Calcium ions affect the hepatitis B virus core assembly

Previous report showed that cytosolic Ca2+ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca2+ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.     

Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Southeast Asia and Hong Kong. Among the four proteins that are encoded by the HBV genome, HBV X (HBx) is the most potentially oncogenic factor. It is known that HBx plays an important role in hepatocarcinogenesis, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In this study, we constructed expression vectors for small hairpin RNAs (shRNA) against HBx and investigated their regulatory effects in PLC/PRF/5 HCC cells, which constitutively produce HBx. Our data show that this tool of RNA interference (RNAi) could successfully reduce the HBx mRNA and protein levels by 50-95%. RNAi targeting HBx in PLC/PRF/5 cells demonstrated significant reduction in cell proliferation, cell growth, anchorage-independent growth in soft agar, and tumour development in nude mice. In addition, depletion of HBx expression increased cell sensitivity to TNFalpha-mediated and serum-free-induced apoptosis, and reduced the expression levels of C-myc and Bcl-X(L). These findings suggest that HBx plays an important role in tumorigenicity and anti-apoptotic mechanisms in HCC.     

Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.     

The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.     

PTEN modulates hepatitis B virus-X protein induced survival signaling in Chang liver cells

PTEN gene, a novel tumor suppressor is frequently mutated or deleted in several malignancies including human hepatocellular carcinoma (HCC). We report previously that human hepatitis B virus-X (HBx) protein achieves protection from apoptotic cell death through-PI3K-Akt-Bad signaling that is p53-independent in liver cells (JBC; 276, 16969 (2000)). In this report, we demonstrated the PTEN effect on HBx induced anti-apoptotic signaling in Chang liver cells (CHL). Expression of PTEN in CHL cells downregulate HBx induced PI3K, Akt activities, Akt, Bad phosphorylations, decreased caspase 3 activity and protection from DNA fragmentations. PTEN suppression of CHL cell growth at G1 phase (JBC;278,4057(2003)) in cell cycle analysis, which is overcome by HBx activated Akt/PKB further confirmed that same PI3K/Akt pathway is involved in cell survival and apoptosis by HBx and PTEN. PTEN suppression of HBx-mediated cell survival through PI3K pathway is specific, since PTEN does not suppress the effect of HBx on the protection from Fas-mediated apoptosis. Taken together, these findings demonstrate that PTEN potently modulate HBx-mediated signaling and is a viable target in therapeutic approaches to inhibit the formation of HCC caused by HBV infections.     

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on (“Tet-on”) gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.     

Concurrent hepatitis C virus and hepatitis delta virus superinfection in patients with chronic hepatitis B virus infection.

Since hepatitis C virus (HCV) and hepatitis delta virus (HDV) are transmitted by the same routes as hepatitis B virus (HBV), simultaneous or concurrent HCV and HDV infection in patients with chronic HBV infection may occur. To test this hypothesis and to examine the clinicohistological and immunopathological presentations of such multiple hepatitis virus infections, acute and/or convalescent serum specimens from 86 patients with acute HDV superinfection were tested by enzyme immunoassay for antibodies to HCV. Of the 86 patients, 18 (20.9%) were associated with HCV infection. Although patients with early mortality cannot be evaluated by the HCV markers used in this study, the results showed that the clinical and histologic features were similar except that patients with HCV infection were older than those without HCV infection (P less than 0.01). Immunopathological studies carried out within 2 months after the onset of acute HDV superinfection demonstrated that hepatitis B core antigen (HBcAg) was not detected in any patient and HDV antigen was detected in 18.2% of the patients with HCV infection whereas HBcAg and HDAg were found in 7% and 65.1%, respectively, of those without HCV coinfection (P less than 0.02). It is concluded that concurrent HCV and HDV superinfections can and do occur in patients with chronic HBV infection. In these triple viral infections, HCV may even transiently suppress HDV and HBV.     

Intrahepatic expression of hepatitis B virus antigens: effect of hepatitis C virus infection.

Coinfection with hepatitis B and C viruses (HBV, HCV) is not uncommon, but the expression of HBV antigens in the liver of patients with concomitant HCV infection has not been investigated. This study aimed to evaluate the effects of concomitant HCV infection on the intrahepatic expression of HBV antigens in chronic hepatitis. HBV surface and core antigens (HBsAg, HBcAg) were immunohistochemically evaluated and semiquantitatively scored in liver biopsy specimens from patients with chronic hepatitis, comprising 17 cases with dual HBV/HCV infection and 25 with HBV infection alone. The prevalence of HBV Ag expression proved significantly lower in the group with dual infection. In the presence of active HBV replication (HBV DNA-positive serum) the prevalence of HBsAg and HBcAg immunoreaction was similar in the two groups, though a significantly lower percentage of cells expressed HBcAg in the group of coinfected patients. HBV Ag was not detected at all among HBV DNA-negative/HCV RNA-positive cases. In conclusion, these observations suggest that HCV might influence HBV antigen expression in the liver and that either partial or complete suppression might occur.     

乙型肝炎病毒X蛋白对肝癌细胞生长的影响

目的探讨乙型肝炎病毒X蛋白（hepatitis Bvirus Xprotein，HBx）对肝癌细胞恶性程度的影响。方法构建携带HBV。基因真核表达载体pcDNA用Bx，以脂质体介导转染HepG2肝癌细胞，建立可稳定表达HBx的肝癌细胞系HepG2-HBx细胞，同时设空载体pcDNA，转染细胞HepG2-pcDNA，及未转染HepG2细胞为对照组。PCR法扩增Neo基因检测插入的质粒DNA片段．免疫荧光检测HBx的表达。通过生长曲线测定、平板克隆形成实验、MTr比色实验、Hoechst33342核形态学染色观察及流式细胞仪测定．了解稳定转染细胞的生物学行为变化。结果与对照组相比，转染pcDNA√IBx的HepG2-HBx细胞生长速度加快．其倍增时间缩短（28h对32．5h或34h，P〈0．05），克隆形成率增加[（10．12±0．23）％对（5．33±O．19）％或（5．19±0．28）％，P〈0．05]，细胞周期分析显示由GdG，期-S期的进程明显加快。细胞凋亡检测显示HepG2-HB。细胞可抵抗放线菌素D（ActD）诱导的凋亡作用。结论HBx可提高肝癌细胞的增殖活性，并增强肝癌细胞的抗凋亡能力．增加了肝癌细胞的恶性表型。     

A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein

A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions.     

Impact of hepatitis B virus (HBV) x gene mutations on hepatocellular carcinoma development in chronic HBV infection

The hepatitis B virus (HBV) PreS mutations C1653T, T1753V, and A1762T/G1764A were reported as a strong risk factor of hepatocellular carcinoma (HCC) in a meta-analysis. HBV core promoter overlaps partially with HBx coding sequence, so the nucleotide 1762 and 1764 mutations induce HBV X protein (HBx) 130 and 131 substitutions. We sought to elucidate the impact of HBx mutations on HCC development. Chronically HBV-infected patients were enrolled in this study: 42 chronic hepatitis B (CHB) patients, 23 liver cirrhosis (LC) patients, and 31 HCC patients. Direct sequencing showed HBx131, HBx130, HBx5, HBx94, and HBx38 amino acid mutations were common in HCC patients. Of various mutations, HBx130+HBx131 (double) mutations and HBx5+HBx130+HBx131 (triple) mutations were significantly high in HCC patients. Double and triple mutations increased the risk for HCC by 3.75-fold (95% confidence interval [CI] = 1.101 to 12.768, P = 0.033) and 5.34-fold (95% CI = 1.65 to 17.309, P = 0.005), respectively, when HCC patients were compared to CHB patients. Functionally, there were significantly higher levels of NF-κB activity in cells with the HBx5 mutant and with the double mutants than that of wild-type cells and the triple-mutant cells. The triple mutation did not increase NF-κB activity. Other regulatory pathways seem to exist for NF-κB activation. In conclusion, a specific HBx mutation may contribute to HCC development by activating NF-κB activity. The HBx5 mutation in genotype C2 HBV appears to be a risk factor for the development of HCC and may be used to predict the clinical outcomes of patients with chronic HBV infection.     

Identification and functional studies of HLA-A0201 restricted CTL epitopes in the X protein of hepatitis B virus

Cytotoxic T lymphocyte (CTL) epitopes in the X protein (HBx) of hepatitis B virus (HBV) may play a key role in the viral control and liver damage. The aim of this study was to identify and study the function of HLA-A0201 restricted CTL epitopes in HBx of HBV genotypes B and C that are epidemic in China. Four nonapeptides signed HBx1: VLCLRPVGA, HBx2: CLFKDWEEL, HBx3: VLHKRTLGL, and HBx4: HLSLRGLPV were predicated by computational analysis and manually confirmed by defining the peptide supermotif, extended motif, and quantitative motif. Synthesized peptides were examined for their affinity and binding stability with HLA-A0201. After being analyzed by enzyme-linked immunospot (ELISPOT) and cytolytic activity assays, the HBx2 epitope was selected for a construction of HLA-A0201-peptide tetramers. The tetramer staining method was used to analyze peripheral blood mononuclear cells (PBMCs) isolated from HBV-infected patients at different disease stages (chronic hepatitis, liver cirrhosis, and hepatoma). Compared with CTL epitopes in the HBV envelope or polymerase, HBx2 is also a potential HLA-A0201 restricted CTL epitope, what may have a clinical implication.     

The hepatitis B virus HBx protein modulates cell cycle regulatory proteins in cultured primary human hepatocytes

There are over 350 million people chronically infected with the Hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). While the precise mechanism of HBV-associated HCC remains undefined, it is believed to involve a combination of the host immune response to infection and activities of HBV proteins including the nonstructural X protein (HBx). HBx is a multifunctional protein that can modulate various cellular processes including cell proliferation. The exact effect of HBx on cell proliferation has varied depending on the cell line and exact conditions used in the study. Our previously published reports have demonstrated that HBx modulates the levels of cell cycle regulatory proteins in primary rat hepatocytes; however, the effect of HBx on cell cycle regulatory proteins in primary human hepatocytes, the natural host for HBV infection, has not been studied. Here we have examined the effect of HBx on cell cycle regulatory proteins in cultured, primary human hepatocytes. We demonstrate that HBx decreases the levels of cell cycle proteins that prevent progression into G1 phase and increases the levels of cell cycle proteins active in G1 phase. We have also shown that HBx modulation of cell cycle regulatory proteins requires cytosolic calcium, similar to the results we previously obtained in primary rat hepatocytes. Cumulatively, our results are the first demonstration that HBx modulates the levels of cell cycle regulatory proteins in a calcium-dependent manner in primary human hepatocytes.