Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.     

Altered DNA mutation spectrum in aflatoxin b1-treated transgenic mice that express the hepatitis B virus x protein

Humans chronically infected with hepatitis B virus (HBV) are at further risk of liver cancer upon exposure to dietary aflatoxin B1 (AFB1), a carcinogenic product of the mold Aspergillus flavus. For the present study, we utilized double-transgenic mice (ATX mice) that express the HBV X protein (HBx) and possess a bacteriophage lambda transgene to evaluate the in vivo effect of HBx expression on AFB1-induced DNA mutations. The expression of HBx correlated with a 24% increase in mutation frequency overall and an approximately twofold increase in the incidence of G/C-to-T/A transversion mutations following AFB1 exposure. These results are consistent with a model in which expression of HBx during chronic HBV infection may contribute to the development of hepatocellular carcinoma following exposure to environmental carcinogens.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Hepatitis B virus X protein stimulates viral genome replication via a DDB1-dependent pathway distinct from that leading to cell death

The hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been implicated in the development of liver cancer associated with chronic infection. HBx can interact with a number of cellular proteins, and in cell culture, it exhibits pleiotropic activities, among which is its ability to interfere with cell viability and stimulate HBV replication. Previous work has demonstrated that HBx affects cell viability by a mechanism that requires its binding to DDB1, a highly conserved protein implicated in DNA repair and cell cycle regulation. We now show that an interaction with DDB1 is also needed for HBx to stimulate HBV genome replication. Thus, HBx point mutants defective for DDB1 binding fail to complement the low level of replication of an HBx-deficient HBV genome when provided in trans, and one such mutant regains activity when directly fused to DDB1. Furthermore, DDB1 depletion by RNA interference specifically compromises replication of wild-type HBV, indicating that HBx produced from the viral genome also functions in a DDB1-dependent fashion. We also show that HBx in association with DDB1 acts in the nucleus and stimulates HBV replication mainly by enhancing viral mRNA levels, regardless of whether the protein is expressed from the HBV genome itself or supplied in trans. Interestingly, whereas HBx induces cell death in both HepG2 and Huh-7 hepatoma cell lines, it enhances HBV replication only in HepG2 cells, suggesting that the two activities involve distinct DDB1-dependent pathways.     

The Hepatitis B Virus X Protein Inhibits Thymine DNA Glycosylase Initiated Base Excision Repair

The hepatitis B virus (HBV) genome encodes the X protein (HBx), a ubiquitous transactivator that is required for HBV replication. Expression of the HBx protein has been associated with the development of HBV infection-related hepatocellular carcinoma (HCC). Previously, we generated a 3D structure of HBx by combined homology and ab initio in silico modelling. This structure showed a striking similarity to the human thymine DNA glycosylase (TDG), a key enzyme in the base excision repair (BER) pathway. To further explore this finding, we investigated whether both proteins interfere with or complement each other’s functions. Here we show that TDG does not affect HBV replication, but that HBx strongly inhibits TDG-initiated base excision repair (BER), a major DNA repair pathway. Inhibition of the BER pathway may contribute substantially to the oncogenic effect of HBV infection.     

双表达小干扰RNA抑制小鼠体内HBV感染

目的运用水压法复制HBV小鼠急性模型,用于研究双表达siRNA在小鼠体内对HBV复制的抑制作用。方法使用HBV质粒DNA对BALB/c小鼠进行尾静脉大剂量快速推注,获得急性HBV体内表达模型。同时使用相同方法对推注后的小鼠进行RNAi干预。对不同处理组动物的HBsAg、HBeAg、HBcAg和HBV DNA进行ELISA、免疫组织化学和实时定量RT-PCR检测。结果水压法复制HBV小鼠急性模型与HBV DNA感染剂量无明显相关。与单表达siRNA相比较,双表达siRNA对HBV抗原表达抑制较为明显。结论双表达siRNA不失为一种体内抑制HBV感染的一种策略。     

Hepatitis B Virus X Protein Interferes with Cellular DNA Repair

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell’s capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.     

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.     

Enhancement of hepatitis B virus replication by the regulatory X protein in vitro and in vivo

The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.     

Evidence of finely tuned expression of DNA polymerase beta in vivo using transgenic mice

DNA polymerase (Pol) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol mRNA and protein, supporting the idea of a tight regulation of Pol in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol overexpression event is not sufficient to initiate tumorigenesis in vivo.     

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication

Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. During the HBV life cycle, HBV hijacks various host factors to assist viral replication. In this research, we find that the HBV regulatory protein X (HBx) can induce the upregulation of DExH‐box RNA helicase 9 (DHX9) expression by repressing proteasome‐dependent degradation mediated by MDM2. Furthermore, we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization. In addition, the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98. Our findings reveal that HBx‐mediated DHX9 upregulation is essential for HBV DNA replication.     

Hepatitis B virus core protein stimulates the proteasome-mediated degradation of viral X protein

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the ability to transactivate the expression of all HBV proteins, including the viral core protein HBc. Consistent with its regulatory role, HBx is relatively unstable and is present at low levels in the cell. We report here that the level of HBx was significantly reduced by the coexpression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. Moreover, the inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of the proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated, mutational analysis indicated that the C-terminal half of HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through control of the HBx level via an inhibitory feedback type of mechanism.     

乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。