Wild-type HBx Research Articles

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

HBx Protein Potentiates Hepatitis B Virus Reactivation

Hepatitis B virus (HBV) can cause chronic hepatitis B, one of the most prevalent infectious diseases in the world. Global estimates suggest that over 2 billion people are affected by HBV, with over 250 million people developing chronic infection. Upon treatment of comorbidities, patients with chronic infection may develop an abrupt increase of viral replication-HBV reactivation-leading to liver decompensation and, in some cases, death. HBV reactivation occurs mostly due to suppression of antiviral immune response and activation of intracellular pro-viral signaling. Defining the mechanisms of HBV reactivation is necessary for the rational use of drugs and reduction of mortality rates in patients with chronic infection. In this study, for the first time we analyzed the effects of HBx protein on HBV reactivation, described reactivation of HBV from the transcriptionally inactivated state at the methylated recombinant HBV genome model, and investigated HBV reactivation upon treatment with genotoxic agents (doxorubicin and hydrogen peroxide) and targeted drug therapies (sunitinib and bortezomib). We report that both wild-type HBx protein and, to a greater extent, the mutant form of HBx protein lacking the nuclear exportation signal, potentiate viral replication and promote HBV reactivation. For the first time, we demonstrate that HBV can reactivate from the transcriptionally inactive state. Doxorubicin and hydrogen peroxide induce HBV reactivation at models of both transcriptionally active and transcriptionally silenced viral genome. Sunitinib weakly reactivates HBV, while bortezomib does not affect HBV replication in vitro.

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.

Effect of carboxyl-terminal truncated HBx mediated down-regulation of RhoGDIα expression on the metastasis and invasion of hepatocellular carcinoma

Objective To investigate the mechanism of natural carboxyl-terminal truncated HBx (with 31 amino acids deleted at the C-terminal end) (HBxΔ31)-dependent down-regulation of Rho GDP dissociation inhibitor alpha (RhoGDIα) expression and its role in enhancing hepatocellular carcinoma (HCC) metastasis. Methods HepG2 cells with stable expression of wild type HBx and its deletion mutant HBxΔ31 protein were selected as the study subjects. The effects of HBx and HBxΔ31 on RhoGDIα expression in HepG2 cells was detected using real-time quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. A series of deleted and mutated variants of the RhoGDIα promoter were made and individually co-transfected with HBx-and HBxΔ31-expressing vectors or control empty vector into HepG2 cells. The luciferase reporter assay was used to identify the cis-regulatory elements of the RhoGDIα promoter in response to HBxΔ31 regulation. The interaction between Myc-associated zinc finger protein (MAZ) and HBxΔ31 was examined by co-immunoprecipitation experiment. Eelectrophoretic mobility shift assay (EMSA) was performed to determine interaction of MAZ with the RhoGDIα promoter in HBxΔ31-expressing HepG2 cells. The impact of reduced RhoGDIα expression by HBxΔ31 on cell-invasive activity was analyzed by the Matrigel cell invasion assay. In addition, the effects of silencing of shRNA-mediated RhoGDIα in HepG2 or introduction of RhoGDIα by transfection in HBxΔ31-expressing HepG2 cells on cell-invasion were investigated. Results HBxΔ31, but not HBx, suppressed RhoGDIα expression at transcriptional levels. Analysis of the deletion and mutation of RhoGDIα promoter showed that the HBxΔ31 repressive element localized between nt-460 and-242 bp of RhoGDIα promoter and that the transcription factor MAZ binding sites was required for RhoGDIα promoter inactivation regulated by HBxΔ31. In addition, HBxΔ31 represses RhoGDIα expression by enhancing MAZ binding to its promoter through directly associating with MAZ. The cell-migratory and cell-invasive activity were significantly increased in sh-RhoGDIα-expressing HepG2 cells, as compared to control cells (migrated cells number: 58 ± 5 vs. 98 ± 7, invaded cells number: 55 ± 6 vs. 113 ± 6, t= 18.91 and t= 20.12, both P < 0.01). However, ectopic expression of RhoGDIα in HBxΔ31-expressing HepG2 cells significantly decreased cell migration and invasion (migrated cells number: 40 ± 4 vs. 115 ± 5, invaded cells number: 42 ± 4 vs. 102 ± 4, t= 18.14 and t= 16.31, both P < 0.001). Conclusion Carboxyl-terminal truncated HBx deregulates RhoGDIα expression through MAZ, in turn which promotes the invasion and metastasis of HCC. Key words: Carcinoma, hepatocellular; Sequence deletion; Hepatitis B virus; Gnanine nucleotide dissociation inhibitors; Neoplasm metastasis

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B.

AIMTo determine the role of hepatitis B virus X protein (HBx), HBx in regulating hepatic progenitor cell (HPC)-like features in hepatocellular carcinoma (HCC) and the underlying molecular mechanisms.METHODSWe used a retrovirus vector to introduce wild type HBx or empty vector into HepG2 cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Gene expression profiling was carried out on Affymetrix GeneChip Human U133A2.0 ver.2 arrays according to the manufacturer’s protocol. Unsupervised hierarchical clustering analysis and Class Comparison analysis were performed by BRB-Array Tools software Version 4.2.2. A total of 238 hepatitis B virus (HBV)-related HCC patients’ array data were used for analyzing clinical features.RESULTSThe histone demethylase KDM5B was significantly highly expressed in HBV-related HCC cases (P < 0.01). In HBV proteins, only HBx up-regulated KDM5B by activating c-myc. Hepatic stem cell (HpSC) markers (EpCAM, AFP, PROM1, and NANOG) were significantly highly expressed in KDM5B-high HCC cases (P < 0.01). KDM5B played an important role in maintaining HpSC-like features and was associated with a poor prognosis. Moreover, inhibition of KDM5B suppressed spheroid formation and cell invasion in vitro.CONCLUSIONHBx activates the histone demethylase KDM5B and induces HPC-like features in HCC. Histone demethylases KDM5B may be an important therapeutic target against HBV-related HCC cases.

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.

Wild type HBx and truncated HBx: Pleiotropic regulators driving sequential genetic and epigenetic steps of hepatocarcinogenesis and progression of HBV-associated neoplasms.

Hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma. The molecular mechanisms of tumorigenesis are complex. One of the host factors involved is apparently the long-lasting inflammatory reaction which accompanies chronic HBV infection. Although HBV lacks a typical viral oncogene, the HBx gene encoding a pleiotropic regulatory protein emerged as a major player in liver carcinogenesis. Here we review the tumorigenic functions of HBx with an emphasis on wild type and truncated HBx variants, and their role in the transcriptional dysregulation and epigenetic reprogramming of the host cell genome. We suggest that HBx acquired by the HBV genome during evolution acts like a cellular proto-onc gene that is activated by deletion during hepatocarcinogenesis. The resulting viral oncogene (v-onc gene) codes for a truncated HBx protein that facilitates tumor progression. Copyright © 2015 John Wiley & Sons, Ltd.

Stronger activation of SREBP-1a by nucleus-localized HBx

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of the nucleus-localized HBx in regulating host lipogenic pathway and HBV replication.

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

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.

Effect of microRNA on proliferation caused by mutant HBx in human hepatocytes

To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein, HBx, in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect. The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation. The differential miRNA expression profiles were determined by microarray analysis and confirmed by real-time PCR. Two miRNAs, miR-338-3p and miR-551b, that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure. The cell survival rate was analyzed by MTT assay, and cell cycles were assessed by flow cytometry. Expressions of cyclinD1, cyclinG1, and E2F1 were assessed by real-time PCR and Western blotting. Compared with the microarray miRNA profile of L02/pcDNA3.0 cells, six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells, while four miRNAs were up-regulated and 12 were down-regulated in the L02/HBx cells. The microarray results were consistent with real-time PCR results. Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P less than 0.001) and induced G0/G1 phase cycle arrest. According to MTT results: for L02/HBx-d382 cells, compared with lipofectamine or non-transfected (NC) controls, the t value of miR-338-3p was 10.402, 9.133 and the t value of miR-551b was 8.763, 7.403; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 9.105, 8.074 and the t value of miR-551b was 7.673, 7.52. According to flow cytometry results: for L02/HBx-d382 cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 12.173, 11.107 and the t value of miR-551b was 15.364, 13.377; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 15.416, 13.378, and the t value of miR-551b was 13.276, 13.109. The protein levels of cyclinD1, cyclinG1, and E2F1 were significantly reduced by both miR-338-3p and miR-551b ( P less than 0.001). For L02/HBx-d382 cells, compared with lipofectamine or NC controls: E2F1 had t = 11.132, 10.031 and 12.017, 10.973, respectively; cyclinD1 had t = 15.654, 15.013 and 15.447, 14.733, respectively; cyclinG1 had t = 8.017, 7.661 and 7.402, 7.417, respectively. For L02/HBx cells, compared with lipofectamine or NC controls: E2F1 had t = 14.244, 13.331 and 15.022, 14.468, respectively; cyclinD1 had t = 8.695, 8.137 and 7.877, 7.503, respectively; cyclinG1 had t = 7.73, 7.471 and 7.596, 7.41, respectively. In contrast, the mRNA levels for E2F1, cyclinD1, and cylcinG1 showed no significant differences between the miRNA transfected cells and controls. Wild-type HBx and the high proliferation-inducing mutant HBx can influence the miRNA expression profile of L02 cells. HBx down-regulates miR-338-3p and miR-551b in L02 cells, and the high proliferation-inducing mutant has a more robust effect. The mechanism of miR-338-3p- or miR-551b-mediated cell growth inhibition appears to be related to the direct modulation of cyclinD1, cyclinG1, and E2F1.

A natural mutation of the hepatitis B virus X gene affects cell cycle progression and apoptosis in Huh7 cells

Hepatocellular carcinoma (HCC), one of the most prevalent cancers, and HCC-associated diseases are caused by the hepatitis B virus (HBV). Viral sequences often contain mutations in the basal core promoter (BCP), which overlaps with the open reading frame encoding HBV X protein (HBx). HBx protein plays a very important role in the development of HCC. Influence of naturally-occurring mutation of HBx (BCP1) on cell cycle progression was investigated in the Huh7 HCC line. BCP1 mutation was cloned from clinical samples into recombinant green fluorescent protein-fusion expression plasmid constructs that were then transfected into Huh7 cells for either transient expression studies or isolation of stable transfectants, along with wild-type HBx and empty vector controls. The mutant protein was more effective than wild-type in reducing expression of cyclindependent kinase (CDK) inhibitor p27kip1, promoting cell cycle progression, inhibiting production of cleaved poly ADP ribose polymerase (PARP) (a marker for apoptosis), and down-regulating the multifunctional cytokine transforming growth factor-β (TGF-β). Since TGF-controls the cell cycle and apoptosis, the effect of BCP1 mutation on the enhanced activity of HBx to promote cell cycle progression and to inhibit apoptosis in Huh7 cells may result from down-regulation of TGF-β.

Hepatitis B Virus Core Promoter Mutations Contribute to Hepatocarcinogenesis by Deregulating SKP2 and Its Target, p21

Clinical studies have associated hepatitis B virus core promoter (CP) mutations with an increased risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been implicated in hepatocarcinogenesis. The cyclin kinase inhibitor p21WAF1/CIP1 is an important regulator of cell cycle progression and proliferation. We determined whether HBx mutants that result from mutations in the CP deregulate p21 and these processes. We constructed a series of HBx mutants with changes in the CP region that correspond to A1762T/G1764A (TA), T1753A, T1768A, or a combination of these (combo) and expressed them, along with wild-type HBx under control of its endogenous promoter, in primary human hepatocytes (PHHs) and HepG2 cells. We then analyzed the effects of CP mutations on expression and degradation of p21 and the effects on cell cycle progression and proliferation. The combo mutant decreased levels of p21 and increased cyclin E expression in PHHs and HepG2 cells. The combo mutant, but not HBx with single or double CP mutations, accelerated p21 degradation in HepG2 cells. The combo mutant increased expression of S-phase kinase-associated protein 2 (SKP2) in PHHs and Huh7 cells. Silencing of SKP2 abrogated the effects of CP mutations on p21 expression. The kinetics of p21 expression correlated with changes in cell cycle distribution. The combo mutant accelerated cell cycle progression; p21 overexpression restored G1 arrest. HBx mutants with changes that correspond to a combination of CP mutations up-regulate SKP2, which then down-regulates p21 via ubiquitin-mediated proteasomal degradation. CP mutations might increase the risk of hepatocellular carcinoma via this pathway.

A mutant of hepatitis B virus X protein (HBxΔ127) enhances hepatoma cell migration via osteopontin involving 5-lipoxygenase

To explore a novel function of a mutant of the hepatitis B virus X protein (HBx Delta 127) in the promotion of hepatoma cell migration. The effect of HBx Delta 127 and wild type HBx on the migration ability of hepatoblastoma HepG2 cells were examined using wound healing assays in stable transfection systems. The full-length osteopontin(OPN) promoter sequence was cloned into the pGL3-Basic plasmid. The promoter activities of OPN in stably HBx Delta 127-transfected hepatoblastoma HepG2 (HepG2-X Delta 127) and hepatocellular carcinoma H7402 (H7402-X Delta 127) cells were determined using luciferase reporter gene assays. The mRNA expression levels of OPN were detected by RT-PCR. And the effect of MK886, a specific inhibitor of 5-lipoxygenase (5-LOX), on OPN promoter activity and mRNA expression in HepG2-X Delta 127 and H7402-X Delta 127 cells were examined using luciferase reporter gene assays and RT-PCR, respectively. Finally, the migration ability of HepG2-X Delta 127 was observed after treatment with siRNA targeting OPN mRNA and HBx mRNA using wound healing assays. HepG2-X Delta 127 cells exhibited a greater capacity for wound repair compared to HepG2-X cells. The promoter activity and mRNA expression levels of OPN were also increased in HepG2-X Delta 127 and H7402-X Delta 127 cells. Moreover, MK886 abolished the HBx Delta 127-mediated upregulation of OPN. Wound healing assays demonstrated that the migration ability of HepG2-X Delta 127 cells can be suppressed by treatment with siRNA targeting OPN mRNA and siRNA targeting HBx mRNA. HBx Delta 127 strongly promotes hepatoma cell migration via activation of OPN involving 5-LOX.

Interaction of Hepatitis B Viral Oncoprotein with Cellular Target HBXIP Dysregulates Centrosome Dynamics and Mitotic Spindle Formation

Hepatitis B virus infection is associated with hepatocellular carcinoma, claiming 1 million lives annually worldwide. To understand the carcinogenic mechanism of hepatitis B virus-encoded oncoprotein HBx, we explored the function of HBx interaction with its cellular target HBXIP. Previously, we demonstrated that viral HBx and cellular HBXIP control mitotic spindle formation, regulating centrosome splitting. By using various fragments of HBx, we determined that residues (137)CRHK(140) within HBx are necessary for binding HBXIP. Mutation of the (137)CRHK(140) motif in HBx abolished its ability to bind HBXIP and to dysregulate centrosome dynamics in HeLa and immortal diploid RPE-1 cells. Unlike wild-type HBx, which targets to centrosomes as determined by subcellular fractionation and immunofluorescence microscopy, HBx mutants failed to localize to centrosomes. Overexpression of viral HBx wild-type protein and knockdown of endogenous HBXIP altered centrosome assembly and induced modifications of pericentrin and centrin-2, two essential proteins required for centrosome formation and function, whereas HBXIP nonbinding mutants of HBx did not. Overexpression of HBXIP or fragments of HBXIP that bind HBx neutralized the effects of viral HBx on centrosome dynamics and spindle formation. These results suggest that HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation.

Gene expression profiles and proteomics analysis of the cell transfected with the mutant type of COOH-terminal deleted of hepatitis B virus X

To investigate the mechanism of the different biological impacts of HBx3'-40, an engineered deletion mutant lacking the last 40 C-terminal amino acids. Human hepatocellular cells of the line Huh7 were transfected with HBx3'-40 or wtHBx (wild type HBx) constructs. An oligo cDNA microarray containing 21074 human genes and Ests was utilized to examine the different gene expression between HBx3'-40 and paired control wtHBx cells. A series of methods, including immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), silver staining, and PDQuest 2-D analysis software were used to analyze the differential protein-spots between HBx3'-40 and wtHBx cells. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF-MS) and database searching. 165 (0.82%) candidate genes showing aberrant expression under HBx3'-40 induction were identified, of which 144 were up-regulated while 21 were down-regulated. Compared with wtHBx group, there were 135 +/- 13 differently protein spots in HBx3'-40 group by 2-DE. Of them, 7 significantly different protein spots were identified using mass spectrometry and computer matching with protein database. Most of the differently expressed genes and proteins were involved in transcription, oncogenes and tumor suppressor genes or protein, cell adhesion, signal transduction pathways, metabolisms, etc. The mutant HBx affects cell genes and proteins involved in various processes, especially in regulation of liver metabolism.

Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein.

Various functions are ascribed to the HBx regulatory protein of the hepatitis B virus (HBV). Due to the low expression level of HBx, it has been difficult to correlate spatial and temporal HBx expression levels with specific functions. Based on a novel cell-permeable peptide, known as the translocation motif (TLM), cell-permeable HBx fusion proteins were generated. The TLM-HBx fusion protein is rapidly internalized from the medium into almost all cells, whereas no significant internalization was seen with wild-type HBx. The major fraction of internalized HBx protein moves from the cytoplasm to the nucleus. The cytosolic fraction, however, activates c-RAF1/extracellular-signal-related kinase 2 signalling and causes activation of activator protein 1 (AP1) and nuclear factor-kappaB. The TLM-HBx protein rescues HBV gene expression from an activator-deficient HBV genome. These results indicate that cell-permeable regulatory proteins provide a novel, efficient tool for a clearly defined, dose-dependent analysis of regulatory protein function, without affecting the integrity of the cell, and can be used for the safe reconstitution of virus production from a regulatory-protein-deficient virus genome.

A carboxy-terminal region of the hepatitis B virus X protein promotes DNA interaction of CREB and mimics the native protein for transactivation function.

Earlier we had shown that the conserved region E (residues 120-140) of HBV X protein (HBx) is crucial for transactivation. To investigate this region further, its oligomerisation was considered necessary to augment intracellular biochemical stability. Two to ten unit long tandem repeats of the E region (X16-n) were generated and their expression vectors constructed. Transient transfection of the E expression vectors along with different CAT constructs showed increase in the reporter activity. Interestingly a direct correlation was observed between the number of E repeat units in an expression vector and the level of transactivation. The transactivation levels with decameric X16 on different reporter constructs were comparable to those of the wild type HBx. Co-expression of X16 in a stable CHO-K1 cell line expressing the native HBx, showed co-operativity for transactivation. Further, X16 facilitated the binding of cAMP response element binding protein (CREB) to its responsive element just like the native HBx. The present study suggests that the C-terminal 'E' region of HBx represents its transactivation domain that acts by promoting the interaction of transcription factors to their cognate response elements.

Human hepatitis B virus X protein is detectable in nuclei of transfected cells, and is active for transactivation

Subcellular localization and transactivation of human hepatitis B virus X protein (HBx), a plausible causative factor for hepatocellular carcinogenesis, were studied in transiently transfected cells. The transactivation was detected not only by the cis-element driven chloramphenicol acetyltransferase (CAT) assay but also by immunostaining of CAT protein cotransfected into human hepatoma cell line HepG2. Scanning fluorescence microscopy showed the majority of immunological signals of HBx to be at the perinuclear region of transfected cytoplasm. HBx was also clearly detectable in the nucleus, though less intensely expressed. This was confirmed by Western analysis and coimmunoprecipitation of HBx with transcription factor IIB (TFIIB) in subcellular fractionations. The percentage of HBx-positive cells coincided with that of CAT-positive cells, and confocal laser microscopy revealed the coexistence of CAT signals in GFP-HBx positive cells. The SV40 large T antigen nuclear localization signal (NLS) appended HBx, regardless of whether NLS was added to the N- or C-terminus, transactivated all the examined X-responsive elements (XRE) similarly as did wild-type HBx. Similar results were obtained in p53 negative Saos-2 cells. The detected nuclear HBx may be involved in modulating the transcription at the promoter level whereas the HBx in cytoplasm may be working through signal transduction pathways.

Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication.

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.