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Hatooka H, Shimomura Y, Imamura M, Teraoka Y, Morio K, Fujino H, Ono A, Nakahara T, Murakami E, Yamauchi M, Kawaoka T, Makokha GN, Miki D, Tsuge M, Hiramatsu A, Abe-Chayama H, Hayes CN, Aikata H, Tanaka S, Chayama K. Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody. J Infect 2021; 84:391-399. [PMID: 34953905 DOI: 10.1016/j.jinf.2021.12.025] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 12/06/2021] [Accepted: 12/13/2021] [Indexed: 11/30/2022]
Abstract
OBJECTIVES The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. METHODS Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. RESULTS Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3 log IU/mL or alanine aminotransferase ≤30 U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). CONCLUSIONS We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.
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Affiliation(s)
- Haruna Hatooka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Yumi Shimomura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Yuji Teraoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Kei Morio
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hatsue Fujino
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Atsushi Ono
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takashi Nakahara
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Eisuke Murakami
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Masami Yamauchi
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Tomokazu Kawaoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Grace Naswa Makokha
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Daiki Miki
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Masataka Tsuge
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan; Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan
| | - Akira Hiramatsu
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan; Center for Medical Specialist Graduate Education and Research, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - C Nelson Hayes
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Aikata
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Shinji Tanaka
- Department of Endoscopy, Hiroshima University Hospital, Hiroshima, Japan
| | - Kazuaki Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan; Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan; Institute of Physical and Chemical Research (RIKEN) Center for Integrative Medical Sciences, Yokohama, Japan.
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2
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Cao X, Zao X, Xue B, Chen H, Zhang J, Li S, Li X, Zhu S, Guo R, Li X, Ye Y. The mechanism of TiaoGanYiPi formula for treating chronic hepatitis B by network pharmacology and molecular docking verification. Sci Rep 2021; 11:8402. [PMID: 33863948 PMCID: PMC8052433 DOI: 10.1038/s41598-021-87812-9] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 03/30/2021] [Indexed: 12/14/2022] Open
Abstract
The Chinese herbal formula TiaoGanYiPi (TGYP) showed effective against chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection. Hence, we aimed to clarify the mechanisms and potential targets between TGYP and CHB. The active compounds and related putative targets of TGYP, and disease targets of CHB were obtained from the public databases. The key targets between TGYP and CHB were identified through the network construction and module analysis. The expression of the key targets was detected in Gene Expression Omnibus (GEO) dataset and normal hepatocyte cell line LO2. We first obtained 11 key targets which were predominantly enriched in the Cancer, Cell cycle and HBV-related pathways. And the expression of the key targets was related to HBV infection and liver inflammation verified in GSE83148 database. Furthermore, the results of real-time quantitative PCR and CCK-8 assay indicated that TGYP could regulate the expression of key targets including CCNA2, ABL1, CDK4, CDKN1A, IGFR and MAP2K1, and promote proliferation of LO2 cells. In coclusion, we identified the active compounds and key targets btween TGYP and CHB, and found that the TGYP might exhibite curative effect on CHB via promoting hepatocyte proliferation and inhibiting the liver inflammatory processes.
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Affiliation(s)
- Xu Cao
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaobin Zao
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China
| | - Baiquan Xue
- The First People's Hospital of Jinzhou District, Dalian, 116100, China
| | - Hening Chen
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Jiaxin Zhang
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Shuo Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaobin Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Shun Zhu
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Rui Guo
- Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaoke Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. .,Institute of Liver Diseases, Beijing University of Chinese Medicine, Beijing, 100700, China.
| | - Yong'an Ye
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. .,Institute of Liver Diseases, Beijing University of Chinese Medicine, Beijing, 100700, China.
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Bender D, Hildt E. Effect of Hepatitis Viruses on the Nrf2/Keap1-Signaling Pathway and Its Impact on Viral Replication and Pathogenesis. Int J Mol Sci 2019; 20:ijms20184659. [PMID: 31546975 PMCID: PMC6769940 DOI: 10.3390/ijms20184659] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Revised: 09/16/2019] [Accepted: 09/17/2019] [Indexed: 12/15/2022] Open
Abstract
With respect to their genome and their structure, the human hepatitis B virus (HBV) and hepatitis C virus (HCV) are complete different viruses. However, both viruses can cause an acute and chronic infection of the liver that is associated with liver inflammation (hepatitis). For both viruses chronic infection can lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Reactive oxygen species (ROS) play a central role in a variety of chronic inflammatory diseases. In light of this, this review summarizes the impact of both viruses on ROS-generating and ROS-inactivating mechanisms. The focus is on the effect of both viruses on the transcription factor Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2). By binding to its target sequence, the antioxidant response element (ARE), Nrf2 triggers the expression of a variety of cytoprotective genes including ROS-detoxifying enzymes. The review summarizes the literature about the pathways for the modulation of Nrf2 that are deregulated by HBV and HCV and describes the impact of Nrf2 deregulation on the viral life cycle of the respective viruses and the virus-associated pathogenesis.
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Affiliation(s)
- Daniela Bender
- Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straβe 51-59, D-63225 Langen, Germany.
| | - Eberhard Hildt
- Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straβe 51-59, D-63225 Langen, Germany.
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Abdoli A, Nakhaie M, Feizi N, Salimi Jeda A, Ramezani A. Harmonized Autophagy Versus Full-Fledged Hepatitis B Virus: Victorious or Defeated. Viral Immunol 2019; 32:322-334. [PMID: 31483214 DOI: 10.1089/vim.2019.0042] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Autophagy is a finely tuned process in the regulation of innate immunity to avoid excessive inflammatory responses and inflammasome signaling. In contrast, the results of recent studies have shown that autophagy may disease-dependently contribute to the pathogenesis of liver diseases, such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) during hepatitis B virus (HBV) infection. HBV has learned to subvert the cell's autophagic machinery to promote its replication. Given the great impact of the autophagy mechanism on the HBV infection and HCC, recognizing these factors may be offered new hope for human intervention and treatment of chronic HBV. This review focuses on recent findings viewing the dual role of autophagy plays in the pathogenesis of HBV infected hepatocytes.
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Affiliation(s)
- Asghar Abdoli
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Mohsen Nakhaie
- Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Neda Feizi
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy
| | - Ali Salimi Jeda
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Amitis Ramezani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
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5
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HBV infection increases the risk of macular degeneration: the roles of HBx-mediated sensitization of retinal pigment epithelial cells to UV and blue light irradiation. J Transl Med 2018; 16:221. [PMID: 30097062 PMCID: PMC6086029 DOI: 10.1186/s12967-018-1594-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2018] [Accepted: 08/02/2018] [Indexed: 11/29/2022] Open
Abstract
Background Hepatitis B virus (HBV) infection is strongly associated with hepatocellular carcinoma due to the main pathogenic X protein of HBV (HBx). Whether HBV infection and the HBx protein could result in macular degeneration (MD) is not known. The aim of this study is to assess the association and underlying mechanisms between HBV infection and MD. Methods The National Health Research Institutes in Taiwan built a large database, the National Health Insurance Research Database (NHIRD), which includes the claims data from the Taiwan National Health Insurance (NHI) program. The Taiwan NHI is a single-payer, compulsory health insurance program for Taiwan citizens. The data for the present study were derived from the Longitudinal Health Insurance Database, which contains the claims data of 1 million insured people within the NHIRD, including beneficiary registration, inpatient and outpatient files, drug use, and other medical services. In this study, we first investigated the association of HBV infection and the risk of MD by a population-based cohorts study enrolling 39,796 HBV-infected patients and 159,184 non-HBV-infected patients. Results After adjustment of age, sex, and comorbidities, the risk of MD was significantly higher in the HBV-infected cohort than in the non-HBV-infected cohort (adjusted HR = 1.31; 95% CI = 1.17–1.46). In vitro, we provided evidence to demonstrate that overexpression of HBx in the human retinal pigment epithelial (RPE) cell line, ARPE19, significantly reduced cell viability and clonogenic survival upon UV and blue light irradiation. By gene microarray analysis, we further showed that almost all genes in DNA repair pathways including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination were significantly down-regulated in the UV-induced cell death of HBx-transfected ARPE19 cells. Conclusions The HBx protein may sensitize RPE cells to UV and blue light irradiation and increase the risk of HBV-infection-associated MD through down-regulation of multiple DNA repair pathways. Electronic supplementary material The online version of this article (10.1186/s12967-018-1594-4) contains supplementary material, which is available to authorized users.
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6
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Ziogas IA, Tsoulfas G. Evolving role of Sorafenib in the management of hepatocellular carcinoma. World J Clin Oncol 2017; 8:203-213. [PMID: 28638790 PMCID: PMC5465010 DOI: 10.5306/wjco.v8.i3.203] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2017] [Revised: 04/03/2017] [Accepted: 04/23/2017] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignant diseases worldwide and comes third in cancer-related mortality. Although there is a broad spectrum of treatment options to choose from, only a few patients are eligible candidates to receive a curative therapy according to their stage of disease, and thus palliative treatment is implemented in the majority of the patients suffering from liver cancer. Sorafenib, a multikinase inhibitor, is the only currently approved agent for systemic therapy in patients with advanced stage HCC and early stage liver disease. It has been shown to improve the overall survival, but with various side effects, while its cost is not negligible. Sorafenib has been in the market for a decade and has set the stage for personalized targeted therapy. Its role during this time has ranged from monotherapy to neoadjuvant and adjuvant treatment with surgical resection, liver transplantation and chemoembolization or even in combination with other chemotherapeutic agents. In this review our aim is to highlight in depth the current position of Sorafenib in the armamentarium against HCC and how that has evolved over time in its use either as a single agent or in combination with other therapies.
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7
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Peiffer KH, Akhras S, Himmelsbach K, Hassemer M, Finkernagel M, Carra G, Nuebling M, Chudy M, Niekamp H, Glebe D, Sarrazin C, Zeuzem S, Hildt E. Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level. J Hepatol 2015; 62:791-8. [PMID: 25445396 DOI: 10.1016/j.jhep.2014.11.028] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2014] [Revised: 10/06/2014] [Accepted: 11/23/2014] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aimed at (1) comparing HBV genotype G and A2 with respect to morphogenesis and release of HBV-derived particles, (2) characterizing factors contributing to HBV/G-associated pathogenesis. METHODS HBV/G- and HBV/A-expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, Western blot, real-time PCR, density gradient centrifugation, and electron microscopy. Modulation of the transcription factors Nrf2 and AP-1 was analyzed. RESULTS While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired, although they are produced in high amounts. These subviral particles, which display an increased density and a predominantly filamentous morphology, accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G, which forms aggregates, causes the block of HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROIs. This results in activation of JNK and as a consequence in Ser-phosphorylation of IRS-1, which is known to impair insulin signaling, a key factor for liver regeneration. CONCLUSIONS Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. In parallel, HBV-induced Nrf2 activation diminishes, which causes a decrease of the capacity to inactivate ROIs. This might be related to genotype-specific pathogenesis.
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Affiliation(s)
- Kai-Henrik Peiffer
- Goethe-University Hospital Frankfurt, Department of Gastroenterology and Hepatology, D-60590 Frankfurt am Main, Germany; Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany.
| | - Sami Akhras
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | | | - Matthias Hassemer
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | - Malin Finkernagel
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | - Gert Carra
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | - Michael Nuebling
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | - Michael Chudy
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany
| | - Hauke Niekamp
- Justus-Liebig University, Institute of Medical Virology, National Reference Centre for Hepatitis B and D Viruses, D-35392 Giessen, Germany; DZIF, German Center for Infection Research, Germany
| | - Dieter Glebe
- Justus-Liebig University, Institute of Medical Virology, National Reference Centre for Hepatitis B and D Viruses, D-35392 Giessen, Germany; DZIF, German Center for Infection Research, Germany
| | - Christoph Sarrazin
- Goethe-University Hospital Frankfurt, Department of Gastroenterology and Hepatology, D-60590 Frankfurt am Main, Germany
| | - Stefan Zeuzem
- Goethe-University Hospital Frankfurt, Department of Gastroenterology and Hepatology, D-60590 Frankfurt am Main, Germany
| | - Eberhard Hildt
- Paul Ehrlich Institut, Division of Virology, D-63325 Langen, Germany; DZIF, German Center for Infection Research, Germany.
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8
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Hu Z, Li M, Liu J, Yu L, Xue Y, Chen Y. Detection of Hepatitis B Virus Large Surface Protein Using a Time-Resolved Immunofluorometric Assay. J Clin Lab Anal 2014; 29:498-504. [PMID: 25277704 DOI: 10.1002/jcla.21800] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2013] [Revised: 06/17/2014] [Accepted: 08/07/2014] [Indexed: 01/28/2023] Open
Abstract
BACKGROUND To establish a novel method based on time-resolved immunofluorometric assay (TR-IFMA) with higher sensitivity and a broader detection range for detecting serum hepatitis B virus large surface protein (L protein). METHODS The precision, sensitivity, specificity, coefficient of recovery, and stability of the assay were evaluated and comparison with the classical enzyme-linked immunosorbent assay (ELISA) was also executed. RESULTS The precision, specificity, and sensitivity of the TR-IFMA were clearly better than ELISA. Particularly, the sensitivity was 0.1 ng/ml; moreover, the specificity was 100%, 96%, 92.5%, 96.9%, 97.8%, and 100% in the sera of healthy blood donors, systemic lupus erythematosus (SLE) patients, rheumatoid arthritis (RA) patients, hepatitis C virus (HCV) patients, cytomegalovirus (CMV) infection patients, and pregnant patients, respectively. Meanwhile, we observed that the established TR-IFMA kit has a wider acceptable linear range of 0.63-10,367 ng/ml rather than the regular commercial ELISA kit having range of only 10.12-1095.9 ng/ml. Subsequently, correlation coefficient between the TR-IFMA and ELISA was 0.8009. The intra- and interassay precision rates were less than 5% for three different concentrations. The average recovery rate for L protein was 101.17%. In sum, the established assay kit performed better in terms of stability than the commercial ELISA kit. CONCLUSION The TR-IFMA that we developed for L protein presented a higher sensitivity and wider detecting range than regular commercial ELISA. Therefore, this TR-IFMA has promising value both in the screening of HBV and monitoring of antiviral therapy.
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Affiliation(s)
- Zhigang Hu
- Department of Laboratory Medicine, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Mei Li
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Jie Liu
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Lei Yu
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Yifeng Xue
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Yu Chen
- Department of Laboratory Medicine, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
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9
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Ranieri G, Marech I, Lorusso V, Goffredo V, Paradiso A, Ribatti D, Gadaleta CD. Molecular targeting agents associated with transarterial chemoembolization or radiofrequency ablation in hepatocarcinoma treatment. World J Gastroenterol 2014; 20:486-497. [PMID: 24574717 PMCID: PMC3923023 DOI: 10.3748/wjg.v20.i2.486] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/11/2013] [Accepted: 12/13/2013] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer in the world. According to Barcelona Clinic Liver Cancer modified criteria, patients with early stage disease are candidate to radiofrequency ablation (RFA), while patients with intermediate stage HCC are usually treated by transarterial chemoembolization (TACE). TACE and RFA induce a transient devascularisation effect followed by strong neo-angiogenic stimulus. In fact, after these procedures, it has been demonstrated an up-regulation of pro-angiogenic and growth factors such as vascular endothelial growth factor-A, which might contribute to accelerated progression in patients with incomplete response. Several studies have demonstrated that MAP-kinase and AKT pathways, in addition to neo-angiogenesis, have an important role in the development of HCC. In advanced HCC, anti-angiogenic therapy and tyrosine kinases inhibitors showed potential clinical benefit. Actually, a number of clinical studies are ongoing testing these agents in combination with TACE or RFA. In this paper, we have reviewed the most recent preclinical and clinical results of such trials.
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10
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Lupberger J, Schaedler S, Peiran A, Hildt E. Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase. World J Gastroenterol 2013; 19:8000-8010. [PMID: 24307793 PMCID: PMC3848147 DOI: 10.3748/wjg.v19.i44.8000] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2013] [Revised: 09/10/2013] [Accepted: 09/17/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.
METHODS: Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.
RESULTS: We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection.
CONCLUSION: Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.
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11
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Ang C, O'Reilly EM, Abou-Alfa GK. Targeted agents and systemic therapy in hepatocellular carcinoma. Recent Results Cancer Res 2013; 190:225-46. [PMID: 22941024 DOI: 10.1007/978-3-642-16037-0_15] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Cytotoxic chemotherapy, hormonal agents, and immunotherapy have been tested in hepatocellular cancer (HCC) with marginal efficacy to date. Recent insights into the molecular pathogenesis of HCC have identified several aberrant signaling pathways that have served as targets for novel therapeutic agents. These discoveries have been translated into the clinical realm with the use of the antiangiogenic and the Raf kinase inhibitor, sorafenib, and have revealed the potential of targeted agents to produce clinically meaningful survival benefits in patients with advanced HCC. Efforts continue in the quest to improve the outcome of HCC patients through the development and evaluation of other targeted agents, and to better understand the interactions between the underlying disease biology and response to therapy. Several pathways are now implicated in hepatocarcinogenesis and agents that target these pathways continue to be developed.
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Affiliation(s)
- Celina Ang
- Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
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12
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HBx induces HepG-2 cells autophagy through PI3K/Akt-mTOR pathway. Mol Cell Biochem 2012; 372:161-8. [PMID: 23001846 DOI: 10.1007/s11010-012-1457-x] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Accepted: 09/07/2012] [Indexed: 12/18/2022]
Abstract
Chronic hepatitis B virus infection is the dominant global cause of hepatocellular carcinoma (HCC), especially hepatitis B virus-X (HBx) plays a major role in this process. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumor suppressor gene and other tumor suppressor genes and senescence-related factors. However, the relationship between HBx and autophagy during the HCC development is poorly known. Previous studies found that autophagy functions as a survival mechanism in liver cancer cells. We suggest that autophagy plays a possible role in the pathogenesis of HBx-induced HCC. The present study showed that HBx transfection brought about an increase in the formation of autophagosomes and autolysosomes. Microtubule-associated protein light chain 3, Beclin 1, and lysosome-associated membrane protein 2a were up-regulated after HBx transfection. HBx-induced increase in the autophagic level was increased by mTOR inhibitor rapamycin and was blocked by treatment with the PI3K-Akt inhibitor LY294002. The same results can also be found in HepG2.2.15 cells. These results suggest that HBx activates the autophagic lysosome pathway in HepG-2 cells through the PI3K-Akt-mTOR pathway.
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GUO PENGTAO, YANG DONG, SUN ZHE, XU HUIMIAN. Hepatitis B virus X protein plays an important role in gastric ulcers. Oncol Rep 2012; 28:1653-8. [DOI: 10.3892/or.2012.2011] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2012] [Accepted: 07/09/2012] [Indexed: 11/06/2022] Open
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Sorafenib for treatment of hepatocellular carcinoma: a systematic review. Dig Dis Sci 2012; 57:1122-9. [PMID: 22451120 PMCID: PMC3596114 DOI: 10.1007/s10620-012-2136-1] [Citation(s) in RCA: 97] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2011] [Accepted: 03/02/2012] [Indexed: 02/08/2023]
Abstract
BACKGROUND Sorafenib, a drug that inhibits Raf serine/threonine kinases mediating cell proliferation and receptor tyrosine kinases involved in angiogenesis, is approved for treatment of advanced hepatocellular carcinoma. AIMS To explore the efficacy and safety of sorafenib for treating advanced HCC, and to identify clinical factors that might affect that efficacy and safety. METHODS We conducted a systematic review using the PRISMA guidelines to identify prospective studies on sorafenib used alone or in combination with systemic and/or loco regional anti-tumor therapy for treating advanced HCC. RESULTS We identified 21 prospective trials of sorafenib treatment alone (7) or combined with other treatment (14). In randomized, placebo-controlled trials, sorafenib prolonged overall survival by 2.3-2.8 months, extended the time to tumor progression by 1.4-2.7 months, and increased disease control by 11-19 %. OS and DCRs were lowest for studies with the highest percentage of hepatitis B patients. Most studies reported major side effects (diarrhea, fatigue, and hand-foot syndrome) in <15 % of patients, with greater incidence in patients with advanced cirrhosis and those treated with sorafenib in combination with 5-FU drugs. CONCLUSIONS Treatment with sorafenib results in statistically significant, but clinically modest, improvements in OS, TTP, and DCR. For patients with hepatitis B, response seems to be poorer than for those with hepatitis C. The frequency of hand-foot syndrome seems to be higher when sorafenib is used in advanced cirrhosis and is combined with 5-FU drugs. It is not clear that sorafenib combined with other treatments is more effective than sorafenib alone.
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The MAPK MEK1/2-ERK1/2 Pathway and Its Implication in Hepatocyte Cell Cycle Control. Int J Hepatol 2012; 2012:328372. [PMID: 23133759 PMCID: PMC3485978 DOI: 10.1155/2012/328372] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2012] [Revised: 09/06/2012] [Accepted: 09/10/2012] [Indexed: 12/15/2022] Open
Abstract
Primary cultures of hepatocytes are powerful models in studying the sequence of events that are necessary for cell progression from a G0-like state to S phase. The models mimic the physiological process of hepatic regeneration after liver injury or partial hepatectomy. Many reports suggest that the mitogen-activated protein kinase (MAPK) ERK1/2 can support hepatocyte proliferation in vitro and in vivo and the MEK/ERK cascade acts as an essential element in hepatocyte responses induced by the EGF. Moreover, its disregulation has been associated with the promotion of tumor cell growth of a variety of tumors, including hepatocellular carcinoma. Whereas the strict specificity of action of ERK1 and ERK2 is still debated, the MAPKs may have specific biological functions under certain contexts and according to the differentiation status of the cells, notably hepatocytes. In this paper, we will focus on MEK1/2-ERK1/2 activations and roles in normal rodent hepatocytes in vitro and in vivo after partial hepatectomy and in human hepatocarcinoma cells. The possible specificity of ERK1 and ERK2 in normal and transformed hepatocyte will be discussed in regard to other differentiated and undifferentiated cellular models.
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von Hahn T, Schulze A, Chicano Wust I, Heidrich B, Becker T, Steinmann E, Helfritz FA, Rohrmann K, Urban S, Manns MP, Pietschmann T, Ciesek S. The novel immunosuppressive protein kinase C inhibitor sotrastaurin has no pro-viral effects on the replication cycle of hepatitis B or C virus. PLoS One 2011; 6:e24142. [PMID: 21909416 PMCID: PMC3164709 DOI: 10.1371/journal.pone.0024142] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2011] [Accepted: 07/31/2011] [Indexed: 01/12/2023] Open
Abstract
The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients.
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Affiliation(s)
- Thomas von Hahn
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
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Ma J, Sun T, Park S, Shen G, Liu J. The role of hepatitis B virus X protein is related to its differential intracellular localization. Acta Biochim Biophys Sin (Shanghai) 2011; 43:583-8. [PMID: 21693548 DOI: 10.1093/abbs/gmr048] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma. HBV encodes an oncogenic hepatitis B virus X protein (HBx), which is a multifunctional regulator that modulates signal transduction, transcription, cell cycle progress, protein degradation, apoptosis, and genetic stability through direct and indirect interaction with host factors. The subcellular localization of HBx is primarily cytoplasmic, with a small fraction in the nucleus. In addition, high levels of HBx expression lead to an abnormal mitochondrial distribution. The dynamic distribution of HBx could be important to the multiple functions of HBx at different stages of the HBV life cycle. This short review presents an overview of the differential roles of HBx as a function of its intracellular localization.
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Affiliation(s)
- Jingwei Ma
- Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Schaedler S, Krause J, Himmelsbach K, Carvajal-Yepes M, Lieder F, Klingel K, Nassal M, Weiss TS, Werner S, Hildt E. Hepatitis B virus induces expression of antioxidant response element-regulated genes by activation of Nrf2. J Biol Chem 2010; 285:41074-86. [PMID: 20956535 DOI: 10.1074/jbc.m110.145862] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated induction of cytoprotective genes by HBV results in a better protection of HBV-positive cells against oxidative damage as compared with control cells. Furthermore, there is a significantly increased expression of the Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is associated with a decreased level of the immunoproteasome subunit PSMB5i. In accordance with this finding, HBV-positive cells display a higher constitutive proteasome activity and a decreased activity of the immunoproteasome as compared with control cells even after interferon α/γ treatment. The HBV-dependent induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell, shape the immune response to HBV, and thereby promote establishment of the infection.
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Affiliation(s)
- Stephanie Schaedler
- Institute of Infection Medicine, Molecular Medical Virology, University of Kiel, D-24105 Kiel, Germany
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Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene 2010; 29:4989-5005. [PMID: 20639898 DOI: 10.1038/onc.2010.236] [Citation(s) in RCA: 671] [Impact Index Per Article: 44.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Hepatocellular carcinoma (HCC) is a highly prevalent, treatment-resistant malignancy with a multifaceted molecular pathogenesis. Current evidence indicates that during hepatocarcinogenesis, two main pathogenic mechanisms prevail: (1) cirrhosis associated with hepatic regeneration after tissue damage caused by hepatitis infection, toxins (for example, alcohol or aflatoxin) or metabolic influences, and (2) mutations occurring in single or multiple oncogenes or tumor suppressor genes. Both mechanisms have been linked with alterations in several important cellular signaling pathways. These pathways are of interest from a therapeutic perspective, because targeting them may help to reverse, delay or prevent tumorigenesis. In this review, we explore some of the major pathways implicated in HCC. These include the RAF/MEK/ERK pathway, phosphatidylinositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, WNT/beta-catenin pathway, insulin-like growth factor pathway, hepatocyte growth factor/c-MET pathway and growth factor-regulated angiogenic signaling. We focus on the role of these pathways in hepatocarcinogenesis, how they are altered, and the consequences of these abnormalities. In addition, we also review the latest preclinical and clinical data on the rationally designed targeted agents that are now being directed against these pathways, with early evidence of success.
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Affiliation(s)
- S Whittaker
- Dana-Farber Cancer Institute, Boston, MA, USA
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Kim SY, Lee PY, Shin HJ, Kim DH, Kang S, Moon HB, Kang SW, Kim JM, Park SG, Park BC, Yu DY, Bae KH, Lee SC. Proteomic analysis of liver tissue from HBx-transgenic mice at early stages of hepatocarcinogenesis. Proteomics 2010; 9:5056-66. [PMID: 19813210 DOI: 10.1002/pmic.200800779] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The hepatitis B virus X-protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx-transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2-DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC-MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3-ketoacyl-CoA thiolase, intestinal fatty acid-binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx-mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.
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Affiliation(s)
- Sun-Young Kim
- Medical Proteomics Research Center, KRIBB, 52 Eoeun-Dong, Yusung-Gu, Daejeon, South Korea
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HBV life cycle: entry and morphogenesis. Viruses 2009; 1:185-209. [PMID: 21994545 PMCID: PMC3185491 DOI: 10.3390/v1020185] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2009] [Revised: 07/31/2009] [Accepted: 08/13/2009] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.
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Zhu AX, Raymond E. Early development of sunitinib in hepatocellular carcinoma. Expert Rev Anticancer Ther 2009; 9:143-50. [PMID: 19105714 DOI: 10.1586/14737140.9.1.143] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Sunitinib malate is an oral, multitargeted receptor tyrosine kinase inhibitor of VEGF receptors 1, 2 and 3; PDGF receptors alpha and beta, and other receptor tyrosine kinases implicated in tumor growth, angiogenesis and metastasis. Hepatocellular carcinoma (HCC) is a highly vascular tumor that overexpresses several angiogenic factors; VEGF and PDGF signaling pathways play a key role in HCC. Until recently, treatment options for advanced HCC were limited and conventional therapies have met with poor response rates. Sorafenib provided proof-of-concept for molecularly targeted therapy in advanced HCC and has recently been approved for treatment. However, not all patients can tolerate sorafenib and patients may experience tumor progression; therefore, additional treatment options are warranted. Sunitinib has shown early evidence of anti-tumor activity in Phase II trials in US, European and Asian patients with locally advanced, unresectable and metastatic HCC. A Phase III trial of sunitinib in HCC is ongoing.
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Affiliation(s)
- Andrew X Zhu
- Tucker Gosnell Center for Gastrointestinal Cancers, Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.
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Chang WW, Su IJ, Chang WT, Huang W, Lei HY. Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide. J Viral Hepat 2008; 15:490-7. [PMID: 18221299 DOI: 10.1111/j.1365-2893.2007.00968.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The role of the p38 mitogen-activated protein kinase (MAPK) pathway in hepatitis B virus (HBV) replication was investigated in this study. After transient transfection with HBV plasmid, p38 MAPK, but not JNK or ERK1/2, was significantly phosphorylated in human hepatoma cell Huh7. Interestingly, HBV proteins and RNA synthesis were significantly inhibited by a specific inhibitor of p38 MAPK, SB203580, in a dose-dependent manner. Intracellular core-associated DNA, extracellular virion-associated DNA and covalently closed circular DNA were also significantly inhibited by SB203580. Further results showed the antiviral role of nitric oxide (NO) on the suppression of HBV replication and downregulation of p38 MAPK phosphorylation. In conclusion, these results suggested that suppression of phosphorylation of p38 MAPK by inhibitor or NO could inhibit intracellular HBV replication.
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Affiliation(s)
- W-W Chang
- Department of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
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Chang WW, Su IJ, Lai MD, Chang WT, Huang W, Lei HY. Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide. J Viral Hepat 2008. [DOI: 10.1111/j.1365-2893.2008.00968.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
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Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation. Biochim Biophys Acta Mol Basis Dis 2007; 1772:1150-7. [DOI: 10.1016/j.bbadis.2007.08.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2007] [Revised: 07/27/2007] [Accepted: 08/08/2007] [Indexed: 01/09/2023]
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Abstract
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with an annual incidence of more than 500 000 in the year 2000. Its incidence is rising in many countries. Recently, it has been estimated that about 53% of HCC cases in the world are related to hepatitis B virus (HBV). The epidemiological association of HBV with HCC is well established. In recent studies, it was revealed that HBsAg carriers have a 25-37 times increased risk of developing HCC as compared to non-infected people. At present, HBV-associated carcinogenesis can be seen as a multi-factorial process that includes both direct and indirect mechanisms that might act synergistically. The integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion. The integration has been shown in a number of cases to affect a variety of cancer-related genes and to exert insertional mutagenesis. The permanent liver inflammation, induced by the immune response, resulting in a degeneration and regeneration process confers to the accumulation of critical mutations in the host genome. In addition to this, the regulatory proteins HBx and the PreS2 activators that can be encoded by the integrate exert a tumor promoter-like function resulting in positive selection of cells producing a functional regulatory protein. Gene expression profiling and proteomic techniques may help to characterize the molecular mechanisms driving HBV-associated carcinogenesis, and thus potentially identify new strategies in diagnosis and therapy.
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Affiliation(s)
- Joachim Lupberger
- University of Freiburg, Department of Internal Medicine II, Hugstetter Strasse 55, Freiburg D-79106, Germany
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Lupberger J, Mund A, Kock J, Hildt E. Cultivation of HepG2.2.15 on Cytodex-3: higher yield of hepatitis B virus and less subviral particles compared to conventional culture methods. J Hepatol 2006; 45:547-52. [PMID: 16879893 DOI: 10.1016/j.jhep.2006.05.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2005] [Revised: 05/16/2006] [Accepted: 05/18/2006] [Indexed: 01/19/2023]
Abstract
BACKGROUND/AIMS Several novel systems are available to study human hepatitis B virus (HBV) replication in cell culture demanding for efficient cell culture based systems for HBV production. The aim was to enhance HBV production of the HBV stably producing cell line HepG2.2.15 by cultivation on spherical micro substrate. METHODS HepG2.2.15 was cultivated on microcarrier substrate Cytodex-3. HBV specific transcripts, viral protein and genome secretion, cell proliferation and MAP kinase signaling were analyzed. Infectivity of HBV particles was analyzed using primary tupaia hepatocytes. RESULTS Compared to stationary flask cultures, HepG2.2.15 on Cytodex-3 secreted 18-fold more HBV genomes, more HBeAg per culture volume and less HBV surface antigen per extracellular viral genome equivalent. This was reflected by a significantly higher infectivity of supernatant derived from carrier grown HepG.2.2.15 cells tested by infection of primary tupaia hepatocytes. The amount of phosphorylated ERK-2 was significantly elevated in cells cultivated on microcarrier. CONCLUSIONS The cultivation of HepG2.2.15 on Cytodex-3 increased production of infectious HBV particles and decreased secretion of subviral particles compared to the stationary cell cultivation. Microcarrier cultivation activates MAP kinase signaling that is crucial for HBV replication.
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Brandenburg B, Stockl L, Gutzeit C, Roos M, Lupberger J, Schwartlander R, Gelderblom H, Sauer IM, Hofschneider PH, Hildt E. A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle. Hepatology 2005; 42:1300-9. [PMID: 16317706 DOI: 10.1002/hep.20950] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell-permeable virus-like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)-PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA-encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA-packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM-PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles.
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Friedrich B, Wollersheim M, Brandenburg B, Foerste R, Will H, Hildt E. Induction of anti-proliferative mechanisms in hepatitis B virus producing cells. J Hepatol 2005; 43:696-703. [PMID: 15922479 DOI: 10.1016/j.jhep.2005.02.026] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2004] [Revised: 12/10/2004] [Accepted: 02/02/2005] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Hepatitis B virus (HBV) preferentially replicates in quiescent cells. It was analyzed whether HBV affects cell cycle control. METHODS The amount of EGF-receptor (EGFR) and the binding capacity for 125I-EGF was determined. Expression of mdm2 and p21 and relevance of p53 for it were analyzed by reporter gene assays and western blotting. Cyclin A/E associated cdk2 activities were determined by immunocomplex assays. Cell proliferation was quantified by measurement of BrdU incorporation. RESULTS In HBV producing cells a significant reduction of EGFR expression, diminished 125I-EGF-binding capacity and insensitivity to EGF-stimulation were observed as compared to the control. Moreover, c-Raf-1-dependent induction of mdm2-P2 and p21cip1/waf1-promoter and elevated amounts of the respective proteins were observed in HBV producing cells. Whereas activation of mdm2-P2-promoter requires p53, activation of p21cip1/waf1-promoter is mediated partially by a p53-independent process. Induction of p21cip1/waf1 is reflected by a reduction of cyclin A associated cdk2 activity and an increase of cyclin E associated cdk2 activity. In accordance with this proliferation rate of HBV-producing hepatocytes is reduced as compared to control cells. CONCLUSIONS These results describe novel cell-cycle inhibitory functions of HBV that correlate well with the general concept of enhanced HBV replication in quiescent cells.
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Ji D, Cheng J, Chen GF, Liu Y, Wang L, Guo J. Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization. World J Gastroenterol 2005; 11:5438-43. [PMID: 16222733 PMCID: PMC4320350 DOI: 10.3748/wjg.v11.i35.5438] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection.
METHODS: pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.
RESULTS: The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein.
CONCLUSION: The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
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Affiliation(s)
- Dong Ji
- The 7th Department of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China.
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Ji D, Cheng J, Lu YY, Dong J, Guo J, Liu Y. Autonomous activation of hepatitis B virus large surface protein. Shijie Huaren Xiaohua Zazhi 2004; 12:2321-2324. [DOI: 10.11569/wcjd.v12.i10.2321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct the yeast expression vector of hepatitis B virus large surface protein (LHBs), and to study its autonomous activation.
METHODS: The Matchmaker GAL4 two-hybrid technique was used. The LHBs, pre-S1, pre-S2 and SHBs genes were amplified by polymerase chain reaction (PCR) with respective primers. The amplified PCR fragments were then subcloned into the EcoR I/BamH I sites (5'ands) of pGBKT7 vector to obtain the expression vectors including pGBKT7(-)-LHBs, pGBKT7(-)-preS1, pGBKT7(-)-preS2 and pGBKT7(-)-SHBs. This vectors were identifed by PCR and digestion of EcoR I/BamH I. After the constructed vectors were transformed into yeast AH109, the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing x-a-gal for testing their autonomous activation.
RESULTS: The yeast expression vectors were constructed. The yeast cells transformed with pGBKT7-LHB and pGBKT7-preS1 vectors could grow well on both of the media. However, cells transformed with pGBKT7-preS2 and pGBKT7-SHBs vectors could only grow on the SD/-Trp medium.
CONCLUSION: The LHBs functions as a transcriptional transactivator, and serves as the functional GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). The autonomous activation of LHBs roots in its pre-S1 domain.
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Ji D, Cheng J, Dong J, Liu Y, Wang JJ, Guo J. Screening and identification of genes trans-regulated by HBV pre-S2 protein with cDNA microarray. Shijie Huaren Xiaohua Zazhi 2004; 12:1559-1563. [DOI: 10.11569/wcjd.v12.i7.1559] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To understand the target genes up-regulated or down-regulated by HBV pre-S2 protein, we compared the differentially expressed genes between the hepatoblastoma cell line HepG2 transfected by pcDNA3.1(-) and pcDNA3.1(-)-preS2, respectively with cDNA microarray technique.
METHODS: The HBV pre-S2 coding DNA fragment was amplified with polymerase chain reaction (PCR) technique by using G376-7 plasmid containing the full length of HBV genome as the template. The expressive vector of pcDNA3.1-preS2 was constructed by routine molecular biological methods. The HepG2 cells were transfected by pcDNA3.1(-) and pcDNA3.1(-)-preS2, respectively, using FuGENE6 transfection reagent. The total RNA was isolated and reversely transcribed. The cDNAs were subjected for microarray screening with 1 152 cDNA probes.
RESULTS: The expressive vector was constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. High quality mRNA and cDNA were prepared and successful microarray screening conducted. From the scanning results, it was found 42 genes were up-regulated and 36 genes down-regulated by pre-S2 protein of HBV.
CONCLUSION: HBV pre-S2 protein is a transactivator. The expression of pre-S2 protein affects the expression spectrum of HBV infected hepatocyte.
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Meier P, Scougall CA, Will H, Burrell CJ, Jilbert AR. A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus. Virology 2004; 317:291-8. [PMID: 14698667 DOI: 10.1016/j.virol.2003.08.025] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i). their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii). the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii). the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.
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Affiliation(s)
- P Meier
- School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia.
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Robek MD, Boyd BS, Wieland SF, Chisari FV. Signal transduction pathways that inhibit hepatitis B virus replication. Proc Natl Acad Sci U S A 2004; 101:1743-7. [PMID: 14757813 PMCID: PMC341846 DOI: 10.1073/pnas.0308340100] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The replication of hepatitis B virus (HBV) in hepatocytes is strongly inhibited in response to IFN-alpha/beta and IFN-gamma. Although it has been previously demonstrated that IFN-alpha/beta eliminates HBV RNA-containing capsids from the cell in a proteasome-dependent manner, the precise cellular pathway that mediates this antiviral effect has not been identified. Because IFN-induced signal transduction involves kinase-mediated activation of gene expression, we used an immortalized hepatocyte cell line that replicates HBV in an IFN-sensitive manner to investigate the role of cellular kinase activity and the cellular transcription and translation machinery in the antiviral effect. Our results indicate that Janus kinase activity is required for the antiviral effect of IFN against HBV, but that phosphatidylinositol 3-kinase, cyclin-dependent kinase, mitogen-activated protein kinase, and NF-kappaB activity are not. Additionally, we found that inhibitors of cellular transcription and translation completely abolish the antiviral effect, which also appears to require cellular kinase activity downstream of signal transduction and gene expression. Collectively, these results identify IFN-regulated pathways that interrupt the HBV replication cycle by eliminating viral RNA-containing capsids from the cell, and they provide direction for discovery of the terminal effector molecules that ultimately mediate this antiviral effect.
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Affiliation(s)
- Michael D Robek
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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Hafner A, Brandenburg B, Hildt E. Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein. EMBO Rep 2003; 4:767-73. [PMID: 12872136 PMCID: PMC1326343 DOI: 10.1038/sj.embor.embor903] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2003] [Revised: 05/13/2003] [Accepted: 06/18/2003] [Indexed: 11/09/2022] Open
Abstract
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.
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Affiliation(s)
- Angela Hafner
- Robert Koch-Institut, Nordufer 20,
D-13353 Berlin, Germany
| | | | - Eberhard Hildt
- Robert Koch-Institut, Nordufer 20,
D-13353 Berlin, Germany
- Tel: +49 304547 2528; Fax: +49 304547 2328;
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