HBx Activation Research Articles

Hepatitis B virus X protein induces p16 gene promoter methylation through upregulation of DNA methylation transferases DNMT1 and DNMT3A.

As a tumor suppressor, p16 can competitively block the cyclin D1-CDK4/6 complex to arrest the cell cycle in the G1 phase. Lack of p16 gene expression can lead to infinite cell proliferation and malignant transformation. To determine whether the hepatitis B virus X protein (HBx) regulates the methylation and expression of the p16 gene. We constructed a eukaryotic expression vector carrying the HBx gene and green fluorescent protein (GFP), and transfected it into HepG2 cells to build cell lines stably expressing GFP and GFP-HBx. The p16 protein level and p16 gene methylation were measured in these cell lines. We further detected the mRNA expression of DNA methyltransferases (DNMTs) 1, 3A and 3B. The DNMT1, DNMT3A and DNMT3B gene promoter sequences were inserted into a reporter vector (pGL3-Basic) to build recombinant vectors, which were then transiently transfected into different cell lines. After 48 h, the luciferase activity was measured. The level of p16 protein in GFP-HBx/HepG2 cells was significantly lower than in HepG2 and GFP/HepG2 cells. The CpG methylation was present in the p16 gene promoter region of GFP-HBx/HepG2 cells. The DNMT1 and DNMT3A mRNA levels in GFP-HBx/HepG2 cells were significantly elevated compared to that in the HepG2 cells (p = 0.0495). The luciferase activity in GFP-HBx/HepG2 cells transfected with the pGL3-DNMT1/3A pro plasmid was significantly higher than in HepG2 and GFP/HepG2 cells (both p < 0.05). The HBx can induce p16 gene promoter methylation and inhibit the expression of p16 in HepG2 cells. This occurs due to HBx activation of DNMT1 and DNMT3A promoters and the induction of p16 promoter methylation, which downregulates the expression of p16 protein.

The mutations frequency of enhancer II/HBx regions of hepatitis B virus in acutely infected Iranian patients: a cross-sectional study.

The viral transactivator HBx protein affect cellular, viral and pregenomic factors pathway. Mutations in this protein can produce new viruses with new antigenic determinants that are generally related to developing cancerous. In this cross-sectional study, 33 serum samples of patients diagnosed with acute HBV infection were investigated for HBeAg and HBV DNA viral load and HBx gene mutations. mutation in the HBx protein detected by sequencing analysis. Out of the 33 samples, 19 samples were males (57.6%), and 14 samples were females. 15 (45.5%) were positive for HBx DNA and 18 patients were negative for HBx DNA (54.5%). After sequencing, three mutations were recognized in HBx at nucleotide positions 147, 148, and 391 that were stationed to G1524A, G1525A, and G1767C mutations. The analysis result of this study shows G1524A and G1525A mutations that an important role in altering the inhibition function of the HBx activity domain. The G1767C mutation inactivates HBx transactivation activity. These mutations have a critical role in the pathogenicity of the virus, and the intensity of hepatic tissue demolition and the development of cirrhosis or carcinoma in patients can be understood.

Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) is an intermediate in the life cycle of HBV. HBV-encoded X protein (HBx), a key viral oncoprotein, can be specifically ubiquitylated by male specific lethal 2 (MSL2), which causes upregulation of HBx activity and promotes transcription, cell proliferation and tumor growth. The present study compared the levels of cccDNA, MSL2 mRNA and HBx mRNA in tumor and peri-tumor tissues, and clarified the effect of antiviral therapy on these indicators. Levels of intrahepatic cccDNA, MSL2 mRNA and HBx mRNA were determined using quantitative PCR in patients with HBV-associated HCC who had undergone liver surgery. A total of 50 patients were included in the present study. Prior to surgery, 31 patients had undergone antiviral treatment. Intrahepatic cccDNA levels were significantly higher in the tumor tissues compared with the peri-tumor tissues (P=0.001), particularly in the hepatitis B e antigen-positive (P=0.008), tumor recurrence (P=0.002) and <3 cm tumor size (P=0.003) groups. Furthermore, in patients with preoperative cirrhosis, levels of cccDNA and MSL2 mRNA were significantly higher in tumor tissues compared with that in peri-tumor tissues (P<0.001 and P=0.023, respectively). The expression levels of HBx mRNA in antiviral-treated tumors and peri-tumor tissues were significantly lower compared with those in untreated tissues (P=0.026 and P=0.035). The levels of cccDNA and MSL2 mRNA in the HBx-positive group were significantly higher in tumor tissues compared with those in peri-tumor tissues (P=0.026 and P=0.013). In conclusion, cccDNA participated in the tumorigenesis of HBV-associated HCC, and antiviral therapy was found to modulate hepatocarcinogenesis by decreasing the levels of HBx to inhibit the tumorigenic effect of MSL2 and cccDNA.

The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B

Chronic infection of hepatitis B virus (HBV) is associated with an increased incidence of hepatocellular carcinoma (HCC). HBV encodes an oncoprotein, hepatitis B x protein (HBx), that is crucial for viral replication and interferes with multiple cellular activities including gene expression, histone modifications, and genomic stability. To date, it remains unclear how disruption of these activities contributes to hepatocarcinogenesis. Here, we report that HBV exhibits antiresection activity by disrupting DNA end resection, thus impairing the initial steps of homologous recombination (HR). This antiresection activity occurs in primary human hepatocytes undergoing a natural viral infection–replication cycle as well as in cells with integrated HBV genomes. Among the seven HBV‐encoded proteins, we identified HBx as the sole viral factor that inhibits resection. By disrupting an evolutionarily conserved Cullin4A–damage‐specific DNA binding protein 1–RING type of E3 ligase, CRL4WDR70, through its H‐box, we show that HBx inhibits H2B monoubiquitylation at lysine 120 at double‐strand breaks, thus reducing the efficiency of long‐range resection. We further show that directly impairing H2B monoubiquitylation elicited tumorigenesis upon engraftment of deficient cells in athymic mice, confirming that the impairment of CRL4WDR70 function by HBx is sufficient to promote carcinogenesis. Finally, we demonstrate that lack of H2B monoubiquitylation is manifest in human HBV‐associated HCC when compared with HBV‐free HCC, implying corresponding defects of epigenetic regulation and end resection. Conclusion: The antiresection activity of HBx induces an HR defect and genomic instability and contributes to tumorigenesis of host hepatocytes.

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.

HBx promotes the proliferative ability of HL‑7702 cells via the COX‑2/Wnt/β‑catenin pathway.

Hepatitis B virus X protein (HBx) has been termed a viral oncoprotein, and is involved in the initiation and progression of hepatocellular carcinoma (HCC). Cyclooxygenase‑2 (COX‑2) and β‑catenin have been attributed to the oncogenic activity of HBx in HBV‑associated HCC. The present study aimed to determine whether there is crosstalk between COX‑2 and the Wnt/β‑catenin signaling pathway during HL‑7702‑HBx cell proliferation, and to investigate the associated underlying molecular mechanism. In the present study, cell proliferation assay, colony formation assay and flow cytometric analysis were used to detect the proliferative ability of cells. Reverse transcription‑quantitative polymerase chain reaction and western blotting were performed to examine the mRNA and protein expression of COX‑2, β‑catenin, cyclin‑D1 and c‑myc. The results demonstrated that HL‑7702‑HBx exhibited increased cell proliferation, higher colony formation efficiency and a shortened G1period of the cell cycle. In addition, the mRNA and protein expression levels of COX‑2 were increased, and this was associated with HL‑7702‑HBx cell growth. Furthermore, the expression of β‑catenin and its target genes, cyclin‑D1 and c‑myc proto‑oncogene protein, was upregulated by HBx via COX‑2. Finally, HBx promoted HL‑7702 cell proliferation through the Wnt/β‑catenin signaling pathway. In conclusion, the primary finding of the present study was that HBx may promote HL‑7702 cell proliferation via the COX‑2/Wnt/β‑catenin pathway. Thus, it may be helpful to further investigate the molecular mechanism of HBV‑associated hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication

Chronic HBV infection is a risk factor for hepatocellular carcinoma (HCC). The HBV HBx protein stimulates HBV replication and likely influences the development of HBV-associated HCC. Whether HBx affects regulators of metabolism in normal hepatocytes has not been addressed. We used an ex vivo, cultured primary rat hepatocyte system to assess the interplay between HBV replication and mechanistic target of rapamycin complex 1 (mTORC1) signaling. HBx activated mTORC1 signaling; however, inhibition of mTORC1 enhanced HBV replication. HBx also decreased ATP levels and activated the energy-sensing factor AMP-activated protein kinase (AMPK). Inhibition of AMPK decreased HBV replication. Inhibition of AMPK activates mTORC1, and we showed that activated mTORC1 is one factor that reduces HBV replication when AMPK is inhibited. HBx activation of both AMPK and mTORC1 suggests that these activities could provide a balancing mechanism to facilitate persistent HBV replication. HBx activation of mTORC1 and AMPK could also influence HCC development.

E3 Ubiquitin Ligase Siah-1 is Down-regulated and Fails to Target Natural HBx Truncates for Degradation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common worldwide malignancy with high morbidity and mortality. Hepatitis B viral (HBV)-encoded X protein (HBx) and natural HBx variants play important roles in HBV-associated HCC development. HBx is an unstable protein that can be degraded in vivo. Our previous study found that the E3 ubiquitin ligase Siah-1 could target HBx for poly-ubiquitylation and proteasomal degradation and attenuate the transcriptional activity of HBx. However, in HCC patients, high expression levels of HBx and HBx variants are frequently observed and are associated with HCC progression. The mechanism underlying their up-regulation is largely unknown. In this study, we screened for Siah-1 mutations in 270 HCC samples and 9 HCC cell lines, and examined Siah-1 mRNA and protein expression in a subset of paired HCC specimens. Our results demonstrate that Siah-1 is highly conserved, as no somatic mutation was identified, with the exception of one synonymous transition from G to A at codon 67. Both the mRNA and protein levels of Siah-1 were significantly down-regulated in HCC tissues compared with their adjacent normal counterparts. We constructed three natural HBx truncates that were identified in our HCC cases. We found that Siah-1 failed to decrease the stability of these HBx variants and was unable to inhibit the transcriptional activity of these HBx truncates at heat shock elements (HSEs). Moreover, Siah-1 had weaker association with three HBx mutants than full length HBx. Therefore, our findings suggest that down-regulation of Siah-1, but not its mutations, and natural HBx variants resistant to Siah-1-induced degradation may be a novel mechanism for HCC development.

Technical standards for hepatitis B virus X protein (HBx) research.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies.

Downregulation of mPGES‐1 Expression via EGR1 Plays an Important Role in Inhibition of Caffeine on PGE2 Synthesis of HBx(+) Hepatocytes

We investigated the mechanism of caffeine in influencing HBx(+) hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200–800 μM) and treatment times (1–7 days), which was first observed at the second test time point (caffeine treatment for 4 days). The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800 μM caffeine and at caffeine treatment for 7 days. The PGE2 secretion and the expression of mPGES-1 and EGR1 were downregulated, whereas PPARγ expression was upregulated. The mPGES-1 promoter activity of HBx(+) hepatocytes decreased more significantly than that of HBx(−) hepatocytes. Moreover, the expression of EGR1 and PPARγ changed more significantly in HBx(+) hepatocytes cultured for 12 to 24 hours in the presence of 5 mM caffeine. This limited success may be attributed to caffeine releasing the binding of HBx and PPARγ and furthermore affecting the mPGES-1 expression by EGR1 in HBx(+) hepatocytes. The results indicate that caffeine could effectively reduce PGE2 synthesis in HBx(+) hepatocytes by specifically blocking the PPARγ-EGR1-mPGES-1 pathway, thereby providing a new evidence of molecular biology for the hypothesis that drinking coffee is beneficial to HBV-infected patients.

The hepatitis B virus (HBV) HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an essential role of HBx during HBV replication, HBx activation of AKT inhibits hepatocyte apoptosis, and this may facilitate persistent, noncytopathic HBV replication. AKT regulates HBV replication by reducing the activity of the transcription factor hepatocyte nuclear factor 4α (HNF4α). HBx activation of AKT may contribute to the development of liver cancer by facilitating persistent HBV replication, augmenting the dedifferentiation of hepatocytes by inhibiting HNF4α functions, and activating AKT-regulated oncogenic pathways. AKT-regulated factors may provide therapeutic targets for inhibiting HBV replication and the development of HBV-associated liver cancer.

La protéine X du virus de l'hépatite B et son rôle dans le développement du carcinome hépatocellulaire.

Hepatocellular carcinoma (HCC) is the most frequent form of liver cancer worldwide, and represents the third cause of death. While epidemiological studies have clearly established that hepatitis B virus (HBV) infection is a major risk factor for the development of HCC, the molecular mechanisms underlying virally-induced tumourigenesis are not fully understood. The transcriptional regulatory HBx protein has been described as a multifunctional protein exhibiting numerous activities affecting gene transcription, intracellular signal transduction, cell proliferation, apoptosis and DNA repair. While any or all of the multiple activities of HBx could contribute to hepato-carcinogenesis, HBx is not considered as an oncogene. HBx rather acts as a co-factor of carcinogenesis, through the up-regulation of a large number of cellular genes involved in oncogenesis, proliferation, inflammation and immune response. In this review, we will summarize the current knowledge on the mechanisms involving HBx protein in liver carcinogenesis.

Modulation of apoptotic signaling by the hepatitis B virus X protein.

Worldwide, an estimated 350 million people are chronically infected with the Hepatitis B Virus (HBV); chronic infection with HBV is associated with the development of severe liver diseases including hepatitis and cirrhosis. Individuals who are chronically infected with HBV also have a significantly higher risk of developing hepatocellular carcinoma (HCC) than uninfected individuals. The HBV X protein (HBx) is a key regulatory HBV protein that is important for HBV replication, and likely plays a cofactor role in the development of HCC in chronically HBV-infected individuals. Although some of the functions of HBx that may contribute to the development of HCC have been characterized, many HBx activities, and their putative roles during the development of HBV-associated HCC, remain incompletely understood. HBx is a multifunctional protein that localizes to the cytoplasm, nucleus, and mitochondria of HBV‑infected hepatocytes. HBx regulates numerous cellular signal transduction pathways and transcription factors as well as cell cycle progression and apoptosis. In this review, we will summarize reports in which the impact of HBx expression on cellular apoptotic pathways has been analyzed. Although various effects of HBx on apoptotic pathways have been observed in different model systems, studies of HBx activities in biologically relevant hepatocyte systems have begun to clarify apoptotic effects of HBx and suggest mechanisms that could link HBx modulation of apoptotic pathways to the development of HBV-associated HCC.

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.

Overexpression of hepatitis B x-interacting protein in HepG2 cells enhances tumor-induced angiogenesis

Hepatocellular carcinoma (HCC) is a common malignancy and a leading cause of cancer death worldwide. Hepatitis B x-interacting protein (HBXIP), a cofactor of survivin, was originally identified by binding with the C-terminus of the HBx and negatively regulated the activity of HBx. In this study, the effect of HBXIP on the hepatoma cells-induced angiogenesis was investigated. Proliferation and migration of human umbilical vein endothelial cells (HUVECs) were detected by MTT and transwell assay, respectively. Tube formation and chick chorioallantoic membrane model were used to observe the angiogenesis. Vascular endothelial growth factor activity was assayed using ELISA kits. Western blotting was performed to examine the protein expression. Our results indicated that overexpression of HBXIP increased HepG2 cell-induced endothelial cells migration, proliferation, and angiogenesis, which may be related to increasing phosphorylation of endothelial NO synthase in HUVECs. These results suggest that HBXIP may play an important role in tumorigenesis by enhancing angiogenesis in HCC.

The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication.

The C-terminal region of the hepatitis B virus X protein is essential for its stability and function

More than 350 million people worldwide are chronically infected with the human hepatitis B virus (HBV). Chronic HBV infections are associated with the development of hepatocellular carcinoma. While the mechanism of HBV-associated carcinoma remains undefined, it is thought to involve a combination of a continuous inflammatory response to HBV-infected hepatocytes and activities of HBV proteins such as the HBV X protein (HBx). HBx stimulates HBV replication; however, the mechanism by which HBx stimulates HBV replication remains incompletely understood. Studies performed with the woodchuck hepatitis virus (WHV) in woodchucks demonstrated that a C-terminally truncated mutant of the WHV X protein could not stimulate WHV replication. However, whether the C-terminus of HBx is important for HBx-stimulation of HBV replication is unclear. We have constructed C-terminal truncation mutants of HBx and have demonstrated that the C-terminus of HBx impacts HBx stability, HBx activation of transcription, and HBx stimulation of HBV replication. These observations highlight the impact of the HBx C-terminus on HBx activities and the importance of directly analyzing HBx expression and functions in HBV-associated tumors that contain chromosomal integrants of HBV with truncations of the HBx gene.

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.

The Hepatitis B Virus X Protein Modulates Hepatocyte Proliferation Pathways To Stimulate Viral Replication

Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G(1) phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G(1) phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.

Pro-apoptotic function of hepatitis B virus X protein

Infection of hepatitis B virus (HBV) is a main cause of liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Among the HBV-encoded proteins, the HBV X protein (HBx) has been suspected to be strongly involved in HBV-associated liver pathogenesis. HBx, a virally encoded multifunctional regulator, has been shown to induce apoptosis, anti-apoptosis, proliferation, and transformation of cells depending on the cell lines, model systems used, assay protocols, and research groups. Among the several activities of HBx, the pro-apoptotic function of HBx will be discussed in this review. Given that the disruption of apoptosis pathway by HBx contributes to the liver pathogenesis, a better understanding of the molecular interference in the cellular pro-apoptotic networks by HBx will provide useful clues for the intervention in HBV-mediated liver diseases.