Transactivation of human hepatitis B virus X protein, HBx, operates through a mechanism distinct from protein kinase C and okadaic acid activation pathways

Human hepatitis B virus (HBV) X protein, HBx, transactivates virus and host genes through a wide variety of cis-elements. Expression of HBx is controlled by HBV enhancer 1 (Enh1). Both Enh1 and the core sequence of Enh1, which consists of an AP-1 related site (cFAP1) and a C stretch, respond to HBx and a phorbol ester (TPA). Biochemical pathways of the responses to HBx and TPA are still controversial. We therefore asked whether HBx and TPA stimulate Enh1 core activity through a common process. Protein kinase C (PKC) inhibitors, H-7 and staurosporin, did not inhibit HBx transactivation at concentrations sufficient to abolish the TPA effects in HepG2 cells. Although HBx transactivation synergized independently with TPA or a phosphoprotein phosphatase inhibitor, okadaic acid (OA), the PKC inhibitors eliminated only the TPA contribution. HBx transactivation required both the cFAP1 and the C stretch of the Enh1 core region; however, mutations in either or both of the two cis-elements demonstrated that TPA augmentation required only cFAP1. These results imply that HBx transactivation operates through a mechanism distinct from the PKC and OA activation pathways.     

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.     

Hepatitis B virus with X gene mutation is associated with the majority of serologically "silent" non-b, non-c chronic hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically "silent" non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically "silent" NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.     

Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.     

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.