The hepatitis B virus-encoded HBx protein coactivates transcription of viral and cellular genes, and it is believed to play an important role in hepatitis B virusrelated liver cancer. HBx has been shown to alter the coordinated balance between proliferation and programmed cell death, being able to either induce or block apoptosis. Here, we demonstrate for the first time that the HBx is a potent caspase 3 inhibitor. Rat fibroblasts (REV2) and hepatoma cells (Hep) synthesizing the HBx protein were resistant to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor ␣, or anti-Fas antibodies administration. In these cells, HBx prevented DNA fragmentation and cell death in the absence of de novo protein synthesis, with a similar efficiency as the competitive caspase 3 substrates inhibitors VAD-FMK and DEVD-FMK. Protein extracts obtained from the HBx positive cells contained a very low caspase activity, and addition of anti-HBx antibody restored the endogenous caspase activity. To obtain a functional map of the anti-caspase activity of HBx, various cell lines were established that synthesized either N-terminally or C-terminally truncated HBx molecules. These gene dissection experiments revealed that the regions required for the anti-caspase activity overlap with the two known transactivation domains of HBx.Caspase 3 (CPP32) is a member of the cysteine protease family, activating downstream substrates of the apoptosis pathway by proteolytic cleavage (e.g.: poly(ADP)-polymerase (PARP)), 1 which leads to DNA fragmentation and to cell death (1). In mammalian cells, the CPP32 activity and apoptosis are regulated by various endogenous and exogenous factors including different virus gene products (2, 3).HBx has been reported to sensitize rodent fibroblasts as well as human hepatoma cells to TNF␣ cytotoxicity and to DNA damage-induced p53-dependent apoptosis (4, 5). When overexpressed, HBx directly kills both fibroblasts and hepatoma cells (4 -6), which can be rescued in analogy to what has been also observed for c-myc-and N-myc-induced apoptosis by insulinlike growth factors 1 and 2, respectively (4, 8).2 On the other hand, HBx also inhibits apoptosis induced by p53 overexpression in human primary fibroblasts (9, 10). Therefore, the fate of infected cells expressing HBx is likely to be determined by the balance between apoptotic and antiapoptotic signals of viral cellular and environmental origin. The ability of HBx to modulate cell survival is potentially relevant for both viral pathogenicity in acute and chronic HBV infection as well as for the late development of hepatocellular carcinoma. In the long term, it is the anti-apoptotic function of HBx that is likely to be the major determinant for manifestation of the transformed phenotype.From this perspective, it is of central importance to define the molecular mechanisms underlying HBx-induced dysregulation of the cell death program. HBx has been reported to contain two Kunitz-type serine protease inhibitor consensus sequences that overlap with its...

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