Reactive oxygen species modulates the intracellular level of HBx viral oncoprotein

HBx (hepatitis B virus X) viral oncoprotein is a multifunctional protein of which the cellular level may be one of the important factors in determining HBV-mediated pathological progression of liver diseases, chronic hepatitis, and hepatocellular carcinoma. Our previous work revealed that adriamycin, a chemotherapeutic agent, caused a marked increase in the intracellular level of HBx by retarding its rapid degradation. In the present study, modulation of HBx expression was found to be confined to adriamycin but not to other chemotherapeutic agents, cisplatin and 5-fluorouracil. Interestingly, adriamycin caused a rapid increase of reactive oxygen species (ROS) and its accumulation continued until 24h. In contrast, two other agents had little effect on ROS generation, suggesting the possible involvement of ROS in the HBx regulation. In fact, direct addition of H(2)O(2) to the cells significantly increased the level of HBx protein in HBx-expressing ChangX-34 cells as well as in hepatitis B virus-related hepatoma cells, PLC/PRF/5 and HepG2.2.15 cells. Furthermore, antioxidants, N-acetyl-cysteine and pyrrolidinedithiocarbamate (PDTC), completely abolished the increase of HBx protein induced by adriamycin, indicating that adriamycin modulates the intracellular HBx level via ROS generation. Together, these findings provide a novel aspect of HBx regulation by cellular ROS level. Therefore, intracellular microenvironments generating ROS such as severe inflammation may aggravate the pathogenesis of liver disease by accumulating the HBx level.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

Involvement of hepatitis B virus X gene (HBx) integration in hepatocarcinogenesis via a recombination of HBx/Alu core sequence/subtelomeric DNA

The significance of hepatitis B virus (HBV) DNA-based integration in hepatocarcinogenesis is poorly understood. In the present study, we investigated whether the integration of HBV X gene (HBx) is involved in the event. Our finding showed that the integration of HBx fragment (316-462 bp/262-462 bp) was able to transform human immortalized normal liver LO2 cells using a cell model of HBx-integration. We identified that the recombination, HBx/Alu core sequence/subtelomeric DNA, was required for the transformation, which could be detected in 5 out of 44 clinical HBx-positive hepatocellular carcinoma tissues. Thus, we conclude that HBx integration is involved in the hepatocarcinogenesis.     

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.     

Expression of Jagged1 and its association with hepatitis B virus X protein in hepatocellular carcinoma

Jagged1 is one of the ligands of Notch signaling pathway, which controls cellular proliferation and differentiation, and also plays important roles in various malignant tumors. However, the expression of Jagged1 in hepatocellular carcinoma (HCC) has not been elucidated, nor whether it is associated with hepatitis B virus X protein (HBx). In this study, we found that Jagged1 was highly expressed in 79.2% (42/53) of HCC tissues compared with adjacent nontumor liver (P <0.05), and its expression was found to be closely related with HBx (rs=0.522, P <0.001) in HCC tissues. Our in vitro study also showed that alteration of HBx expression in HCC cell lines led to a consistent change of Jagged1. Moreover, Jagged1 was found to co-localize and directly interact with HBx in HCC tissues and HBx expressed HCC cell lines. Our results reveal that Jagged1, which is regulated by HBx, may contribute to the development of HCC.     

Expression,purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-x_L, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-x_L, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-x_L fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-x_L and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-x_L at the atomic level.     

Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18

Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2'-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.     

Polymorphism analyses of hepatitis B virus X gene in hepatocellular carcinoma patients from southern China

Chronic hepatitis B virus(HBV)infection is one of the major causes of hepatocellularcarcinoma(HCC),and the HBV X(HBx)gene plays a critical role in the molecular pathogenesis ofHBV-related HCC.We have investigated whether there are particular HBx gene mutations associated withHCC in patients from southern China.The HBx gene was examined in 51 paraffin-embedded tumor tissuesamples from patients with HCC and 25 serum samples from the HBV carrier by nested polymerase chainreaction(PCR),single-stranded conformational polymorphism and heteroduplex analysis.The HBx geneswith potentially important mutations from tumor tissue samples were cloned,sequenced and aligned withthe published HBx gene sequence.HBV genotypes in tumor tissue samples were analyzed by nested PCR.Analyses of HBx gene polymorphism showed that 31.3% of HBx gene fragments in tumor tissue sampleshad a special pattern.A common deletion at nt 382-400 of the HBx gene accompanied by 29 point mutationswas detected in four randomly selected tumor tissue samples with this pattern which caused a frame-shiftin the HBx open reading frame with a new stop codon at nt 1818,resulting in an HBx polypeptide chaintruncated at the C end in these cases.Among the four randomly selected samples,three were HBV genotypeB,and one was not detected by our present assay.In another tumor tissue sample,amplification of thefull-length HBx gene yielded a shorter fragment.Sequencing of this fragment revealed a 264 bp deletionbetween nt 1577 and 1840 of the HBV gene.These results suggest that HBx gene mutation occurs frequentlyin HCC samples,and the deletion at nt 382-400 of the HBx gene might play a role in carcinogenesis of HCCin southern China.     

Analysis of hepatitis B virus X gene (HBx) mutants in tissues of patients suffered from hepatocellular carcinoma in China

Hepatitis B virus (HBV) X (HBx) gene multi-site mutations are a frequent event in the clinical hepatocellular carcinoma (HCC) tissues. It has been reported that the mutation of the HBx plays a crucial role in the development of HBV-related HCC. To identify the novel mutations of HBx in the HCC tissues, we examined and analyzed the sequences of HBx gene in 60 cases of HCC tumor tissues and paratumor tissues from China by polymerase chain reaction (PCR). The mutation patterns of HBx were analyzed by comparing the tumor tissues with non-tumor tissues. The data showed that 44 cases of tissues out of 60 patients were HBV-positive. Our results showed that the mutations at amino acid 30, 88, 144 from tumor samples and at amino acid 31, 43, 87, 94 from non-tumor samples were highly frequent events. Interestingly, we found that a novel type of HBx linked-mutations, such as at aa L30F/S144A, was 29.5% (13/44) positive in the tumor tissues. However, the role of HBx gene mutations at aa L30F/S144A relative to wild type HBx gene is unclear in hepatocarcinogenesis. The novel HBx linked-mutations may be significant in the development of HCC.     

Detection and expression of hepatitis B virus X gene in one and two-cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.     

Self-association of the hepatitis B virus X protein in the yeast two-hybrid system

Self-association of the transactivator HBx protein of hepatitis B virus was investigated using the yeast two-hybrid system. Expression vectors for the full-length HBx (X0) and its truncated mutants (X15 and X16) were constructed by separately ligating the DNA-binding (BD) and transactivation domains (AD) of Gal4. Co-transformants of the BD and AD constructs of HBx were selected using defined minimal medium and analyzed for the reconstitution of beta-galactosidase activity. No two-hybrid interaction was observed either between the full-length HBx molecules or its highly truncated mutant X16. However, a strong functional interaction between X0 and X15, X0 and X16, and X15 and X16 suggested that HBx could self-associate in a cellular environment through its carboxy-terminal region.     

Transcriptional activation of gammaherpesviral oncogene promoters by the hepatitis B viral X protein (HBx)

The latent membrane protein-1 (LMP1) of Epstein-Barr Virus (EBV), saimiri transformation protein (STP) of Herpesvirus saimiri (HVS), and K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) are potent gammaherpesvirus oncogenes. To study the possible effects of double viral infection, we investigated the effects of oncogenic early proteins of DNA viruses E1A and E1B (adenovirus-5), E6 and E7 (human papillomavirus-16), HBx (hepatitis B virus), Tag (SV40), and gammaherpesviral oncogene during co-infection in human B-lymphoma (Ramos) and human T-cell leukemia (Jurkat) cell lines. HBx transactivated the promoters of LMP1, STP, and K1 the most, by about six-, three-, and twofold, respectively. Analyses of site-directed mutation and the heterologous promoter system showed that HBx activated the promoter activity of these genes via the NF-kappaB site. These results suggest that HBV (HBx) infection of cells previously infected by gammaherpesviruses transactivates their oncogenes, resulting in possible virus-related disease pathogenesis.     

Recent advances in the study of hepatitis B virus covalently closed circular DNA

Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.

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