Effect of hepatitis B virus X protein on the expression of retinoblastoma gene product

Consistent reports from several recent studies suggest that users of third generation oral contraceptives (OCs) containing gestodene and desogestrel may be at increased risk of venous thromboembolic disease (VTE). Paradoxically, other reports indicate that these users may be at decreased risk of acute myocardial infarction (MI) compared with users of second generation OCs. To determine whether the potentially increased risk of VTE would outweigh the potentially reduced risk of MI in users of third generation OCs, we conducted an analysis to quantify the trade-offs providers and users may be faced to make between these formulations. The baseline rates of VTE and MI among non-users were calculated using US data on incidence and mortality of these conditions and estimates of the proportion of women exposed to these formulations in the US. These were multiplied by relative risks published in recent studies on third generation progestins to produce age- and formulation-specific risks. Results indicate that there would be small differences in disease burden between users of second and third generation OCs under the model assumptions at younger ages. However, among women 35-44 years of age, modeling results indicate that the potentially decreased incidence of MI among users of third generation OCs more than offsets the potentially increased risk of VTE at this age.     

Pro-apoptotic effect of the hepatitis B virus X gene

The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.     

An antisense promoter within the hepatitis B virus X gene

Sequences of the Hepatitis B virus X (HBx) gene are preferentially retained on chromosomally integrated viral DNA and thereby the precore/core promoter as a part of its reading frame. The existence of a second promoter mapping to the same DNA region is suggested by an antisense (AS) RNA which has been described earlier by Standring's group. Here, the capacity of sequences upstream to this AS RNA to function as a bidirectional promoter was analyzed. On a cloned monomer of viral DNA a segment spanning the start codon of the HBx gene and a site within the HBx frame was replaced by a luciferase reporter gene (Photinus pyralis) plus a downstream polyadenylation signal of SV40 origin. Insertion in HBx and AS orientation allowed to compare the apparent strengths of the respective promoter activities. Both DNA constructs expressed luciferase to levels above the one induced by a reference plasmid expressing the gene under control of the SV40 promoter. In the context of a reporter plasmid a 241-bp subregion of the HBx gene with enhancer II in its center part functioned bidirectionally as AS and as core promoter. For the expression of a putative AS factor two effector plasmids driven by the autologous and a heterologous promoter, respectively, were established which stimulated in co-transfection experiments a c-myc target gene to a higher degree than a corresponding HBx effector construct.     

乙肝病毒X蛋白对肝细胞生物钟基因的影响及其意义

Objective: The aim of this study was to investigate the influence of hepatitis B virus X protein (HBx) on the clock genes in LO2 cells and its significance. Methods: A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Results: The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx (P < 0.05), while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx (P < 0.05). Conclusion: The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer.     

Mimicry between the hepatitis B virus DNA polymerase and the antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis B virus infection

Autoantibodies to nuclear and smooth muscle are common in hepatitis B virus (HBV) infection. To understand their origin, we scanned protein databases and found that HBV-DNA polymerase (HBV-pol) shares 7-9 amino acid sequences with nuclear (MHC II trans-activator, nuclear pore core protein, nuclear mitotic apparatus, and polymyositis sclerosis Ag) and smooth muscle proteins (caldesmon and myosin). Twenty-mer peptides with relevant homologues and an irrelevant control peptide were constructed and ELISAs were established. Sixty-five children with chronic HBV infection, 104 patients with other chronic liver diseases (CLD), 36 patients with extrahepatic autoimmune diseases, and 24 healthy controls were investigated. Double reactivity to HBV-pol peptides and corresponding self homologues was observed in 40% of HBV-positive patients as compared with four (4%) with other chronic liver diseases, two (6%) with extrahepatic autoimmune diseases, and in none of the healthy controls (p < 0.001 for all). Double reactivity to myosin or caldesmon peptides and their HBV-pol homologues was associated with anti-smooth muscle Ab positivity by immunofluorescence (p < 0.05 for both). HBV-positive sera double reactive for myosin or caldesmon and their homologous HBV-pol peptides also reacted with the native proteins on immunoblot. Fifty to ninety percent Ab inhibition to individual HBV-pol and HBV-pol99-118 peptides was noted by preincubation with individual HBV-pol/self homologue peptide and native proteins, respectively, but not with control peptide. Our results show that cross-reactive immunity targeting homologous sequences of viral and self proteins may partly account for autoantibody production in HBV infection.     

Effect of mutations in the small envelope protein of hepatitis B virus on assembly and secretion of hepatitis delta virus

The gene coding for the small (S) envelope protein of hepatitis B virus was mutated to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. This study was focused on a domain of the S protein that is exposed in the cytoplasm during synthesis and thereby represented a good candidate for interaction with the viral nucleocapsid during virion assembly. The mutations consisted of deletion/insertions spanning the entire cytosolic domain of S between amino acid residues 24 and 80. Although the expression of mutants clustered between residues 59 and 80 could not be obtained, we demonstrated that a large part of the cytosolic loop, from residues 29-47 and 49-59, does not contain motifs essential for production of hepatitis B virus subviral particles or HDV virions. However, deletion of residues 24-28 led to the synthesis of S protein mutant, which was competent for secretion of subviral particles but deficient for production of HDV. We concluded that the sequence between Arg-24 and Ile-28 located at the carboxyl boundary of the transmembrane signal I for S contains residue or residues important for HDV particle assembly.     

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Distinguishing between acute and symptomatic chronic hepatitis B virus infection

BACKGROUND/AIMS: Differentiating between an acute hepatitis B (AH-B) infection and an acute exacerbation of a chronic hepatitis B (CH-B) infection can present a problem for the clinician. The only current serological method of distinguishing between acute and symptomatic chronic hepatitis B virus (HBV) infection is the immunoglobulin M antibody to hepatitis B core antigen (anti-HBc) assay, which can be problematic. Therefore, in an attempt to better distinguish between acute and chronic HBV infection, sera from 26 patients with AH-B and 53 patients with CH-B were compared in a variety of experimental immunoassays. METHODS: Experimental assays have been designed to detect free antibody to hepatitis B e antigen (anti-HBe), hepatitis B e antigen (HBeAg)/anti-HBe immune complexes (ICs), and hepatitis B surface antigens (HBsAg)/antibody to hepatitis B surface antigen (anti-HBs) in the presence of excess antigen. An additional assay was developed to detect a novel anti-HBc specificity, designated antibody to woodchuck hepatitis virus (anti-HBcW), which cross-reacts with the core antigen of the woodchuck hepatitis virus. RESULTS: Sera from patients with CH-B showed significantly higher levels of free anti-HBe, HBeAg/anti-HBe ICs, and HBsAg/anti-HBs ICs compared with AH-B patient sera. Furthermore, patients with CH-B consistently produced high titer anti-HBcW, whereas patients with AH-B produced little or no anti-HBcW antibody. CONCLUSIONS: The serology of AH-B infection and symptomatic CH-B infection can be distinguished using a variety of experimental immunoassays in addition to the immunoglobulin M anti-HBc assay.     

Mapping of homologous interaction sites in the hepatitis B virus core protein

Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.     

Long-term therapy with the guanine nucleoside analog penciclovir controls chronic duck hepatitis B virus infection in vivo

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the antiviral guanine nucleoside analog penciclovir for 12 or 24 weeks at a dosage of 10 mg/kg of body weight per day. By the completion of both 12 and 24 weeks of therapy, molecular hybridization studies of the liver tissue revealed that the viral DNA, RNA, and protein levels were significantly reduced compared to those in the placebo-treated controls. Penciclovir treatment for 12 or 24 weeks was not associated with any toxicity, establishing the efficacy and safety of long-term penciclovir therapy in chronic DHBV infection.     

Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework

In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.