Chromosome remodeling related to hepatitis B virus replication in HepG2 cells

Hepatitis B Virus (HBV) covalently closed circular DNA (cccDNA) is the main replicative intermediate of HBV and is organized into minichromosomes by the interaction with histone and nonhistone proteins. The remodeling of HBV minichromosomes such as post-translational modifications of histone proteins plays an important role in regulating HBV replication. To determine whether other remodeling occurs in addition to acetylation of cccDNA-bound H3 histones in the presence of HBV replication, a cell culture replication model of HBV was used to assess the dynamic status of acetylation, phosphorylation, and methylation of cccDNA-bound H3 histones at various times after transient transfection of linear HBV DNA into human hepatoma, HepG2 cells. H3 histones bound to cccDNA were found to be phosphorylated, mono-methylated, and acetylated in HepG2 cells containing replicating HBV. The acetylation and methylation status of H3 histones bound to cccDNA paralleled HBV replication. Our results demonstrate that phosphorylation and methylation occur in the remodeling of HBV minichromosomes during HBV replication. The modifications of cccDNA-bound H3 histones were associated with the level of HBV replication. These findings suggest that alterations in the extent of minichromosome remodeling might be a potential target to inhibit HBV replication in the development of effective novel antiviral agents.     

Mucroporin-M1 Inhibits Hepatitis B Virus Replication by Activating the Mitogen-activated Protein Kinase (MAPK) Pathway and Down-regulating HNF4α in Vitro and in Vivo

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.     

CGK733 enhances multinucleated cell formation and cytotoxicity induced by taxol in Chk1-deficient HBV-positive hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers. Chronic hepatitis B virus (HBV) infection is one of the predominant risk factors associated with the development of HCC and complicates the treatment of HCC. In this study, we demonstrate that a HBV-positive HCC cell line HepG2.2.15, was more resistant to chemotherapy agents than its parental HBV-negative cell line HepG2. HBV-positive HCC cells exhibited defective Chk1 phosphorylation and increased chromosomal instability. CGK733, a small molecule inhibitor reportedly targeting the kinase activities of ATM and ATR, significantly enhanced taxol-induced cytotoxicity in HBV-positive HepG2.2.15 cells. The mechanism lies in CGK733 triggers the formation of multinucleated cells thus promotes the premature mitotic exit of taxol-induced mitotic-damaged cells through multinucleation and mitotic catastrophe in HBV-positive HepG2.2.15 cells. These results suggest that CGK733 could potentially reverse the taxol resistance in HBV-positive HCC cells and may suggest a novel strategy to treat HBV-infected HCC patients.     

SIRT3激动剂对乙肝病毒转录和复制的影响

该文探讨了SIRT3激动剂(Honokiol,HKL)对乙肝病毒(Hepatitis B virus,HBV)转录和复制的影响。培养HepG2-NTCP和人原代肝细胞(primary human hepatocytes,PHH),感染HBV颗粒后,用Honokiol(5μmol/L、10μmol/L、20μmol/L)处理细胞后继续培养10天,通过荧光定量PCR检测细胞内HBV DNA、cccDNA和HBV RNAs水平,Southern blot实验进一步检测胞内HBV DNA水平。构建SIRT3-KO细胞,检测敲除SIRT3后,Honokiol对细胞内HBV DNA、cccDNA和HBV RNAs的影响。通过小鼠尾静脉高压注射pCMV-KRAB-Cre质粒和precccDNA质粒构建持续感染小鼠模型,一周后腹腔注射Honokiol持续20天。荧光定量PCR检测小鼠血清中HBV DNA拷贝数,肝组织内HBV DNA、cccDNA和HBV RNAs水平。结果表明,Honokiol浓度依赖性地抑制HepG2-NTCP和PHH细胞内HBV DNA以及HBV RNAs水平,此外,Honokiol可以降低cccDNA的转录活性;敲除SIRT3后,Honokiol不能发挥抗病毒作用;小鼠模型中,Honokiol能够降低血清中HBV DNA和肝组织内HBV DNA拷贝数,以及能够显著抑制肝组织内HBV RNAs水平和cccDNA的转录活性。该研究结果表明,Honokiol能够抑制乙肝病毒转录和复制。     

Inhibition of Hepatitis B Virus and Induction of Hepatoma Cell Apoptosis by ASGPR-Directed Delivery of shRNAs

Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.     

Glucose-regulated Protein 78 Is an Intracellular Antiviral Factor against Hepatitis B Virus

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2′,5′-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2′,5′-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.Hepatitis B virus infection is a global public health problem. An estimated 2 billion (one-third of the world''s population) people are infected with HBV¹ worldwide, and more than 400 million are chronic hepatitis B (CHB) carriers (1). Epidemiological studies have shown that HBV infection is one of the major risk factors for chronic hepatitis, liver fibrosis, and hepatocellular carcinoma (HCC). Every year, over 1 million people die of HBV-related liver diseases, 30–50% of which are attributed to HCC (2). In China, more than 130 million (10% of the national population) people are suffering from CHB (3), and HCC has been ranked as the second major cause of cancer-related death since 1990 (4). However, the limited efficacy of antiviral therapies, high rates of post-treatment HBV relapse, and the emergence of drug-resistant viral mutants have greatly hindered the effective management of CHB infection. Therefore, it is of prime importance to understand the mechanisms of HBV-host interactions during malignant transformation in CHB infection to identify novel therapeutic anti-HBV targets.Because human HBV is incapable of infecting hepatocytes in vitro efficiently and the availability of reliable in vitro culture systems that favor HBV replication is limited, the pathogenetic studies of HBV and the development of anti-HBV drugs have long been hampered. HepAD38 and HepG2.2.15, both of which are derived from HepG2 cells and integrated with a greater than 1-unit-length HBV genome, have been widely accepted and are well established cell lines for the study of the HBV life cycle and screening potential HBV inhibitors since the late 1990s (5, 6). Recently comparative proteomics analysis of the HBV-expressing HepG2.2.15 cells and the parental HepG2 cells has been performed in two independent laboratories to characterize the altered proteome profile induced by HBV (7, 8). However, the different genetic backgrounds of HepG2.2.15 and HepG2 may lead to an inaccurate evaluation of the impact of HBV replication on host genes. When compared with HepG2.2.15 cells, which produce HBV particles in a continuous manner, HepAD38 cells produce higher levels of HBV DNA in a controllable and inducible way (5). HBV production in HepAD38 is under the strict control of a tetracycline-responsive promoter; therefore, a direct comparison of cellular characteristics with or without HBV replication in HepAD38 is easily achieved. To date, changes in the proteome profile of HepAD38 induced by HBV replication have not been reported. In this study, we performed comparative proteomics to globally analyze the host response to HBV by using an inducible HBV-producing cell line, HepAD38. The combination of two-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS revealed that 23 cellular proteins were differentially expressed when HBV replicated. Among them, GRP78, which was one of the most highly up-regulated proteins, was further selected for functional assessment.