Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20

Hepatitis B virus (HBV) infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx) is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD), and the formation of death-inducing signaling complex (DISC), thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.     

HBx蛋白与Hepsin共表达可促进HBV复制

运用RT-PCR技术从原发性肝癌(Hepatocellular carcinoma,HCC)患者的癌旁组织中扩增了Hepsin基因编码区序列,该序列已被克隆并构建了pCMV-tag-HS表达质粒。将pCMV-tag-HS表达质粒和表达HBx蛋白的载体共转染HepG2．2．1．5细胞,观察HBx蛋白和Hepsin蛋白相互作用对HBV病毒复制和病毒蛋白表达的影响。研究结果表明共表达HBx蛋白、Hepsin蛋白可以协同提高HBV病毒颗粒在培养液中的拷贝数,并提高HBV病毒蛋白HBs、HBe的表达水平。这说明HBx蛋白和Hepsin蛋白相互作用可以增强HBV病毒的复制。     

Hepatitis B virus X protein upregulates alpha-fetoprotein to promote hepatocellular carcinoma by targeting miR-1236 and miR-329

Hepatitis B virus (HBV) infection is the most common cause of hepatocellular carcinoma (HCC) worldwide, wherein the expression of alpha-fetoprotein (AFP) is reactivated to promote tumorgenesis. Hepatitis B virus X protein (HBx) protein encoded by the HBV virus X gene has been considered to be oncogenic and implicated in hepatocarcinogenesis. However, the relationship between HBx and abnormal AFP expression in HCC is yet to be fully understood. To explore the potential regulation of HBx on AFP re-expression in HCC, 97 HCC samples of different etiologies were analyzed, and extremely higher serum AFP levels were found in patients with HBsAg⁺. Analyses of HBV-related HCC specimens showed that the expression of AFP was negatively correlated with the levels of miR-1236 and miR-329. Further analyses indicated that HBx promotes the expression of AFP by orchestrating the levels of miR-1236 and miR-329 both in vitro and in vivo. Specifically, miR-1236 and miR-329 bind to the potential target sequences in AFP mRNA 3′-untranslated region to suppress its expression. HBx transfection resulted in the significant decrement of these microRNAs and increment of AFP expression. Moreover, AFP promotes the proliferation of hepatoma cells and attenuates the proapoptotic effect of chemotherapy agents. These findings revealed a novel regulatory mechanism of HBx on the abnormal AFP expression in HCC, which may provide a therapeutic approach for combating HBV-related HCC by targeting the regulation of AFP expression.     

Hepatitis B Virus-Specific miRNAs and Argonaute2 Play a Role in the Viral Life Cycle

Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion

These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.     

The X-linked tumor suppressor TSPX interacts and promotes degradation of the hepatitis B viral protein HBx via the proteasome pathway

Hepatitis B virus (HBV) infection is a major risk for hepatocellular carcinoma (HCC), and it is a serious global health problem with two billion people exposed to it worldwide. HBx, an essential factor for viral replication and a putative oncoprotein encoded by the HBV genome, has been shown to promote oncogenic properties at multiple sites in HBV-infected liver cells. The expression level of HBx closely associates with the development and progression of HCC, therefore the mechanism(s) regulating the stability of HBx is important in oncogenesis of HBV-infected cells. We demonstrate that the X-linked tumor suppressor TSPX enhances the degradation of HBx through the ubiquitin-proteasome pathway. TSPX interacts with both HBx and a proteasome 19S lid subunit RPN3 via its C-terminal acidic tail. Most importantly, over-expression of RPN3 protects HBx from, and hence acts as a negative regulator for, proteasome-dependent degradation. TSPX abrogates the RPN3-depedent stabilization of HBx, suggesting that TSPX and RPN3 act competitively in regulation of HBx stability. Since mutation and/or epigenetic repression of X-located tumor suppressor gene(s) could significantly predispose males to human cancers, our data suggest that TSPX-induced HBx degradation could play key role(s) in hepatocarcinogenesis among HBV-infected HCC patients.     

The miR-545/374a Cluster Encoded in the Ftx lncRNA is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Previous studies have shown several long noncoding RNAs (lncRNAs) play various roles in HCC progression, but no research has focused on the expression pattern of microRNA clusters encoded in lncRNAs. The Ftx gene encodes a lncRNA which harbors 2 clusters of microRNAs in its introns, the miR-374b/421 cluster and the miR-545/374a cluster. To date, no research has focused on the role of the miR-545/374a and miR-374b/421 clusters in HBV-related HCC. In this study, 66 pairs of HBV-related HCC tissue and matched non-cancerous liver tissue specimens were analyzed for the expression of the Ftx microRNA clusters. Our results showed that the miR-545/374a cluster was upregulated in HBV-HCC tissue and significantly correlated with prognosis-related clinical features, including histological grade, metastasis and tumor capsule. Transfection studies with microRNA mimics and inhibitors revealed that miR-545/374a expression promoted in vitro cell proliferation, cell migration and invasion. The wild-type HBV-genome-containing plasmid or full-length HBx protein encoding plasmid was transfected into the Bel-7402 cell line and observed for their influence on miR-545/374a expression. We found that transfection of the HBV genome or HBx alone resulted in an increase in miR-545/374a expression. Next, by monitoring the expression of sera miR-545/374a before and after surgical tumor excision, we found serum miR-545/374a was tumor-derived and exhibited a sharp decrease 25 days after tumor excision. We also examined the gender-based difference in miR-545/374a expression among HCC patients and utilized microRNA target prediction software to find the targets of miR-545/374a. One of these targets, namely estrogen-related receptor gamma (ESRRG) was inversely correlated with miR-545 expression. In conclusion, the overexpression of miR-545/374a cluster located in the Ftx lncRNA is partially responsible for a poor prognosis, and monitoring sera levels of miR-545/374a may be a useful diagnostic marker for HCC.     

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

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

A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication

Regulated gene expression and progeny production are essential for persistent and chronic infection by human pathogens, such as hepatitis B virus (HBV), which affects >400 million people worldwide and is a major cause of liver disease. In this study, we provide the first direct evidence that a liver-specific microRNA, miR-122, binds to a highly conserved HBV pregenomic RNA sequence via base-pairing interactions and inhibits HBV gene expression and replication. The miR-122 target sequence is located at the coding region of the mRNA for the viral polymerase and the 3' untranslated region of the mRNA for the core protein. In cultured cells, HBV gene expression and replication reduces with increased expression of miR-122, and the expression of miR-122 decreases in the presence of HBV infection and replication. Furthermore, analyses of clinical samples demonstrated an inverse linear correlation in vivo between the miR-122 level and the viral loads in the peripheral blood mononuclear cells of HBV-positive patients. Our results suggest that miR-122 may down-regulate HBV replication by binding to the viral target sequence, contributing to the persistent/chronic infection of HBV, and that HBV-induced modulation of miR-122 expression may represent a mechanism to facilitate viral pathogenesis.     

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host–virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110–154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419–525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.     

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

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

The Viral Oncoprotein HBx of Hepatitis B virus Promotes the Growth of Hepatocellular Carcinoma through Cooperating with the Cellular Oncoprotein RMP

The smallest gene HBx of Hepatitis B virus (HBV) is recognized as an important viral oncogene (V-oncogene) in the hepatocarcinogenesis. Our previous work demonstrated that RMP is a cellular oncogene (C-oncogene) required for the proliferation of hepatocellular carcinoma (HCC) cells. Here we presented the collaboration between V-oncogene HBx and C-oncogene RMP in the development of HCC. The coexpression of HBx and RMP resulted in the cooperative effect of antiapoptosis and proliferation of HCC cells. In vivo, overexpression of RMP accelerated the growth of HBx-induced xenograft tumors in nude mice and vice versa HBx promoted the growth of RMP-driven xenograft tumors. Although HBx didn''t regulate the expression of RMP, HBx and RMP interact with each other and collocalized in the cytoplasm of HCC cells. HBx and RMP collaboratively inhibited the expression of apoptotic factors and promoted the expression of antiapoptotic factors. This finding suggests that HBV may induce, or at least partially contributes to the carcinogenesis of HCC, through its V-oncoprotein HBx interacting with the C-oncoprotein RMP.