The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.

Hepatitis B virus (HBV) is regarded as the main aetiologic factor in the development of human hepatocellular carcinoma (HCC), one of the most frequent fatal malignancies worldwide. Detection of integrated HBV sequences in the cellular DNA of almost all HCCs studied, and the recent finding that the integrated HBV open reading frame (orf) X encodes a transactivating activity, supports the notion that integrated HBV DNA could contribute to liver carcinogenesis by activation of cellular genes in trans. But not all HCCs seem to harbour a functional orf X. We report here that 3'-truncated preS2/S sequences in integrated HBV DNA of liver cell carcinomas encode a so far unidentified transcriptional trans-activation activity. This activity is also produced by an artificially 3'-truncated preS2/S gene of the wild-type HBV genome. Besides the simian virus 40 promoter of the reporter plasmid pSV2CAT, the promoter of the human c-myc oncogene can also be activated. These results, taken together with the fact that preS/S is the only HBV gene found to be integrated in almost every HBV-related HCC analysed so far, indicate that trans-activation by integrated preS2/S sequences is a possible mechanism for HBV-associated oncogenesis.

The hepatitis B virus (HBV) genome encodes a 154 amino acid protein termed X (HBx, hepatitis B x protein), which is a promiscuous transcriptional activator of polymerase II and III promoters. HBx upregulates a wide range of cellular and viral genes and is thought to facilitate viral pregenome and mRNA transcription; however, its precise role in the viral replication cycle remains to be elucidated. The functional mechanisms of HBx appear very complex. It was shown to activate transcription factors AP-1 and NF-kappa B vis cytoplasmic pathways including ras-MAP kinase. In contrast, nuclear HBx is thought to activate the transcriptional machinery directly. A second transcriptional activator protein (Mst, middle s transactivator) is encoded by 3'-truncated preS2/S sequences of integrated HBV DNA, but not by the intact viral gene. HBx and Mst may contribute to the pathogenicity of chronic hepatitis B and are suggested to promote hepatocyte transformation via upregulation of cellular proto-oncogenes. Further, HBx may enhance HBV related carcinogenesis by inactivation of the tumour suppressor gene product p53.

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