HBx Mutations Research Articles

Sphondin efficiently blocks HBsAg production and cccDNA transcription through promoting HBx degradation.

Hepatitis B surface antigen (HBsAg) loss and seroconversion, which is considered as functional cure of chronic Hepatitis B virus(HBV) infection, is rarely achieved even after long-term antiviral treatments. Therefore, new antiviral strategies interfering with other HBV replication steps are required, especially those that could efficiently inhibit HBsAg production. Here, we identified novel anti-HBV compounds that could potently block HBsAg expression from cccDNA by screening a natural compound library derived from Chinese traditional medical plants by a novel screening strategy. The combination of ELISA assay detecting the HBsAg and real-time PCR detecting HBV RNAs as indicator for cccDNA transcriptional activity were used. The antiviral activity of a candidate compound and underlying mechanism were evaluated in HBV-infected cells and a humanized liver mouse model. Herein, we selected a highly effective low-cytotoxic compound sphondin, which could effectively inhibit both intracellular HBsAg production and HBV RNAs levels. Moreover, we found that sphondin markedly inhibited cccDNA transcriptional activity without affecting cccDNA level. Mechanistic study found sphondin preferentially bound to HBx protein by residue Arg72, which led to increased 26S proteasome-mediated degradation of HBx. Sphondin treatment significantly reduced the recruitment of HBx to cccDNA, which subsequently led to inhibition of cccDNA transcription and HBsAg expression. The absence of HBx or R72A mutation potently abrogated the antiviral effect induced by sphondin in HBV-infected cells. Collectively, sphondin may be considered as a novel and natural antiviral agent directly targeting HBx protein, which effectively inhibited cccDNA transcription and HBsAg expression.

T118N Substitution of Hepatitis B X Protein Reduces Colony Formation of HepG2 Cells

BACKGROUND: The acute Hepatitis B virus (HBV) infection usually ceases before six months, but chronic infection that lasts for more than six months might develop into liver cirrhosis and hepatocellular carcinoma (HCC). Viral particle load, HBV genotypes and association to the HBV x (HBx) gene mutations are the probable factors related to HCC occurrence. The mutation which leads to HBx T118N was found as the second most common HBx mutation in Indonesia, as compared to the known cancer-related HBx K130M/V131I mutant. However, the effect of T118N mutation and its combination with K130M/V131I on human hepatoma cells has not been elucidated well. Hence, this study was conducted to dissect the role of HBx T118N and its mutant combination in colony formation, as compared to the wild type HBx and cancer-related HBx K130M/V131I.METHODS: In this study, the genes encoding wild type HBx, HBx T118N, and HBx K130M/V131I mutations were obtained as synthetic gene. Meanwhile, the gene encoding HBx T118N/K130M/V131I mutations was successfully generated using site-directed mutagenesis. The optimum condition for colony formation assays was determined through Zeocin sensitivity test of HepG2 cells.RESULTS: Selection of HepG2 cells using Zeocin was determined at 200 µg/mL. Colony formation assays performed upon expression of HBx T118N and HBx T118N/K130M/V131I mutant proteins showed reduced colony numbers as compared to the expression of wild type HBx, similar to the effect from HBx K130M/V131I mutant expression.CONCLUSION: The HBx T118N and HBx T118N/K130M/V131I mutation caused less colony formation of HepG2 cells, similar to the K130/M131I mutation. This indicates a possible role of the T118N mutation in liver cancer development.KEYWORDS: colony formation assay, hepatitis B virus, HBx, T118N, K130M/V131I

Core promoter mutation of nucleotides A1762T and G1764A of hepatitis B virus increases core promoter transactivation by hepatocyte nuclear factor 1.

Hepatitis B virus (HBV) infection highly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). The clinical manifestation of HBV infection is determined by the mutual interplay of the viral genotype, host genetic factors, mode of transmission, adaptive mutations, and environmental factors. Core promoter activation plays a critical role in the pre-genomic RNA transcription of HBV for HBV replication. The mutations of core promoter have been implicated in HCC development. We had obtained HBV genes from Myanmar HBV infectants and identified gene variations at the core promoter region. For measuring the relative transactivation activity on core promoter, we prepared the core-promoter reporter construct. Both of A1762T and G1764A mutation were consistently found in the HBV genes with hepatocellular carcinoma. The A1762T/G1764A mutation was corresponding to K130M/V131I of HBx protein. We prepared the core promoter-luciferase reporter construct containing the double A1762T/G1764A mutation and the K130M/V131I HBx protein expression construct. The A1762T/G1764A mutation highly was responsive to core promoter transactivation by HBx, regardless of HBx mutation. The A1762T/G1764A mutation newly created hepatocyte nuclear factor 1 (HNF1) responsive element. Ectopic expression of HNF1 largely increased the HBV core promoter containing A1762T/G1764A mutation. In addition, hepatic rich fatty acid, palmitic acid and oleic acid, increased K130M/V131I HBx level by core promoter activation. These results provide biological properties and clinical significance of specific HBV core promoter mutants related with HCC development.

Downregulation of GSK3β and Upregulation of URG7 in Hepatitis B-Related Hepatocellular Carcinoma

: Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC). The exact molecular contributors to the development of HBV-related HCC are not yet completely understood. Recent studies demonstrated that the deregulation of the Wnt pathway is highly associated with the development of HCC. Besides, HBV is known to have roles in the deregulation of this pathway. The present study evaluated the molecular aspects of the Wnt pathway in HBV-related HCC in liver tissue samples. Viral characterization was done by identifying the HBx mutations and the assessment of intrahepatic viral load. The expression of Wnt pathway genes was assessed using real-time PCR and methylation-specific PCR. The intrahepatic viral load was significantly higher in tumor samples than in normal tissues (P = 0.0008). Aberrant expression was observed in Wnt-1, Wnt-7a, FZD2, FZD7, β-catenin, URG7, c-Myc, SFRP5, and GSK3β, among which Wnt1, FZD2, SFRP5, Gsk3β, and URG7 were associated with HBV. HBx mutations at positions I88, L116, and I127 + F132 were associated with the decreased expression of GSK3β and overexpression of URG7 and Wnt1. Alterations in the expression level of β-catenin, as well as some mutants of HBx, were correlated with the level of c-Myc. HBV-related HCC seems to be mostly coordinated with epigenetic behaviors of HBx, such a multi-functional peptide with suppressing/trans-activating functions.

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44–77% reduction) and secretory HBsAg (25–81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33–64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55–60 and 121–126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02–0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69–1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01–14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55–60 and 121–126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.

Omics-based Investigation of Diet-induced Obesity Synergized with HBx, Src, and p53 Mutation Accelerating Hepatocarcinogenesis in Zebrafish Model

The primary type of liver cancer, hepatocellular carcinoma (HCC), has been associated with nonalcoholic steatohepatitis, diabetes, and obesity. Previous studies have identified some genetic risk factors, such as hepatitis B virus X antigens, overexpression of SRC oncogene, and mutation of the p53 tumor suppressor gene; however, the synergism between diet and genetic risk factors is still unclear. To investigate the synergism between diet and genetic risk factors in hepatocarcinogenesis, we used zebrafish with four genetic backgrounds and overfeeding or high-fat-diet-induced obesity with an omics-based expression of genes and histopathological changes. The results show that overfeeding and high-fat diet can induce obesity and nonalcoholic steatohepatitis in wild-type fish. In HBx, Src (p53-) triple transgenic zebrafish, diet-induced obesity accelerated HCC formation at five months of age and increased the cancer incidence threefold. We developed a global omics data analysis method to investigate genes, pathways, and biological systems based on microarray and next-generation sequencing (NGS, RNA-seq) omics data of zebrafish with four diet and genetic risk factors. The results show that two Kyoto Encyclopedia of Genes and Genomes (KEGG) systems, metabolism and genetic information processing, as well as the pathways of fatty acid metabolism, steroid biosynthesis, and ribosome biogenesis, are activated during hepatocarcinogenesis. This study provides a systematic view of the synergism between genetic and diet factors in the dynamic liver cancer formation process, and indicate that overfeeding or a high-fat diet and the risk genes have a synergistic effect in causing liver cancer by affecting fatty acid metabolism and ribosome biogenesis.

SAT-195-The novel HBx mutation F30V correlates with HCC in vivo, hampers HBV replicative efficiency and enhances anti-apoptotic activity of HBx N-terminus in vitro

SAT-195-The novel HBx mutation F30V correlates with HCC in vivo, hampers HBV replicative efficiency and enhances anti-apoptotic activity of HBx N-terminus in vitro

The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro

ObjectiveWe aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. MethodsThis study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. ResultsF30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). ConclusionsF30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Hepatitis B virus (HBV) X gene mutations and their association with liver disease progression in HBV-infected patients.

Hepatitis B virus (HBV) is one of the most widespread human pathogens causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). This study investigated the clinical impact of single and combinational mutations in HBx gene on the pathogenesis of HCC during progressive stages of liver disease. The patients were categorized into inactive HBV carriers, active carriers, cirrhosis and HCC groups based on disease severity. Male sex, age > 50 years, and high serum alanine aminotransferase level were associated with risk of progressive liver disease. I127T, V131I, and F132Y/I/R mutations showed a significant increasing trend associated with the disease progression to HCC. H94Y and K130M mutations were also significantly associated with severe liver disease. One double mutation (K130M+V131I) and two triple mutations (I127T+K130M+V131L and K130M+V131I+F132Y) were observed, with significant rising prevalence through progressive clinical phases of liver disease to HCC. Several single and combinational mutations in HBx correlating with severity and progressive clinical phases of HBV infection were identified. The mutational combinations may have a synergistic effect in accelerating the progression to HCC. These specific patterns of HBx mutations can be useful in predicting the clinical outcome of HBV-infected patients and may serve as early markers of high risk of developing HCC.

Contribution of C1485T mutation in the HBx gene to human and murine hepatocarcinogenesis

Although Hepatitis B virus (HBV) X gene mutations are frequently detected in HBV-related human hepatocellular carcinoma (HCC) patients, causative HBx mutations in the development of HCC have not yet been determined. We herein identified C1485T and C1653T mutations in the HBx gene as independent risk of HCC for HBV through the analysis using serum from chronic hepatitis B patients. We generated transgenic mice expressing wild-type (WT-HBxTg) and mutant (C1485T-HBxTg) HBx to assess the carcinogenic potential of mutated HBx. C1485T-HBxTg mice were more susceptible to diethylnitrosamine-induced hepatocarcinogenesis than WT-HBxTg mice and control non-Tg mice. The promotion of hepatocarcinogenesis in C1485T-HBxTg mice was accompanied by the activation of β-catenin and Jun N-terminal kinase (JNK) signaling pathways as well as the production of reactive oxygen species, whereas the activation of nuclear factor-kappa B in the livers of C1485T-HBxTg mice was attenuated. These results demonstrate that the HBx C1485T mutation contributes to human and murine hepatocarcinogenesis.

HBx mutations promote hepatoma cell migration through the Wnt/β-catenin signaling pathway.

HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β‐catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β‐catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV‐associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/β‐catenin signaling in HBx‐transfected HCC cells and HBV‐related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP‐luc activity and increased nuclear translocation of β‐catenin. Moreover, the HBx Combo mutant increased and stabilized β‐catenin levels through inactivation of glycogen synthase kinase‐3β, resulting in upregulation of downstream target genes such as c‐Myc,CTGF, and WISP2. Enhanced activation of Wnt/β‐catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β‐catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV‐associated hepatocarcinogenesis.

X region mutations of hepatitis B virus related to clinical severity.

Chronic hepatitis B virus (HBV) infection remains a major health problem, with more than 240 million people chronically infected worldwide and potentially 650000 deaths per year due to advanced liver diseases including liver cirrhosis and hepatocellular carcinoma (HCC). HBV-X protein (HBx) contributes to the biology and pathogenesis of HBV via stimulating virus replication or altering host gene expression related to HCC. The HBV X region contains only 465 bp encoding the 16.5 kDa HBx protein, which also contains several critical cis-elements such as enhancer II, the core promoter and the microRNA-binding region. Thus, mutations in this region may affect not only the HBx open reading frame but also the overlapped cis-elements. Recently, several types of HBx mutations significantly associated with clinical severity have been described, although the functional mechanism in most of these cases remains unsolved. This review article will mainly focus on the HBx mutations proven to be significantly related to clinical severity via epidemiological studies.

A novel mutant 10Ala/Arg together with mutant 144Ser/Arg of hepatitis B virus X protein involved in hepatitis B virus-related hepatocarcinogenesis in HepG2 cell lines

Hepatitis B virus (HBV) infection-related hepatocellular carcinoma (HCC) represents a major health problem worldwide. HBV X (HBx) protein is the most common open reading frame that may undergo mutations, resulting in the development of HCC. This study aimed to determine specific HBx mutations that differentiate the central- and para-tumor tissues, and identify their association with HCC development. HBx gene from HCC tumor and para-tumor tissues of 47 HCC patients was amplified, sequenced and statistically analyzed. A novel combination of 2 mutations at residues 10 and 144 was identified which might play a significant role in HCC development. Expression vectors carrying HBx with the specific mutations were constructed and transfected into HepG2 and p53-null HepG2 cells. Compared to wild type (WT) and single mutation of HBx at residue 10 or 144, the 10/144 double mutations strongly up-regulated p21 expression and prolonged G1/S transition in WT- and p53-null HepG2 cells. Apoptosis was also inhibited by HBx harboring 10/44 double-mutation. Binding of 10/144 double-mutant HBx to p53 was lower than WT HBx. Conclusively, the 10/144 double mutation of HBx might play a crucial role in HCC formation.

Hidden secret in hepatitis B viral X protein mutation and hypoxia-inducible factor-1α in hepatocarcinoma cancer.

Hepatitis B type virus (HBV) is an old hepato oncogenic and hepatitis agent. Hepatitis B viral X protein (HBx)-induced malignant transformation requires the excess amounts of ATP level, inducing the extremely oxygen-deprived condition in the cancer tissues and vessels. To adapt, cells go to shift the hypoxic responsive state by altered hypoxia-responsive molecules such as HIF-1. In addition, tumors avoid or suppress immune recognition in the energy-deprived condition. The hypoxia-inducible factor-1α (HIF-1α) regulates MAP1, histone deacetylase and MAPK pathway. In the hypoxia, the HIF-1α interacts with HIF-1β, allowing DNA binding at the hypoxia response elements (HREs), while HBx binds with the nHLH/PAS domain of HIF-1α, preventing pVHL and HIF-1α binding capacity and degradation of HIF-1α protein. Recent work of Liu et al. [2013] demonstrated that HBx in hepatocellular carcinoma (HCC) tissues contained mutations, affecting the HBx transactivation capacity and C-terminal HBx mutation. In the HCC tissues, the HBx C-terminal mutation and HIF-1α expression were related and the different C-terminal mutations of HBx exhibit the different functionality of HIF-1α. The C-terminal region of amino acids 119-140 was important for the stability and transactivation, and the point mutations K130M/V131I enhance the functionality of HIF-1α, while C-terminal truncation diminish the HIF-1α function.

HBx mutants differentially affect the activation of hypoxia-inducible factor-1α in hepatocellular carcinoma

Background:Mutations in HBx gene are frequently found in HBV-associated hepatocellular carcinoma (HCC). Activation of hypoxia-inducible factor-1α (HIF-1α) contributes to HCC development and progression. Wild-type HBx has been demonstrated to activate HIF-1α, but the effect of HBx mutations on HIF-1α has not been elucidated.Methods:HBx mutations were identified by gene sequencing in 101 HCC tissues. Representative HBx mutants were cloned and transfected into HCC cells. Expression and activation of HIF-1α were analysed by western blot and luciferase assays, respectively. The relationship between HBx mutants and HIF-1α expression in HCC tissues was also evaluated.Results:The dual mutations K130M/V131I enhanced the functionality of HBx as they upregulated the expression and transcriptional activity of HIF-1α. The C-terminal truncations and deletion mutations, however, weakened the ability of HBx to upregulate HIF-1α. Meanwhile, the C-terminus was further found to be essential for the stability and transactivation of HBx. In the HCC tissues, there was a positive association between the HBx mutants and HIF-1α expression.Conclusion:Different mutations of HBx exert differentiated effects on the functionality of HIF-1α, however, the overall activity of HBx mutants appears to increase the expression and transcriptional activity of HIF-1α.

Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

Accumulation of the Mutations in Basal Core Promoter of Hepatitis B Virus Subgenotype C1 Increase the Risk of Hepatocellular Carcinoma in Southern China

Hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, HBV subgenotype C1 is the major subgenotype in Southern China. The aim of this study was to investigate whether there was the specific mutation patterns in HBV/C1 associated with Southern Chinese patients with HCC. Mutations in HBV basal core promoter (BCP) and their association with HCC were assessed in a matched cross-sectional control study of 102 HCC and 105 chronic hepatitis (CH) patients (from Guangdong, China) infected with HBV/C1. Functional analysis of HBx mutants was performed by the colony formation assay and the luciferase assays. T1762/A1764 double mutations was frequently found in patients infected with HBV/C1, regardless of clinical status (64.7% in HCC and 51.4% in CH, P>0.05). Unexpectedly, the adjacent V1753 or A1768 mutation significantly increased the risk of HCC (P<0.05). Moreover, the prevalence of triple or quadruple mutations in BCP was significantly higher in patients with HCC than those with CH, particularly for HBeAg-positive-carriers (P<0.05). Functional analysis revealed that T1762/A1764 mutation alone did not alter the transcriptional activity and the inhibitory effects on cell proliferation of HBx, but triple or quadruple mutations largely abrogated this effect. Accumulation of mutations involving V1753 or/and A1768 in addition to T1762/A1764 in BCP region were closely related to HCC among the patients infected with HBV/C1, particularly for HBeAg-positive-carriers. The increased risk of HCC caused by BCP variants may be attributable partially to modifying the biological functions of HBx.

Impact of Hepatitis B Virus (HBV) X Gene Mutations on Hepatocellular Carcinoma Development in Chronic HBV Infection

The hepatitis B virus (HBV) PreS mutations C1653T, T1753V, and A1762T/G1764A were reported as a strong risk factor of hepatocellular carcinoma (HCC) in a meta-analysis. HBV core promoter overlaps partially with HBx coding sequence, so the nucleotide 1762 and 1764 mutations induce HBV X protein (HBx) 130 and 131 substitutions. We sought to elucidate the impact of HBx mutations on HCC development. Chronically HBV-infected patients were enrolled in this study: 42 chronic hepatitis B (CHB) patients, 23 liver cirrhosis (LC) patients, and 31 HCC patients. Direct sequencing showed HBx131, HBx130, HBx5, HBx94, and HBx38 amino acid mutations were common in HCC patients. Of various mutations, HBx130+HBx131 (double) mutations and HBx5+HBx130+HBx131 (triple) mutations were significantly high in HCC patients. Double and triple mutations increased the risk for HCC by 3.75-fold (95% confidence interval [CI] = 1.101 to 12.768, P = 0.033) and 5.34-fold (95% CI = 1.65 to 17.309, P = 0.005), respectively, when HCC patients were compared to CHB patients. Functionally, there were significantly higher levels of NF-κB activity in cells with the HBx5 mutant and with the double mutants than that of wild-type cells and the triple-mutant cells. The triple mutation did not increase NF-κB activity. Other regulatory pathways seem to exist for NF-κB activation. In conclusion, a specific HBx mutation may contribute to HCC development by activating NF-κB activity. The HBx5 mutation in genotype C2 HBV appears to be a risk factor for the development of HCC and may be used to predict the clinical outcomes of patients with chronic HBV infection.

Sequential accumulation of the mutations in core promoter of hepatitis B virus is associated with the development of hepatocellular carcinoma in Qidong, China

To investigate the mutations in hepatitis B virus (HBV) that might be related to hepatocellular carcinoma (HCC) in the high-risk area Qidong, China. DNA sequences of HBV basal core promoter (BCP) and the overlapping X gene were determined in 58 HCC and 71 chronic hepatitis (CH) patients. In addition, a consecutive series of plasma samples from 15 HCC cases were employed to compare the CP/X sequences before and after the occurrence of HCC. T1762/A1764 double mutation was frequently found in Qidong patients, regardless of clinical status (65.5% in HCC and 73.2% in CH, P>0.05). Unexpectedly, the adjacent T1766/A1768 mutation significantly increased the risk of HCC (P<0.05). Moreover, the prevalence of triple mutations in BCP was significantly higher in patients with HCC than those with CH (P<0.05). The longitudinal study demonstrated that the mutations in BCP were gradually accumulated during the development of HCC. Colony formation assay showed while A1764 mutation alone did not alter the colony-inhibitory activity of HBx, double or triple mutations largely abrogated this effect. The complex mutation involving T1766/A1768 was closely related to HCC. The enhanced risk of HCC caused by BCP variants could be attributable partially to the aberrant activity of HBx.

Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular carcinoma

Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) in man. The viral transactivator HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC. The aim of this study was to investigate whether there were particular HBx mutations associated with the Chinese Hong Kong patients with HCC. We have examined HBx in 113 tumor tissue samples from patients with HCC and 48 serum samples from the same group. In addition, we also examined the expression of HBx protein and the index of apoptotic cell death in tumor tissues of HCC. The entire coding region of HBx gene from the sample was sequenced and aligned with the published HBx gene sequence. We have identified total 54 different types of mutations in HBx gene. HBx mutations occurred in a very high percentage of samples tested. Mutation of HBx was found in 95.2% and 95.3% of the tumor tissue and serum samples, respectively. Most of samples contained more than one type of the mutation. Relative risk analysis indicated that the mutations in 12 sites of tissue HBx and nine sites of serum HBx were highly associated with HCC, suggesting a potential role of these mutants in carcinogenesis. An insert mutation at position 204: Insert 204AGGCCC, was always found to co-exist with point mutations at 260 (G-->A) and 264 (G/C/T-->A). Furthermore, this particular pattern of HBx mutation was most frequently detected. Immunochemical staining of HBx protein revealed that the nuclear localization of HBx protein in hepatocytes of tumor tissues was highly associated with this particular pattern of HBx mutation. In conclusion, HBx mutation occurs frequently in HCC samples tested and a sample usually has multiple types of mutations. A special pattern of insert at 204 and point mutations at 260 and 264 was identified, and it appears to be associated with the nuclear localization of HBx protein. The development of multiple types of mutations in a given sample may contribute to the process of multiple steps in hepatocarcinogenesis.