Abstract
The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965-6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840-2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68-117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68-117 without causing redistribution of cytochrome c and apoptosis-inducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death.
Highlights
Epidemiological studies show that chronic hepatitis B virus (HBV)1 infection is closely associated with development of hepatocellular carcinoma [3], and the X gene has been implicated in the carcinogenicity of this virus because of its ability to transform rodent cells [4]
The present results provided strong evidences that hepatitis B virus X protein (HBx)-induced cell death is connected to localization of HBx on mitochondria
Several groups have pointed out that the pro-apoptotic effect of HBx correlates with its transactivation activity [23, 36, 37], our result indicates that the transactivation ability of HBx is not required for its cell death activity, suggesting that the mechanism of HBxinduced cell death is not due to transactivation of the cell death-promoting genes, such as bax
Summary
Reagents and Plasmids—The X expression plasmid pCMVX, pCMVX-dAvaI/AvaII, and pCMVX-⌬RsaI were described previously [12]. A -galactosidase expression plasmid, pCMV, was obtained from Clontech. Transfections were carried out using Tfx-20 (Promega) according to the manufacturer’s instructions. Cells were cultured for 2 days after transfection and subjected to cytochemical staining or Western blot analysis unless stated. Cell Death Assay—HuH7 cells were transfected with indicated plasmid along with the -galactosidase expression plasmid, pCMV, at a ratio of 5:1. A 30-l aliquot of mouse liver mitochondria was incubated with the indicated recombinant protein in 30 l of the same buffer for 60 min at 30 °C. At the end of incubation, the reaction mixture was centrifuged, and the pelleted mitochondria fraction was resuspended in 50 l of 1 ϫ SDS sample buffer, and 12 l of 6 ϫ SDS sample buffer was added to the resulting supernatant. The protein concentration was determined by Bradford method [31]
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