing virus production, while molecules targeting the HBV e antigen (HBeAg) open reading frame and the HBV polymerase (POL) gene were ineffective. Oligonucleotides directed against the HBV encapsidation signal/structure (epsilon) comprised some of the most effective antiviral molecules against HBV. None of 5 oligonucleotides complementary (i.e., 'sense' orientation) to the antisense oligonucleotides targeting HBsAg, HBcAg, HBeAg, preS1 and POL had any measurable effect on HBV production. The relative effectiveness of oligonucleotides targeting the S and C genes on HBV replication was highly correlated with an effect on HBsAg or HBcAg levels, respectively. None of the antisense oligonucleotides examined affected either the levels or the sizes of HBV-specific RNA transcripts. Since antisense oligonucleotides can exert biologic effects on HBV in 2.2.15 cell cultures in a sequence-specific manner which are consistent with predicted modes of action, such molecules may have practical applications in the therapy of chronic HBV infection.

Hepatitis B virus core-preS2 particles expressed by recombinant vaccinia virus.

Vaccinia virus (VV) recombinants expressing hepatitis B virus (HBV) surface (HBsAg) or core (HBcAg) antigens (Kunke et al., Virology 195, 132 - 139 (1993)] have been shown to raise specific antibodies in mice, nevertheless the levels of antibodies reactive with the preS2 and S antigens were low. In an attempt to enhance the immunogenicity of HBsAg-preS2, a fused C-preS2 gene was constructed. The fusion protein was expressed in E. coli and displayed both HBcAg and preS2 antigen as demonstrated by enzyme-linked immunosorbent assay (ELISA). The same gene was then expressed using recombinant VV and chimerical particles whose size and density were similar to those of native HBV core particles produced in CV-1 cells infected with recombinant VV. Unlike HBcAg, preS2 antigen could not be detected on these particles by ELISA but was revealed by immunoblot analysis only. The immunogenicity of the recombinant VV was evaluated in mice. Antibodies to HBcAg and VV antigen but not to preS2 antigen were found in sera of animals inoculated with 10(7) PFU of the recombinant VV. Presumably, HBcAg-preS2 particles produced in E. coli and in eukaryotic cells have a different conformation, and the presence of preS2 antigen on the surface of chimerical particle might be necessary for a pronounced antibody response.

[Inhibitory effect of phosphorothioae oligodeoxynucleotides on HBV replication and synthesis of antigen in vitro].

OBJECTIVE: To evaluate the effect of triplex forming oligodeoxynucleotides (TFO) and antisense oligodeoxynucleotides (ODNas) on the replication of HBV. METHODS: A 21mer phosphorothioate TFO (TFO21) directed at 1734nt-1754nt sites in HBV core promoter and a 21 mer phosphorothioate ODNas (ODNas21) complementory to the initiation sites of pre C RNA and pregenomic RNA were synthesized. Effect of TFO21 and ODNas21 on HBV replication and synthesis of antigen were observed in 22.1.5 cells. RESULTS: Both TFO21 and ODNas21 showed the inhibition to HBV replication and synthesis of antigen while, ODNcon (control of 21 mer phosphorothioate oligodeoxynucleotides) showed little inhibitory effects. At concentration of 10 mumol/L, TFO21 and ODNas21 inhibited the synthesis of HBsAg and HBeAg by 57.5% and 77%; 61% and 79.6%, respectively. The mixture of TFO21 and ODNas21 was more effective than TFO21 or ODNas21 alone. The inhibitions were dose-dependent. No toxicity was observed in the 22.1.5 cells treated with those oligodeoxynucleotides. CONCLUSION: Triplex forming oligodeoxynucleotides and antisense oligodeoxynucleotides were both potent inhibitor, for HBV replication and synthesis of antigen.

[Effects of partial deletion in the core promoter of hepatitis B virus genome on viral antigen expressions].

OBJECTIVE: To study the effects of 20/21 bp partial deletion mutation (from nt 1 748 or nt 1 747 to nt 1 767) in the core promoter (CP) region of hepatitis B virus (HBV) genome complicated by precore stop condon mutation at nt 1 896 on the expression of the viral antigens. METHODS: Eukaryotic expression vector containing full-length HBV genome with the above mutations was constructed. After transfection of the recombinant HBV plasmids into HepG2 cells, the expression of the viral antigens was examined with enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: As shown by ELISA and Western blotting analysis, the amount of extracellular secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) along with intracellular hepatitis B core antigen (HBcAg) in the cells transfected with vectors containing HBV genomes with partial deletion in the CP region was markedly reduced compared with that produced by wild-type HBV. CONCLUSION: The mutations in question causes marked reduction in viral antigen production by HBV in comparison that by wild-type HBV.

[The role of hepatitis B virus X gene and p53 on hepatocellular carcinoma cell growth].

OBJECTIVE: To explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth. METHODS: Two kinds of plasmids containing sense and antisense human wild p53 gene respectively were constructed. SMMU-7721 cells were transfected with HBx, sense-wtp53 antisense-wtp53 separately or cotransfected with either HBx and sense-wtp53 or HBx and antisense-wtp53. Flow cytometry was adopted to measure the apoptosis rates and the effects of HBx on cell cycle progression. The activity of p21(Waf1) promoter-luciferase construct was detected. Growth curves for SMMU-7721 stably transfected with pcDNA3 and pcDNA3HBx were analyzed. RESULTS: After doxorubicin administration, HBx was noticed able to initiate apoptosis of the liver cells. The apoptosis rate was 5.32% in the pcDNA3 transfected and 12.66% in the pcDNA3HBx transfected groups respectively. HBx could also abrogate p53-mediated apoptosis. The apoptosis rate in groups transfected with pcDNA3, pcDNA3wtp53 and pcDNA3HBx + pcDNA3wtp53 was 5.32%, 11.72% and 4.67% respectively. In compared with the normal group, the number of cells in transiently HBx-expressed group and HBx-transfected group decreased 4.79% and 10.25% respectively. HBx inhibited the activity of p21(Waf1) promoter-luciferase constructed (P < 0.05) and promoted cell growth. The growth rate of HBx expression cells was faster. CONCLUSION: Under DNA damage, HBx reduced expression of p21(Waf1) by repressing the activity of p53 protein, followed by disturbing the regulation of G(0)-G(1) cell cycle checkpoint, and promoted the growth rate of hepatoma cells.

[Seroconversion of hepatitis B markers in exposed health service workers after 1 year]. / Serokonwersja markerów wzw typu B u eksponowanych pracowników sluzby zdrowia w odstepie 1 roku.

Serum samples were investigated from 213 health service workers in Szczecin with a particular exposure to infection with HBV. In 5 samples (2.34%) the HBs antigen was demonstrated, and 120 workers (56.33%) anti-HBc antibodies were present. History data showed that only one-fifth of them had had a manifest form of virus hepatitis, thus the asymptomatic infection prevailed. After one year the investigations were repeated in 62 subjects who had had no HBV markers in the first determination and seroconversion was demonstrated in 11 of them (17.74%).