Michael Kew

Hepatitis B virus and hepatocellular carcinoma

Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.

Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy

Summary.u2002 Phylogenetic analysis has led to the classification of hepatitis B virus into eight genotypes, designated A to H. The genotypes have differences in biological properties and show heterogeneity in their global distribution. These attributes of the genotypes may account not only for differences in the prevalence of hepatitis B virus mutants in various geographic regions, but also be responsible for differences in the clinical outcome and response to antiviral treatment in different population groups.

Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV‐induced malignant transformation is, however, incompletely understood. HBx, the protein encoded by the X open reading frame, is a transcriptional activator that has been implicated in hepatocarcinogenesis. HBx inhibits the function of the tumour suppressor protein p53 in what is thought to be an early event in hepatocyte transformation before the later accumulation of inactivating p53 point mutations. HBx inhibits apoptosis but also exerts pro‐apoptotic effects. The effects of HBx on apoptosis may be important not only for the development of HCC but also for the establishment of HBV infection. Further implication of HBx in hepatocyte transformation has been the demonstration that it inhibits the repair of damaged hepatocyte DNA. This effect may be mediated by interaction with p53 or through binding to the damaged DNA binding protein (DDB), which plays an accessory role in nucleotide excision repair. In addition, HBx activates cell signalling cascades involving mitogen‐activated protein kinase (MAPK) and Janus family tyrosine kinases (JAK)/signal transducer and activators of transcription (STAT) pathways. The implications of these modulating effects of HBx are not fully understood, but they are likely to have wide‐ranging effects on hepatocyte proliferation, apoptosis and the regulation of cell growth checkpoints. The cellular functions ascribed to HBx are unusually diverse, and defining the biologically important role of HBx during HBV replication will go some way to understanding the sequelae of chronic HBV infection.

Regulatory elements of hepatitis B virus transcription

summary. The precise modulation of hepatitis B virus (HBV) gene expression is essential for replication of the virus. HBV sequences are transcribed under the control of the preC/pregenomic, S1, S2 and X promoters. With the exception of S1, all the HBV promoters lack the orthodox TATA box motifs required for the formation of the transcription initiation complex, and as such they represent a unique model of transcription initiation elements. The presence of two enhancer sequences and negative regulatoryelements in the HBV genome further augments thecontrolled synthesis of HBV‐ RNA. All these transcription cis‐elements are embedded within protein coding regions of the genome. This feature demonstrates the remarkable ability of the virus to maximize the function of its small genome. HBV transcription control elements also display a preference for liver‐specific or liver‐enriched trans‐factors, which contributes to the liver tropism of the virus. This review outlines the major HBV transcription regulatory elements and highlights the reliance of accurate HBV gene modulation on the complex interplay between several trans‐acting factors and their corresponding cis‐ motifs in the viral genome.

The core promoter of hepatitis B virus

The core promoter (CP) of hepatitis B virus (HBV) plays a central role in HBV replication and morphogenesis, directing the transcription of both species of 3.5 kb mRNA: pregenomic (pg) RNA and precore (pre‐C) mRNA. The CP overlaps the 3′ end of the X open‐reading frame (ORF) and the 5′ end of the pre‐C/C ORF. The major functional elements of the CP are the upper regulatory region (URR) and the basic core promoter (BCP). The BCP is sufficient for accurate initiation of both pre‐C mRNA and pgRNA transcription. It contains four AT‐rich regions and the initiators for pre‐C mRNA and pgRNA transcription. The upstream regulatory region consists of the negative regulatory element and the core upstream regulatory sequence. Co‐operative interaction of various liver‐enriched and ubiquitous transcription factors is necessary for liver‐specific expression from the CP. These factors bind to the CP. Sequence conservation within the CP is crucial for maintaining active viral replication, and variation may contribute to the persistence of HBV within the host, leading to chronic infection and, ultimately, hepatocarcinogenesis. The most frequently described mutations within this region are an A to T transversion at position 1762 together with a G to A transition at position 1764. This double mutant is accompanied by a reduced level of hepatitis B e antigen (HBeAg) expression. Deletions, insertions and duplications occur within the CP.

Hepatitis B virus and hepatocellular carcinoma: HBV and HCC

Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100‐fold increased relative risk of developing the tumour. Several mechanisms of HBV‐induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV‐associated HCC.

Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma

Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of β-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated β-catenin. Additional work showed that HBxAg-activated β-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of β-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis.

The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.

The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.

Structure and function of the encapsidation signal of hepadnaviridae

The hepatitis B virus (HBV) and other members of the hepadnaviridae replicate by reverse transcription of an RNA intermediate, pregenomic RNA (pgRNA). pgRNA is also translated into core protein and polymerase (reverse transcriptase) protein. Before being reverse transcribed, pgRNA is sequestrated from the cytoplasm by being packaged, together with polymerase, into subviral particles composed of core protein. For pgRNA to be encapsidated, its 5′ end is folded into a stem–loop structure, known as the encapsidation signal or epsilon (ɛ). This stable bipartite stem–loop structure contains a bulge and an apical loop. Besides encapsidation, ɛ is involved in the activation of polymerase, in template restriction and in the initiation of DNA synthesis by reverse transcription. HBV DNA encoding ɛ forms part of the template that is translated into the precore/core fusion protein that is in turn post‐translationally modified to produce hepatitis B e antigen (HBeAg). The DNA encoding ɛ may be recombinogenic. Mutations within ɛ can affect its function and sequence conservation within ɛ in natural isolates is therefore high. ɛ could provide a practical target for antiviral therapy.

Hammerhead ribozyme-mediated inhibition of hepatitis B virus X gene expression in cultured cells

BACKGROUND/AIMSnChronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia. Common complications of HBV persistence include cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important objective. The HBV X protein (HBx) is a transcriptional activator that is required for the establishment of HBV infection and is implicated in hepatocarcinogenesis. The aim of this study was to assess the ability of two endogenously expressed hammerhead ribozymes to inhibit expression of HBV genes in transfected cultured cells.nnnMETHODSnEukaryotic expression plasmids producing two ribozymes targeted to the HBx open reading frame, as well as their catalytically inactive homologues, were generated. Established cell lines and a primary culture of malignant hepatocytes were transfected to assess ribozyme effects on HBx expression and HBV replication.nnnRESULTSnThe ribozyme-expressing vectors inhibit expression of functional HBx protein and decrease HBV mRNA encoding surface and HBx sequences in transfected cells. Moreover, decreased HBsAg and HBeAg secretion from cells transfected with the ribozymes and an HBV replication competent plasmid provide evidence for an antireplicative effect of the ribozymes. However, the data do not exclude a dominant antisense effect that inhibits HBV gene expression.nnnCONCLUSIONSnInactivation of HBx, a sequence that is conserved in mammalian hepadnaviruses and found in all HBV transcripts, has potential for the treatment of chronic HBV infection.