Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed antigen from either the retroviral long terminal repeat or the mouse metallothionein-I promoter. HBsAgs were synthesized containing no pre-S sequences, pre-S(2) sequences alone, or pre-S(1) plus pre-S(2) sequences. Inclusion of pre-S(2) sequences did not affect the secretion or density of HBsAg particles but did reduce their mass by approximately 30%. Addition of pre-S(1) sequences almost completely abolished secretion of HBsAg and resulted in its localization in an aqueous-nonextractable pre- or early-Golgi cellular compartment. HBsAg was localized to the cytoplasm of the cell. This localization was unaffected by the presence of pre-S sequences in the antigen. Cell lines synthesizing hepatitis B antigens from core DNA fragments, containing or not containing precore sequences, secreted hepatitis B e antigen. However, the absence of precore DNA sequences resulted in additional synthesis of hepatitis core antigen, which was predominantly nuclear in localization.     

In vivo phosphorylation and protein analysis of hepatitis B virus core antigen.

The C open reading frame of the hepatitis B virus contains two in-frame ATG codons that are separated by the precore region and encodes two major polypeptides that are antigenically distinct and that are probably synthesized from individual mRNAs. The precore region directs the secretion of the e antigen, whereas the core antigen can be expressed in the absence of these sequences. In this report a transient expression system was used to study the hepatitis B virus core antigen. By using a chimeric complex of adenovirus major late promoter-simian virus 40 enhancer sequences, we were able to achieve high levels of core antigen expression in transfected cells, permitting characterization of this protein and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core polypeptide is a 20.9-kilodalton protein, and we show in this study that it is phosphorylated in vivo. Cell fractionation studies, the results of which are supported by indirect immunofluorescence, localized the phosphocore in the cytosol and the nucleus and indicated that it is associated with the membrane of transfected cells. Results of Triton X-114 solubilization studies indicated that the phosphocore is peripherally associated with cytoplasmic membranes. Expression of the membrane-associated phosphocore occurred in the absence of the precore sequences. The phosphocore also assembled into particles in the absence of other viral gene products or intact DNA.     

The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.     

Interferon inhibits hepatitis B virus replication in a stable expression system of transfected viral DNA.

The effect of interferon (IFN) on hepatitis B virus (HBV) replication was investigated in a stable expression system, using HepG2 cells transfected with recombinant HBV DNA. IFN was found to cause a marked reduction in the levels of both minus and plus strands of HBV DNA from core particles in the cytoplasm. Neither HBV DNA from virus particles nor the HBV surface antigen in the culture medium primarily underwent change in quantity by treatment with IFN, as was also found for HBV mRNAs and the HBV core antigen/HBV e antigen in the cytoplasm. IFN exerted no influence on HBV DNA synthesis by endogenous DNA polymerase in the core particle fraction. From these findings, it would appear that IFN inhibits HBV replication by blocking some step in the pregenome RNA-primed assembly of core particles.     

Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site.

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.     

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.     

Suppression of hepatitis C virus core protein by short hairpin RNA expression vectors in the core protein expression Huh-7 cells

Short hairpin RNAs (shRNAs) efficiently inhibit gene expression by RNA interference. Here, we report the efficient inhibition by DNA-based vector-derived shRNAs of core protein expression in Huh-7 cells. The shRNAs were designed to target the core region of the hepatitis C virus (HCV) genome. The core region is the most conserved region in the HCV genome, making it an ideal target for shRNAs. We identified an effective site on the core region for suppression of the HCV core protein. The HCV core protein in core protein-expressing Huh-7 cells was downregulated by core protein-shRNA expression vectors (core-shRNA-452, 479, and 503). Our results support the feasibility of using shRNA-based gene therapy to inhibit HCV core protein production.     

Suppression Of Hepatitis C Virus Core Protein By Short Hairpin Rna Expression Vectors In The Core Protein Expression Huh-7 Cells

Short hairpin RNAs (shRNAs) efficiently inhibit gene expression by RNA interference. Here, we report the efficient inhibition by DNA-based vector-derived shRNAs of core protein expression in Huh-7 cells. The shRNAs were designed to target the core region of the hepatitis C virus (HCV) genome. The core region is the most conserved region in the HCV genome, making it an ideal target for shRNAs. We identified an effective site on the core region for suppression of the HCV core protein. The HCV core protein in core protein-expressing Huh-7 cells was downregulated by core protein-shRNA expression vectors (core-shRNA-452, 479, and 503). Our results support the feasibility of using shRNA-based gene therapy to inhibit HCV core protein production.     

HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells.

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.     

HBV prec/c shRNA慢病毒真核表达载体的构建和鉴定

目的构建针对HBV prec/c区的shRNA真核表达载体psiHBV,感染HepG2 2.15细胞后观察其对HBeAg表达的抑制作用,为探讨防治HBV感染的新措施提供实验依据。方法针对HBV prec/c基因序列,构建shRNA表达载体psiHBV1、psiHBV2和无关序列psiHBVc。psiHBV与慢病毒辅助系统质粒共转染293T细胞组装慢病毒颗粒后,感染HepG2 2.15细胞,RT-PCR检测prec/c mRNA的转录,微粒子化学发光分析仪(MEIA)检测细胞上清和细胞裂解液中HBeAg表达。结果重组质粒双酶切和测序鉴定与预期结果相符合;组装慢病毒颗粒感染HepG2 2.15细胞后,prec/c mRNA转录降低;与对照组比较,HBeAg的表达水平也显著降低,病毒颗粒对HBeAg表达的抑制作用差异有统计学意义(P<0.01)。结论成功构建针对HBVprec/c的慢病毒载体psiHBV1、siHBV2,慢病毒介导的RNA i能抑制HBV表达,为应用RNA干扰技术治疗乙型肝炎提供了实验依据。     

Production of infectious duck hepatitis B virus in a human hepatoma cell line.

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.     

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.     

抗HBcAg人源性单链抗体细胞内表达及其抗病毒复制的实验研究

应用人源性抗HBcAg单链抗体细胞内表达技术,探讨抗HBV复制基因治疗的应用价值.应用噬菌体展示和基因重组技术,从HBV感染的外周血淋巴细胞克隆了人源性抗HBcAg单链抗体,并重组至逆转录病毒载体.以人肝癌细胞smmc-7721和PLC/PRF/5为靶细胞进行基因共转染,分别测定实验组细胞上清中的HBsAg和HBeAg,与对照组做比较,观察抗HBcAg单链抗体细胞内表达的抗病毒治疗作用.结果显示,在急性HBV感染的细胞株中,抑制病毒复制效率为49%～61%,在慢性病毒感染细胞,抑制率为41%～54%.实验结果表明,应用单链抗体细胞内表达技术,在抗病毒治疗研究中具有潜在的应用价值.应对HBV的4个开放阅读框架编码产物进行全面的对比研究,以发现抑制效率高、实用价值大的靶基因.     

Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidémiologie Clinique du SIDA en Aquitaine.

OBJECTIVE: To evaluate the prevalence of antibodies to hepatitis C virus and serological markers for hepatitis B virus infection in patients with HIV. DESIGN: Cross sectional survey. SETTING: Aquitaine, southwestern France, 1991-94. SUBJECTS: 1935 HIV positive patients seen at least once since June 1991. MAIN OUTCOME MEASURES: Presence of antibodies to hepatitis C virus were detected by second or third generation enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) and markers for hepatitis B virus detected by ELISA. RESULTS: The prevalence was 42.5% (823) for antibodies to hepatitis C virus, 56.4 (507) for antibodies to hepatitis B core antigen, 6.9% (133) for hepatitis B surface antigen, 30.2% (584) for antibodies to hepatitis B core and surface antigen with no detectable surface antigen, 26.2% (507) for antibodies to core antigen only, and 4.8% (92) for antibodies to surface antigen only. The prevalence of antibodies to hepatitis C virus was 86.1% (726/843) in subjects who had bloodborne HIV infection and 7.3% (66/899) in those with sexually acquired infection. The prevalence of markers for hepatitis B was higher among homosexuals than in the other groups of patients, except for antibodies to surface antigen alone. The relation between markers for hepatitis B and hepatitis C virus was negative among men but positive among women. CONCLUSIONS: The results favour the hypothesis that hepatitis C virus is sexually transmitted much less commonly than either HIV or hepatitis B virus.     

An expression vector for high-level protein synthesis in Vero cells

We have constructed two new multi-purpose cloning vectors, pNI1 and pNI2, that carry the Escherichia coli gene Ecogpt encoding the enzyme xanthine-guanine phosphoribosyl transferase as a dominant selective marker. The Ecogpt gene is under the control of either the long-terminal-repeat promoter of mouse mammary tumor virus, pNI1, or the simian virus 40 early promoter, pNI2. Another feature of the vectors is a polylinker preceded by the human metallothionein IIA promoter. We have used pNI2 for the synthesis of the hepatitis B surface antigen (HBsAg) at a high level in monkey Vero cells. We show that gene amplification and a concomitant stable increase of HBsAg synthesis can be achieved in these cells using modified selective medium containing hypoxanthine, aminopterin and thymidine, i.e., increasing the aminopterin and decreasing the hypoxanthine concentrations.