Involvement of mitochondrial permeability transition in hepatitis B virus replication

The HBx protein of human hepatitis B virus (HBV) activates a calcium-dependent kinase pathway which is essential for the viral replication. In this study, we found that HBx expression in the absence of other HBV proteins and in the context of HBV replication decreased the mitochondrial calcein-AM/CoCl₂ signals by 10% and 14% in HepG2 cells and by 15% and 10% in Huh7 cells, respectively. This indicates that HBx can induce mitochondrial permeability transition (MPT) and cause calcium effusion into the plasma. In addition, RNA interference of Cylophilin D decreased HBx-induced MPT and suppressed HBV DNA replication by 41% in HepG2 cells. Our results suggest that HBx expression can induce MPT and facilitate HBV DNA replication.     

Hepatitis B virus HBx protein localized to the nucleus restores HBx-deficient virus replication in HepG2 cells and in vivo in hydrodynamically-injected mice

Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins was confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.     

Increased growth of permanent mouse fibroblasts in soft agar after transfection with hepatitis B virus DNA

Summary Previously we have shown that a nontumorigenic mouse hepatocyte line harboring simian virus 40 large tumor antigen (SV 40 TAg) could be converted to a full-malignant phenotype by transfection with HBV DNA. Using a permanent SV 40 TAg-negative mouse fibroblast cell line (LTK^–), we studied whether the in vitro-oncogenicity of HBV was dependent on simultaneous expression of SV 40 TAg or not. Three fibroblast lines stably transfected by full-length HBV DNA formed four times more colonies of large size in soft agar than nontransfected LTK^– cells. All three clones expressed high levels of HBx protein, but variable levels of other HBV proteins. A second type of clone that was transfected by a partial HBV genome and that expressed HBV surface but no HBx proteins, did not acquire increased growth in soft agar. These data reveal that HBV DNA can enhance malignant growth independent of SV 40 TAg and suggest that HBx protein may act as an HBV oncogene at least in vitro.     

The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.     

Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes

Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.     

Hepatitis B virus regulates Raf1 expression in HepG2.2.15 cells by enhancing its promoter activity

Raf1 kinase is a central component of many signaling pathways that are involved in normal cell growth and oncogenic transformation. The expression of Raf1 is significantly increased in hepatocellular carcinoma (HCC). HBV is a major risk factor for HCC. HBx protein can increase the expression of Raf1; however, the mechanism of how HBV regulates Raf1 expression is still unknown. In this study, we showed the Raf1 expression was significantly higher in HepG2.2.15 cells than that in HepG2 cells in vitro. HBV could up-regulate Raf1 expression by enhancing the activity of its promoter in a dose-dependent manner, and HBs and HBx may be involved in this process. After silencing HBs and HBx by using RNA interference, the expression of Raf1 in HepG2.2.15 cells could be significantly inhibited. These results might provide useful information for understanding the mechanism of HCC induced by HBV infection.     

Stimulation of Cellular Proliferation by Hepatitis B Virus X Protein

Chronic infection with the hepatitis B virus (HBV) is a known risk factor in the development of human hepatocellular carcinoma (HCC). The HBV-encoded X protein, HBx, has been investigated for properties that may explain its cancer cofactor role in transgenic mouse lines. We discuss here recent data showing that HBx is able to induce hepatocellular proliferation in vitro and in vivo. This property of HBx is predicted to sensitize hepatocytes to other HCC cofactors, including exposure to carcinogens and to other hepatitis viruses. Cellular proliferation is intimately linked to the mechanism(s) by which most tumor-associated viruses transform virus-infected cells. The HBx alteration of the cell cycle provides an additional mechanism by which chronic HBV infection may contribute to HCC.     

HBx蛋白在HBV与HIV超感染过程中的作用

乙型肝炎病毒(hepatitisBvirus,HBV)是一种常见的与人免疫缺陷病毒(humanimmunodeficiencyvirus,HIV)发生超感染的病毒,能激活处于潜伏状态的HIV。HBVx基因编码的X蛋白(HBx)具有多种功能,它通过与多种宿主功能蛋白直接或间接的相互作用调节这些蛋白的功能,具有广泛的反式激活作用。在HBV、HIV超感染过程中,该蛋白起着极为重要的作用。