The Hepatitis B Virus HBx Protein Inhibits Caspase 3 Activity

Abstract

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...

Hepatitis B virus-X protein upregulates the expression of p21wafl/cipl and prolongs G1→S transition via a p53-independent pathway in human hepatoma cells

“…Modulation of the apoptosis by the interactions of HBV-X protein and p53 gene product has also been reported (Elmore et al, 1997;Takada et al, 1997;Wang et al, 1994). Anti-apoptotic actions of HBV-X protein as a potent caspase 3 inhibitor has been described (Gottlob et al, 1998). HBV-X transfected hepatoma cells (Hep) are resistant to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor-a or anti-Fas antibody administration.…”

“…The pro-apoptotic e ects of HBV-X protein have been described (Gottlob et al, 1998). A direct dose-dependent apoptotic function of HBV-X has been demonstrated in transiently transfected liver cell lines (Terradillos et al, 1998).…”

“…In contrast to these pro-apoptotic e ects of HBV-X protein, evidence of the anti-apoptotic e ect of HBV-X protein has also been demonstrated. A recent report on the antiapoptotic e ect of HBV-X protein showed that HBV-X inhibits caspase 3 activity (Gottlob et al, 1998) resulting in resistance to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor-a or anti-Fas antibody administration. In addition to these proapoptotic or anti-apoptotic e ects of HBV-X, a growth arrest e ect was also demonstrated in our experiment.…”

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner

“…A variety of signaling pathways and transcription factors are known to be activated by this viral protein, thus contributing to the induction of a variety of cellular genes, including growth factors and proto-oncogenes (Feitelson and Duan, 1997;Haviv et al, 1998;Kekule et al, 1993;Klein and Schneider, 1997;Lara-Pezzi et al, 1998;Maguire et al, 1991). HBx is also capable of interfering with the DNA repair and apoptosis mechanisms and with normal p53 function (Gottlob et al, 1998a;Lee et al, 1995;Ueda et al, 1995), favoring cell cycle progression and misregulated cell growth (Koike et al, 1994). Although some of the signaling pathways triggered by HBx are known to be involved in the acquisition of metastasic properties, the possible contribution of this viral protein to the ®nal steps of tumor development remains far unknown.…”

Hepatitis B Virus X Protein Induces Expression of Fas Ligand Gene through Enhancing Transcriptional Activity of Early Growth Response Factor

“…While the first 50 aa in the amino terminus are known to be sufficient to transform infected host cells, they have no transactivating potential. In contrast, the HBx mutant lacking the 50 amino-terminal aa has a very low transforming capability but retains an intact transactivating potential (35). The transactivation function is located in the central region (aa 67-69) and in the carboxyl terminus (aa 110 -139) of HBx (Fig.…”