Based on epidemiological data and experimental results, mammalian hepadnaviruses, in particular hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV), have to be considered as a causative factor in the development of hepatocellular carcinoma (HCC), despite the fact that they lack a complete viral oncogene. Integrated viral DNA is found regularly in woodchuck and human HCC. In woodchucks an activation incisof c-myc and N-myc is almost always observed. By contrast, in humans, a pleiotropic activation intransof cellular genes by integrated genes encoding HBV transactivators, namely the X protein (HBx) and the PreS2 activators (the large surface protein (LHBs) and truncated middle surface proteins (MHBst)), has been described as a general mechanism. Mimicking chemical tumour promoters, i.e. TPA, the viral transactivators trigger PKC/Raf-controlled signalling pathways, finally activating transcription factors such as AP-1 and NF-κB which control genes relevant for proliferation. Moreover, ‘HBV-transactivated’ hepatocytes may give rise to a new epigenetic situation in the form of an ‘immortalized’ inflammatory process which may then pave the way for further critical events such as mutations and chromosomal aberrations.

Integrated hepatitis B virus DNA cloned from hepatitis B virus-associated hepatocellular carcinoma frequently contains 3'-truncated middle surface genes (preS2/S(t)), which were recently found to have a transcriptional transactivator function. Because preS2/S(t), among others, is able to transactivate the promoters of the cellular oncogenes c-myc and c-fos, it has been speculated that integrated preS2/S(t) genes might contribute to hepatitis B virus-associated liver carcinogenesis. In this study, we investigated the mechanism of target gene stimulation by preS2/S(t). It was found that deletion of a fragment containing the binding site for transcription factor AP-1 (Jun-Fos) substantially decreases inducibility of the human c-myc promoter by preS2/S(t). A subsequent investigation of AP-1 activation by preS2/S(t) revealed the following: (a) insertion of multimeric AP-1 binding sites confers inducibility to an otherwise unstimulatable test promoter; (b) transactivation of AP-1 sites is dramatically increased when Jun and Fos are overexpressed by cotransfected expression plasmids; and (c) inhibitors of AP-1 activation also impair transactivation by preS2/S(t). Besides AP-1, preS2/S(t) was also able to utilize the unrelated transcription factors NF-kappaB and AP-2 for transactivation, suggesting that the gene product of preS2/S(t) acts indirectly through one or several general cellular pathways rather than as a bona fide transcription factor. Because AP-I conveys induction of a large panel of tumor-relevant genes, its preS2/S(t)-dependent activation implies a possible causative role in hepatitis B virus-associated hepatocarcinogenesis.

Chronic infection with hepatitis B virus (HBV) is accompanied by an increasing risk of developing hepatocellular carcinoma. There are indications that the HBx protein of HBV is involved in the process of tumour formation. HBx also transactivates several transcription factor binding sites. Recently, we reported that HBx can use a tumour promotor pathway for transactivation. In particular, we found that transactivation of the binding motif for transcription factor AP-1 (JUN/FOS) by HBx is dependent on functional protein kinase C (PKC), as indicated by abolition of the transcriptional stimulation following downregulation or inhibition of the enzyme. Moreover, HBx activates PKC, probably via increasing the endogenous PKC activator sn-1,2-diacylglycerol (DAG). Here we extend these data and report on the time course of PKC activation. We found that activation of PKC by HBx is transient and differs from activation of PKC by the ras oncogene product or phorbol ester in that it does not lead to rapid downregulation of the enzyme subsequent to the activation. Moreover, we provide evidence that an increase in cellular DAG is observable not only as an early event in response to HBx but also in cell lines transformed after transfection with HBV DNA and stably expressing HBx. Besides its important role in the regulation of cellular genes, PKC is also the intracellular receptor for tumour-promoting agents and an activator of proto-oncogenes, suggesting that our observations might provide an explanation for the oncogenic properties of HBx.

THE hepatitis B virus (HBV) transactivator protein HBx is enigmatic in that it stimulates a striking variety of promoters which do not share a common cis-regulatory element1-5. As it does not bind to DNA6,7, it has been speculated that HBx acts indirectly through cellular pathways4,6-8. Under certain conditions HBx can have an oncogenic potential, which may be relevant for HBV-associated liver carcinogenesis9-11, but until now the mechanism for transactivation and cell transformation by HBx was unclear. We report here that HBx uses a complex signal transduction pathway for transactivation. An increase in the endogenous protein kinase C (PKC) activator sn-1,2-diacylglycerol and the subsequent activation of PKC give rise to activation of the transcription factor AP-1 (Jun-Fos). As a result, HBx transactivates through binding sites for AP-1 and other PKC-dependent transcription factors (AP-2, NF-kappaB), thereby explaining the as-yet incomprehensible variety of HBx-inducible genes. As the PKC signal cascade also mediates cell transformation by tumour-promoting agents, the mechanism presented here might account for the oncogenic potential of HBx.

Recently, it was reported that 3' truncation of an integrated surface gene (pre-S2/S) cloned from a hepatitis B virus (HBV)-associated hepatoma gave rise to the generation of a C-terminally truncated middle surface protein (MHBst), which surprisingly exerted a transcriptional transactivator function. To define the molecular requirements for the generation of transactivating MHBs(t) proteins, a 3' deletion analysis of the HBV pre-S2/S gene was performed. In cotransfection experiments with reporter plasmid pSV2CAT, full-length pre-S2/S genes or pre-S2/S genes with minor 3'-terminal deletions did not exhibit transactivator activity. In contrast, deletion of C-terminal hydrophobic region III of the S domain generated the transactivator function. Further stepwise 3' deletions, removing hydrophobic region II and both hydrophilic regions of the S domain, did not interfere with the transactivator activity; it was completely abolished, however, after additional deletion of hydrophobic region I. The results of this study define a range within the S open reading frame (between HBV nucleotides 221 and 573), termed the trans-activity-on region, in which 3' deletions give rise to the generation of transactivating MHBs(t) proteins. Within this region, not only 3' deletions but also the introduction of a stop codon activated the transactivator function, indicating that point mutations of integrated HBV DNA also may give rise to the synthesis of transactivating MHBs(t) proteins in vivo.

We have studied the c-myc gene as a possible target of HBV X protein in liver carcinogenesis. Our results indicate that trans-activation by X protein occurs via PKC/AP1 signal transduction, suggesting a possible two-step mechanism in HBV related liver carcinogenesis.