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1
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Tsuge M. The association between hepatocarcinogenesis and intracellular alterations due to hepatitis B virus infection. Liver Int 2021; 41:2836-2848. [PMID: 34559952 DOI: 10.1111/liv.15065] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Revised: 09/13/2021] [Accepted: 09/19/2021] [Indexed: 02/06/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is a worldwide health problem leading to severe liver dysfunction, including liver cirrhosis and hepatocellular carcinoma. Although current antiviral therapies for chronic HBV infection have been improved and can lead to a strong suppression of viral replication, it is difficult to completely eliminate the virus with these therapies once chronic HBV infection is established in the host. Furthermore, chronic HBV infection alters intracellular metabolism and signalling pathways, resulting in the activation of carcinogenesis in the liver. HBV produces four viral proteins: hepatitis B surface-, hepatitis B core-, hepatitis B x protein, and polymerase; each plays an important role in HBV replication and the intracellular signalling pathways associated with hepatocarcinogenesis. In vitro and in vivo experimental models for analyzing HBV infection and replication have been established, and gene expression analyses using microarrays or next-generation sequencing have also been developed. Thus, it is possible to clarify the molecular mechanisms for intracellular alterations, such as endoplasmic reticulum stress, oxidative stress, and epigenetic modifications. In this review, the impact of HBV viral proteins and intracellular alterations in HBV-associated hepatocarcinogenesis are discussed.
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Affiliation(s)
- Masataka Tsuge
- Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan.,Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
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2
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Wu S, Ye S, Lin X, Chen Y, Zhang Y, Jing Z, Liu W, Chen W, Lin X, Lin X. Small hepatitis B virus surface antigen promotes malignant progression of hepatocellular carcinoma via endoplasmic reticulum stress-induced FGF19/JAK2/STAT3 signaling. Cancer Lett 2021; 499:175-187. [PMID: 33249195 DOI: 10.1016/j.canlet.2020.11.032] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 11/18/2020] [Accepted: 11/21/2020] [Indexed: 02/07/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is one of the major global health problems. Although the small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV viral protein, its pathogenic role and molecular mechanism in malignant progression of HBV-related hepatocellular carcinoma (HCC) remain largely unknown. Here we reported that SHBs expression induced epithelial-mesenchymal transition (EMT) process in HCC cells and significantly increased their migratory and invasive ability as well as metastatic potential. Mechanistically, SHBs expression in HCC cells induced endoplasmic reticulum (ER) stress that activated the activating transcription factor 4 (ATF4) to increase the expression and secretion of fibroblast growth factor 19 (FGF19). The autocrine released FGF19 in turn activated JAK2/STAT3 signaling for induction of EMT process in HCC. Notably, SHBs was positively correlated with the expression of mesenchymal markers, the phosphorylation status of JAK2 and STAT3 as well as FGF19 levels in human HCC samples. HCC patients with SHBs positive had a more advanced clinical stage and worse prognosis. These results suggest an important role of SHBs in the metastasis and progression of HCC and may highlight a potential target for preventive and therapeutic intervention of HBV-related HCC and its malignant progression.
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MESH Headings
- Animals
- Carcinoma, Hepatocellular/blood
- Carcinoma, Hepatocellular/immunology
- Carcinoma, Hepatocellular/mortality
- Carcinoma, Hepatocellular/virology
- Cell Proliferation
- Endoplasmic Reticulum Stress/immunology
- Female
- Fibroblast Growth Factors/genetics
- Fibroblast Growth Factors/metabolism
- Gene Knockdown Techniques
- Hep G2 Cells
- Hepatitis B Surface Antigens/blood
- Hepatitis B Surface Antigens/metabolism
- Hepatitis B virus/immunology
- Hepatitis B virus/metabolism
- Hepatitis B, Chronic/blood
- Hepatitis B, Chronic/immunology
- Hepatitis B, Chronic/mortality
- Hepatitis B, Chronic/virology
- Humans
- Janus Kinase 2/genetics
- Janus Kinase 2/metabolism
- Kaplan-Meier Estimate
- Liver/immunology
- Liver/pathology
- Liver/virology
- Liver Neoplasms/blood
- Liver Neoplasms/immunology
- Liver Neoplasms/mortality
- Liver Neoplasms/virology
- Male
- Mice
- Middle Aged
- RNA, Small Interfering/metabolism
- Receptor, Fibroblast Growth Factor, Type 4/metabolism
- STAT3 Transcription Factor/genetics
- STAT3 Transcription Factor/metabolism
- Signal Transduction/genetics
- Signal Transduction/immunology
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Shuxiang Wu
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Shuangshuang Ye
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Xiaohan Lin
- Department of Gastrointestinal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Yan Chen
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Yi Zhang
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Zhentang Jing
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Wei Liu
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China
| | - Wannan Chen
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China; Fujian Key Laboratory of Tumor Microbiology, Department of Medical Microbiology, Fujian Medical University, Fuzhou, China
| | - Xinjian Lin
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China.
| | - Xu Lin
- Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China; Fujian Key Laboratory of Tumor Microbiology, Department of Medical Microbiology, Fujian Medical University, Fuzhou, China.
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3
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Deng F, Xu G, Cheng Z, Huang Y, Ma C, Luo C, Yu C, Wang J, Xu X, Liu S, Zhu Y. Hepatitis B Surface Antigen Suppresses the Activation of Nuclear Factor Kappa B Pathway via Interaction With the TAK1-TAB2 Complex. Front Immunol 2021; 12:618196. [PMID: 33717111 PMCID: PMC7947203 DOI: 10.3389/fimmu.2021.618196] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Accepted: 01/19/2021] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis B is a major health problem worldwide, with more than 250 million chronic carriers. Hepatitis B virus interferes with the host innate immune system so as to evade elimination via almost all of its constituent proteins; nevertheless, the function of HBsAg with respect to immune escape remains unclear. This study aimed to determine the role HBsAg plays in assisting HBV to escape from immune responses. We found that HBsAg suppressed the activation of the nuclear factor kappa B (NF-кB) pathway, leading to downregulation of innate immune responses. HBsAg interacted with TAK1 and TAB2 specifically, inhibiting the phosphorylation and polyubiquitination of TAK1 and the K63-linked polyubiquitination of TAB2. Autophagy is a major catabolic process participating in many cellular processes, including the life cycle of HBV. We found that HBsAg promoted the autophagic degradation of TAK1 and TAB2 via the formation of complexes with TAK1 and TAB2, resulting in suppression of the NF-κB pathway. The expression of TAK1, TAB2, and the translocation of NF-κB inversely correlated with HBsAg levels in clinical liver tissues. Taken together, our findings suggest a novel mechanism by which HBsAg interacts with TAK1-TAB2 complex and suppresses the activation of NF-κB signaling pathway via reduction of the post-translational modifications and autophagic degradation.
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Affiliation(s)
- Feiyan Deng
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Gang Xu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Zhikui Cheng
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yu Huang
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Caijiao Ma
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Chuanjin Luo
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Chen Yu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Jun Wang
- Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xiupeng Xu
- Department of Clinical Laboratory, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic, Huangshi, China
| | - Shi Liu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Ying Zhu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
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Association of the Hepatitis B Virus Large Surface Protein with Viral Infectivity and Endoplasmic Reticulum Stress-mediated Liver Carcinogenesis. Cells 2020; 9:cells9092052. [PMID: 32911838 PMCID: PMC7563867 DOI: 10.3390/cells9092052] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2020] [Revised: 09/05/2020] [Accepted: 09/07/2020] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B is the most prevalent viral hepatitis worldwide, affecting approximately one-third of the world’s population. Among HBV factors, the surface protein is the most sensitive biomarker for viral infection, given that it is expressed at high levels in all viral infection phases. The large HBV surface protein (LHBs) contains the integral pre-S1 domain, which binds to the HBV receptor sodium taurocholate co transporting polypeptide on the hepatocyte to facilitate viral entry. The accumulation of viral LHBs and its prevalent pre-S mutants in chronic HBV carriers triggers a sustained endoplasmic reticulum (ER) overload response, leading to ER stress-mediated cell proliferation, metabolic switching and genomic instability, which are associated with pro-oncogenic effects. Ground glass hepatocytes identified in HBV-related hepatocellular carcinoma (HCC) patients harbor pre-S deletion variants that largely accumulate in the ER lumen due to mutation-induced protein misfolding and are associated with increased risks of cancer recurrence and metastasis. Moreover, in contrast to the major HBs, which is decreased in tumors to a greater extent than it is in peritumorous regions, LHBs is continuously expressed during tumorigenesis, indicating that LHBs serves as a promising biomarker for HCC in people with CHB. Continuing efforts to delineate the molecular mechanisms by which LHBs regulates pathological changes in CHB patients are important for establishing a correlation between LHBs biomarkers and HCC development.
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Molecular Mechanisms Driving Progression of Liver Cirrhosis towards Hepatocellular Carcinoma in Chronic Hepatitis B and C Infections: A Review. Int J Mol Sci 2019. [PMID: 30889843 DOI: 10.3390/ijms] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Almost all patients with hepatocellular carcinoma (HCC), a major type of primary liver cancer, also have liver cirrhosis, the severity of which hampers effective treatment for HCC despite recent progress in the efficacy of anticancer drugs for advanced stages of HCC. Here, we review recent knowledge concerning the molecular mechanisms of liver cirrhosis and its progression to HCC from genetic and epigenomic points of view. Because ~70% of patients with HCC have hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection, we focused on HBV- and HCV-associated HCC. The literature suggests that genetic and epigenetic factors, such as microRNAs, play a role in liver cirrhosis and its progression to HCC, and that HBV- and HCV-encoded proteins appear to be involved in hepatocarcinogenesis. Further studies are needed to elucidate the mechanisms, including immune checkpoints and molecular targets of kinase inhibitors, associated with liver cirrhosis and its progression to HCC.
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6
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Molecular Mechanisms Driving Progression of Liver Cirrhosis towards Hepatocellular Carcinoma in Chronic Hepatitis B and C Infections: A Review. Int J Mol Sci 2019; 20:ijms20061358. [PMID: 30889843 PMCID: PMC6470669 DOI: 10.3390/ijms20061358] [Citation(s) in RCA: 190] [Impact Index Per Article: 31.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2019] [Revised: 02/23/2019] [Accepted: 03/14/2019] [Indexed: 02/07/2023] Open
Abstract
Almost all patients with hepatocellular carcinoma (HCC), a major type of primary liver cancer, also have liver cirrhosis, the severity of which hampers effective treatment for HCC despite recent progress in the efficacy of anticancer drugs for advanced stages of HCC. Here, we review recent knowledge concerning the molecular mechanisms of liver cirrhosis and its progression to HCC from genetic and epigenomic points of view. Because ~70% of patients with HCC have hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection, we focused on HBV- and HCV-associated HCC. The literature suggests that genetic and epigenetic factors, such as microRNAs, play a role in liver cirrhosis and its progression to HCC, and that HBV- and HCV-encoded proteins appear to be involved in hepatocarcinogenesis. Further studies are needed to elucidate the mechanisms, including immune checkpoints and molecular targets of kinase inhibitors, associated with liver cirrhosis and its progression to HCC.
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7
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Hepatitis B e Antigen Inhibits NF-κB Activity by Interrupting K63-Linked Ubiquitination of NEMO. J Virol 2019; 93:JVI.00667-18. [PMID: 30404796 DOI: 10.1128/jvi.00667-18] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2018] [Accepted: 10/23/2018] [Indexed: 12/22/2022] Open
Abstract
Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-κB activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1β (IL-1β)-stimulated NF-κB activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with IκB kinase, which regulates the activation of NF-κB. HBeAg suppressed the IL-1β-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-κB activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-κB signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1β-induced NF-κB activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-κB pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1β-induced activation of NF-κB in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for IκB kinase (IKK) that controls the NF-κB signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-κB activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-κB activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-κB signaling.
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8
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Yen TTC, Yang A, Chiu WT, Li TN, Wang LH, Wu YH, Wang HC, Chen L, Wang WC, Huang W, Chang CW, Chang MDT, Shen MR, Su IJ, Wang LHC. Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability. Oncotarget 2018; 7:23346-60. [PMID: 26992221 PMCID: PMC5029631 DOI: 10.18632/oncotarget.8109] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Accepted: 02/28/2016] [Indexed: 12/14/2022] Open
Abstract
Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis.
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Affiliation(s)
- Tim Ting-Chung Yen
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Anderson Yang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Wen-Tai Chiu
- Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan.,Center of Infectious Diseases and Signal Transduction, National Cheng Kung University, Tainan 701, Taiwan
| | - Tian-Neng Li
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Lyu-Han Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Yi-Hsuan Wu
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Hui-Chen Wang
- Institute of Pharmaceutics, Development Center for Biotechnology, Taipei 22180, Taiwan
| | - Linyi Chen
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan.,Department of Medical Science, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Wen-Ching Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Wenya Huang
- Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
| | - Chien-Wen Chang
- Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Margaret Dah-Tsyr Chang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan.,Department of Medical Science, National Tsing Hua University, Hsinchu 300, Taiwan
| | - Meng-Ru Shen
- Center of Infectious Diseases and Signal Transduction, National Cheng Kung University, Tainan 701, Taiwan.,Department of Pharmacology, National Cheng Kung University, Tainan 701, Taiwan
| | - Ih-Jen Su
- Center of Infectious Diseases and Signal Transduction, National Cheng Kung University, Tainan 701, Taiwan.,National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan 704, Taiwan.,Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan
| | - Lily Hui-Ching Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan.,Department of Medical Science, National Tsing Hua University, Hsinchu 300, Taiwan
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Bettermann K. NF-κB and Its Implication in Liver Health and Cancer Development. MECHANISMS OF MOLECULAR CARCINOGENESIS – VOLUME 1 2017:87-114. [DOI: 10.1007/978-3-319-53659-0_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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10
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Colledge D, Soppe S, Yuen L, Selleck L, Walsh R, Locarnini S, Warner N. Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis. Virology 2017; 501:70-78. [DOI: 10.1016/j.virol.2016.11.007] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2016] [Revised: 11/07/2016] [Accepted: 11/09/2016] [Indexed: 01/08/2023]
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The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis. J Biomed Sci 2014; 21:98. [PMID: 25316153 PMCID: PMC4200140 DOI: 10.1186/s12929-014-0098-7] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2014] [Accepted: 10/07/2014] [Indexed: 02/07/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.
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12
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Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications. J Hepatol 2014; 61:408-17. [PMID: 24801416 DOI: 10.1016/j.jhep.2014.04.041] [Citation(s) in RCA: 188] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2014] [Revised: 04/21/2014] [Accepted: 04/24/2014] [Indexed: 12/16/2022]
Abstract
The emergence and takeover of hepatitis B virus (HBV) variants carrying mutation(s) in the preS/S genomic region is a fairly frequent event that may occur spontaneously or may be the consequence of immunoprophylaxis or antiviral treatments. Selection of preS/S mutants may have relevant pathobiological and clinical implications. Both experimental data and studies in humans show that several specific mutations in the preS/S gene may induce an imbalance in the synthesis of the surface proteins and their consequent retention within the endoplasmic reticulum (ER) of the hepatocytes. The accumulation of mutated surface proteins may cause ER stress with the consequent induction of oxidative DNA damage and genomic instability. Viral mutants with antigenically modified surface antigen may be potentially infectious to immune-prophylaxed patients and may account for cases of occult HBV infection. In addition, preS/S variants were reported to be associated with cases of fulminant hepatitis as well as of fibrosing cholestatic hepatitis, and they are associated with cirrhosis and hepatocellular carcinoma development.
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13
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Kim BK, Choi SH, Ahn SH, Chung AR, Park YK, Han KH, Kim S, Kim HS, Park JH, Kim KS, Lee HS, Cho YS, Kim KH, Ahn SH. Pre-S mutations of hepatitis B virus affect genome replication and expression of surface antigens. J Gastroenterol Hepatol 2014; 29:843-50. [PMID: 24783251 DOI: 10.1111/jgh.12415] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUNDS AND AIMS In chronic hepatitis B virus (HBV) infection, quantitative HBV surface antigen (qHBsAg) is useful for monitoring viral replication and treatment responses. We aimed to determine whether pre-S mutations have any effect on circulating qHBsAg. METHODS Plasmids expressing 1–8 amino acid deletion in pre-S1 ("pre-S1Δ1-8") and 3-25 amino acid deletion in pre-S2 ("pre-S2Δ3-25") were constructed. At 72 h posttransfection into Huh7 cells, qHBsAg were measured using electrochemiluminescence immunoassay analyzer. To mimic milieus of quasispecies, we co-transfected either pre-S1Δ1-8 or pre-S2Δ3-25 with wild type (WT). RESULTS Pre-S mutations affected transcription and replication ability of HBV because of altered overlapping polymerase. Compared with WT, extracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were on average 3.87-fold higher and 0.92-fold lower, respectively, whereas intracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were 0.57-fold lower and 1.60-fold higher, respectively. Immunofluorescence staining of cellular HBsAg showed that pre-S1Δ1-8 had less staining and that pre-S2Δ3-25 had denser staining. As ratios of either pre-S1Δ1-8 or pre-S2Δ3-25:WT increased from 0:10 to 10:0 gradually, relative extracellular qHBsAg increased from 1.0 to 3.85 in pre-S1Δ1-8 co-transfection, whereas those decreased from 1.0 to 0.88 in pre-S2Δ3-25 co-transfection. CONCLUSION Pre-S mutations exhibit different phenotypes of genome replication and HBsAg expression according to their locations. Thus, qHBsAg level for diagnosis and prognostification in chronic HBV infection should be used more cautiously, considering emergences of pre-S deletion mutants.
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14
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Liu W, Cao Y, Wang T, Xiang G, Lu J, Zhang J, Hou P. The N-Glycosylation Modification of LHBs (Large Surface Proteins of HBV) Effects on Endoplasmic Reticulum Stress, Cell Proliferation and its Secretion. HEPATITIS MONTHLY 2013; 13:e12280. [PMID: 24282423 PMCID: PMC3830522 DOI: 10.5812/hepatmon.12280] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/18/2013] [Revised: 07/21/2013] [Accepted: 08/25/2013] [Indexed: 02/07/2023]
Abstract
BACKGROUND The mutations of LHBs in pre-S, especially in pre-S2, are definitive in hepatocellular carcinoma (HCC) associated with HBV. However, the mechanisms of the N-glycosylation modification in LHBs are unclear. The N-glycosylation modification of LHBs affects Endoplasmic Reticulum stress, cell proliferation and its secretion which was further studied. OBJECTIVES The objectives of our studies was to indentified that modification of LHBs by N glycosylation modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation. MATERIALS AND METHODS The LHBs was mutated; then expression of proteins related to endoplasmic reticulum stress and EAED path of L02 cells affected by LHBs and its mutations was evaluated. LHBs proteins bound to multiubiquitin chains and its glycosylation motif were studied. The subcellular localization and secretion of LHBs and its mutations were identified. The effect on cell cycle and proliferation by LHBs and its mutations were detected. RESULTS These data demonstrated that the N-glycosylation motifs of LHBs were associated with ER stress. The N15S, N123S, and N177S mutated LHBs proteins could induce overexpression of EDEM in L02 cells. LHBs and its mutated proteins contained p62-derived UBA domain, which could affect expression of cyclins. The subcellular localization of LHBs in endoplasmic reticulum was similar to its mutations. The secretion of LHBs was blocked by N320K mutation, which could induce an increase in G1 phase and inhibition of S phase, and inhibited mitotic entry. CONCLUSIONS In conclusion, our studies powerfully demonstrated that modification of LHBs by N glycosylation could modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation. The N320K may be the key sites N-linked glycosylation modification of LHBs. It may be a mechanism of HBV-induced HCC.
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Affiliation(s)
- Wenxiang Liu
- Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China
| | - Yongmei Cao
- International Mongolian Hospital, Hohhot of Inner Mongolia, Hohhot, China
| | - Tao Wang
- Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China
| | - Guoan Xiang
- Department of General Surgery, the Second People's Hospital of Guangdong Province, Guangzhou, China
| | - Jiangyang Lu
- Department of Pathology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China
- Corresponding authors: Peng Hou, Department of Digest, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848191, Fax: +86-1066848191, E-mail: ; Jinqian Zhang, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China. Tel:+86-1084322622, Fax: +86-1084322622, E-mail: ; Jiangyang Lu, Department of Pathology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848193, Fax: +86-1066848193, E-mail:
| | - Jinqian Zhang
- Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Corresponding authors: Peng Hou, Department of Digest, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848191, Fax: +86-1066848191, E-mail: ; Jinqian Zhang, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China. Tel:+86-1084322622, Fax: +86-1084322622, E-mail: ; Jiangyang Lu, Department of Pathology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848193, Fax: +86-1066848193, E-mail:
| | - Peng Hou
- Department of Gastroenterology and Hepatology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China
- Corresponding authors: Peng Hou, Department of Digest, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848191, Fax: +86-1066848191, E-mail: ; Jinqian Zhang, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China. Tel:+86-1084322622, Fax: +86-1084322622, E-mail: ; Jiangyang Lu, Department of Pathology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing, China. Tel: +86-1066848193, Fax: +86-1066848193, E-mail:
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15
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Qin D, Li K, Qu J, Wang S, Zou C, Sheng Y, Huang A, Tang H. HBx and HBs regulate RhoC expression by upregulating transcription factor Ets-1. Arch Virol 2013; 158:1773-81. [DOI: 10.1007/s00705-013-1655-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2012] [Accepted: 02/01/2013] [Indexed: 10/27/2022]
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16
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Truncated active human matrix metalloproteinase-8 delivered by a chimeric adenovirus-hepatitis B virus vector ameliorates rat liver cirrhosis. PLoS One 2013; 8:e53392. [PMID: 23301066 PMCID: PMC3536652 DOI: 10.1371/journal.pone.0053392] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2012] [Accepted: 11/27/2012] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Liver cirrhosis is a potentially life-threatening disease caused by progressive displacement of functional hepatocytes by fibrous tissue. The underlying fibrosis is often driven by chronic infection with hepatitis B virus (HBV). Matrix metalloproteinases including MMP-8 are crucial for excess collagen degradation. In a rat model of liver cirrhosis, MMP-8 delivery by an adenovirus (Ad) vector achieved significant amelioration of fibrosis but application of Ad vectors in humans is subject to various issues, including a lack of intrinsic liver specificity. METHODS HBV is highly liver-specific and its principal suitability as liver-specific gene transfer vector is established. HBV vectors have a limited insertion capacity and are replication-defective. Conversely, in an HBV infected cell vector replication may be rescued in trans by the resident virus, allowing conditional vector amplification and spreading. Capitalizing on a resident pathogen to help in its elimination and/or in treating its pathogenic consequences would provide a novel strategy. However, resident HBV may also reduce susceptibility to HBV vector superinfection. Thus a size-compatible truncated MMP-8 (tMMP8) gene was cloned into an HBV vector which was then used to generate a chimeric Ad-HBV shuttle vector that is not subject to superinfection exclusion. Rats with thioacetamide-induced liver cirrhosis were injected with the chimera to evaluate therapeutic efficacy. RESULTS Our data demonstrate that infectious HBV vector particles can be obtained via trans-complementation by wild-type virus, and that the tMMP8 HBV vector can efficiently be shuttled by an Ad vector into cirrhotic rat livers. There it exerted a comparable beneficial effect on fibrosis and hepatocyte proliferation markers as a conventional full-length MMP-8Ad vector. CONCLUSIONS Though the rat cirrhosis model does not allow assessing in vivo HBV vector amplification these results advocate the further development of Ad-HBV vectors for liver-specific gene therapy, including and perhaps particularly for HBV-related disease.
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17
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Lim L, Tran BM, Vincan E, Locarnini S, Warner N. HBV-related hepatocellular carcinoma: the role of integration, viral proteins and miRNA. Future Virol 2012. [DOI: 10.2217/fvl.12.113] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The development of hepatocellular carcinoma during chronic hepatitis B infection is a multifactorial process thought to be a consequence of several direct and indirect mechanisms. In this review we discuss how viral proteins and cycles of ongoing liver damage and regeneration, coupled with HBV DNA integration and aberrant miRNA expression may enhance the risk for the development of hepatocellular carcinoma.
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Affiliation(s)
- Lucy Lim
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
- Austin Liver Transplant Unit, Heidelberg, Victoria, Australia
| | - Bang Manh Tran
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
- Cancer Biology Laboratory, Department of Anatomy & Neuroscience, The University of Melbourne, Parkville, Victoria, Australia
| | - Elizabeth Vincan
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
- Cancer Biology Laboratory, Department of Anatomy & Neuroscience, The University of Melbourne, Parkville, Victoria, Australia
| | - Stephen Locarnini
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
| | - Nadia Warner
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
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18
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ECHS1 acts as a novel HBsAg-binding protein enhancing apoptosis through the mitochondrial pathway in HepG2 cells. Cancer Lett 2012. [PMID: 23178449 DOI: 10.1016/j.canlet.2012.11.030] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
We aimed to confirm the role of ECHS1 as a binding protein of HBsAg (HBs) and investigate its function during the development of hepatocellular carcinoma (HCC). Our results show that both exogenous and endogenous ECHS1 proteins bind to HBs and co-localize in the cytoplasm in vitro. The coexistence of HBs and ECHS1 enhances HepG2 cell apoptosis, affects ECHS1 localization in the mitochondria and induces apoptosis by decreasing the mitochondrial membrane potential (MMP). These findings suggest that ECHS1 may be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.
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19
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Tian Y, Liu Y, Qu J, Li K, Qin D, Huang A, Tang H. HBV regulated RhoC expression in HepG2.2.15 cells by enhancing its promoter activity. J Basic Microbiol 2012; 53:461-8. [PMID: 22736547 DOI: 10.1002/jobm.201200063] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2012] [Accepted: 03/14/2012] [Indexed: 12/11/2022]
Affiliation(s)
- Yuanyuan Tian
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
| | - Yunzhi Liu
- Department of Forensic Medicine; Chongqing Medical University; Chongqing; China
| | - Jialin Qu
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
| | - Kai Li
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
| | - Dongdong Qin
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
| | - Ailong Huang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
| | - Hua Tang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University; Chongqing; China
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20
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Wang LHC, Huang W, Lai MD, Su IJ. Aberrant cyclin A expression and centrosome overduplication induced by hepatitis B virus pre-S2 mutants and its implication in hepatocarcinogenesis. Carcinogenesis 2011; 33:466-72. [PMID: 22159224 DOI: 10.1093/carcin/bgr296] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.
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Affiliation(s)
- Lily Hui-Ching Wang
- Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan
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21
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Pollicino T, Saitta C, Raimondo G. Hepatocellular carcinoma: the point of view of the hepatitis B virus. Carcinogenesis 2011; 32:1122-32. [PMID: 21665892 DOI: 10.1093/carcin/bgr108] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Affiliation(s)
- Teresa Pollicino
- Department of Internal Medicine, Unit of Clinical and Molecular Hepatology, University Hospital of Messina, Via Consolare Valeria, Messina, Italy.
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22
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Yeung P, Wong DKH, Lai CL, Fung J, Seto WK, Yuen MF. Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in chronic hepatitis B. J Infect Dis 2011; 203:646-54. [PMID: 21227916 DOI: 10.1093/infdis/jiq096] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND We aimed to determine whether hepatitis B virus (HBV) pre-S deletion was an independent factor for the development of hepatocellular carcinoma (HCC). METHODS Pre-S deletions were determined in HBV isolates from 115 chronic hepatitis B (CHB) patients with HCC. Sixty-nine patients were further matched with 69 CHB patients without HCC for age, sex, hepatitis B e antigen (HBeAg) status, and HBV genotype. RESULTS HBV pre-S deletions were clustered mainly in the 3' end of pre-S1 and 5' end of pre-S2 regions. Adjusted for confounding risk factors, patients with HCC had a higher prevalence of HBV with pre-S deletions than did patients without HCC (23 [33.3%] of 69 vs 11 [15.9%] of 69; P = .018; odds ratio [OR], 2.64). In particular, only pre-S2 deletions but not pre-S1 deletions were significantly associated with the development of HCC (P = .020). A higher prevalence of pre-S deletions was observed in HBV isolates from HCC patients under the age of 50 years than from those older than 50 years (10 [62.5%] of 16 vs 13 [24.5%] of 53; P = .012; OR, 5.13). Emergence of de novo pre-S deletions was documented before the development of HCC. CONCLUSIONS HBV pre-S2 deletions were an independent factor associated with the development of HCC. Its oncogenic role may be more important in young patients with HCC.
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Affiliation(s)
- Pok Yeung
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Taiwan
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23
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Tian Y, Hu Y, Wang Z, Chen K, Zhang L, Wang L, Ren M, Huang A, Tang H. Hepatitis B virus regulates Raf1 expression in HepG2.2.15 cells by enhancing its promoter activity. Arch Virol 2011; 156:869-74. [DOI: 10.1007/s00705-010-0901-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2010] [Accepted: 12/20/2010] [Indexed: 11/29/2022]
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24
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Pan JS, Cai JY, Xie CX, Zhou F, Zhang ZP, Dong J, Xu HZ, Shi HX, Ren JL. Interacting with HBsAg compromises resistance of jumping translocation breakpoint protein to ultraviolet radiation-induced apoptosis in 293FT cells. Cancer Lett 2009; 285:151-156. [PMID: 19487072 DOI: 10.1016/j.canlet.2009.05.009] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2009] [Revised: 05/05/2009] [Accepted: 05/10/2009] [Indexed: 01/19/2023]
Abstract
Jumping translocation breakpoint protein (JTB) is suppressed in many cancers, implying it plays a role in the neoplastic transformation of cells. In order to explore the role of JTB in the carcinogenesis of liver, we used mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal to verify the interaction between HBs and JTB. According to the results, HBs interacts with JTB. In addition, we further determined that S region within HBs is sufficient for binding JTB. Overexpression of JTB conferred resistance to apoptosis induced by ultraviolet radiation, whereas this effect was compromised by the co-overexpression of HBs.
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Affiliation(s)
- Jin-Shui Pan
- Division of Gastroenterology, Zhongshan Hospital Xiamen University, Xiamen 361004, Fujian Province, China
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25
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Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection. J Virol 2009; 83:8396-408. [PMID: 19535448 DOI: 10.1128/jvi.00700-09] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.
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26
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Wang J, Jiang D, Zhang H, Lv S, Rao H, Fei R, Wei L. Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line HepG2. Proteomics 2009; 9:1672-82. [PMID: 19242931 DOI: 10.1002/pmic.200800621] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) infection is a worldwide health problem and may develop to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. To investigate the global proteome responses of liver-derived cells to HBV infection and IFNalpha treatment, 2-DE and MS-based analysis were performed to compare the proteome changes between HBV stably transfected cell line HepG2.2.15 and its parental cell line HepG2, as well as HepG2.2.15 before and after IFNalpha treatment (5000 IU/mL for 72 h). Compared to HepG2, 12 of 18 down-regulated and 27 of 32 up-regulated proteins were identified in HepG2.2.15. After IFNalpha treatment, 6 of 7 down-regulated and 11 of 14 up-regulated proteins were identified. Differentially expressed proteins caused by HBV infection were involved with cytoskeletal matrix, heat shock stress, kinases/signal transduction, protease/proteasome components, etc. Prohibitin showed a dose-dependent up-regulation during IFNalpha treatment and might play a potent role in anti-HBV activities of IFNalpha by enhancing the crossbinding p53 expression to achieve the apoptosis of HBV infected liver cells. Down-regulation of interferon-stimulated gene 15 (ISG15) in HepG2.2.15 and recovery by IFNalpha suggested its relationship with IFNalpha's anti-HBV effect.
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Affiliation(s)
- Jianghua Wang
- Peking University Hepatology Institute, Peking University People's Hospital, Beijing, China
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27
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Warner N, Locarnini S. Article Commentary: Can Antiviral Therapy for Chronic Hepatitis B Enhance the Progression to Hepatocellular Carcinoma? Antivir Ther 2009. [DOI: 10.1177/135965350901400208] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Affiliation(s)
- Nadia Warner
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
| | - Stephen Locarnini
- Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
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28
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Abstract
The NF-kappaB signaling pathway has particular relevance to several liver diseases including hepatitis (liver infection by Helicobacter, viral hepatitis induced by HBV and HCV), liver fibrosis and cirrhosis and hepatocellular carcinoma. Furthermore, the NF-kappaB signaling pathway is a potential target for development of hepatoprotective agents. Several types of drugs including: selective estrogen receptor modulators (SERMs), antioxidants, proteasome inhibitors, IKK inhibitors and nucleic acid-based decoys have been shown to interfere with NF-kappaB activity at different levels and may be useful for the treatment of liver diseases. However, NF-kappaB also plays an important hepatoprotective function that needs to be taken into consideration during development of new therapeutic regimens.
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Affiliation(s)
- B Sun
- Liver Transplantation Center of the First Affiliated Hospital and Cancer Center, Nanjing Medical University, Nanjing, Jiangsu Province, PR China
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29
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Pre-P is a secreted glycoprotein encoded as an N-terminal extension of the duck hepatitis B virus polymerase gene. J Virol 2008; 83:1368-78. [PMID: 19004940 DOI: 10.1128/jvi.01263-08] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms "pre-P" by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.
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Gurtsevitch VE. Human oncogenic viruses: hepatitis B and hepatitis C viruses and their role in hepatocarcinogenesis. BIOCHEMISTRY (MOSCOW) 2008; 73:504-13. [PMID: 18605975 DOI: 10.1134/s0006297908050039] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Chronic infections caused by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) are the main risk factors for the development of hepatocellular carcinoma (HCC) in humans. Both viruses cause a wide spectrum of clinical manifestations ranging from healthy carrier state to acute and chronic hepatitis, liver cirrhosis, and HCC. HBV and HCV belong to different viral families (Hepadnoviridae and Flaviviridae, respectively); they are characterized by different genetic structures. Clinical manifestations of these viral infections result from the interaction between these viruses and host hepatocytes (i.e. between viral and cell genomes). Proteins encoded by both viruses play an important role in processes responsible for immortalization and transformation of these cells. Chronic inflammation determined by host immune response to the viral infection, hepatocyte death and their compensatory proliferation, as well as modulation of expression of some regulatory proteins of the cell (growth factors, cytokines, etc.) are the processes that play the major role in liver cancer induced by HBV and HCV.
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Affiliation(s)
- V E Gurtsevitch
- Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Moscow 115478, Russia.
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Su IJ, Wang HC, Wu HC, Huang WY. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J Gastroenterol Hepatol 2008; 23:1169-74. [PMID: 18505413 DOI: 10.1111/j.1440-1746.2008.05348.x] [Citation(s) in RCA: 108] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The discovery of "ground glass" hepatocytes (GGH) that contain hepatitis B virus (HBV) surface antigens by Hadziyannis and Popper in 1973 represents a historical landmark in the pathology of chronic HBV infection. Different types of GGH have been correlated to the expression patterns of surface/core antigens and the stages of virus replication. The original two types (designated types I & II) of GGH were found to contain specific pre-S mutants with deletions over either pre-S1 or pre-S2 regions, respectively. Type II GGH consistently harbor pre-S2 deletion mutants, which can escape from immune attack and grow preferentially to form clusters. Both types of pre-S mutants can induce endoplasmic reticulum (ER) stress and oxidative DNA damage. The pre-S2 mutants, albeit inducing a weaker level of ER stress signals, could additionally initiate ER stress-independent retinoblastoma/adenovirus E2 promoter binding factor/cyclin A signaling through their interaction with c-Jun activation domain binding protein 1 to degrade p27, illustrating the growth advantage of type II GGH. The combined effects of genomic instability and the proliferation of hepatocytes harboring pre-S mutants could potentially lead to hepatocarcinogenesis over the decades of chronic HBV infection. The presence of pre-S mutants in sera was reported to carry a high risk of developing hepatocellular carcinoma (HCC). Furthermore, transgenic mice harboring pre-S2 mutant plasmids have been shown to develop a dysplastic change of hepatocytes and HCC. Therefore, in addition to being a histological marker of chronic HBV infection, GGH, particularly type II GGH, may represent the preneoplastic lesions of HBV-related HCC.
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Affiliation(s)
- Ih-Jen Su
- Division of Clinical Research, National Health Research Institutes, Tainan, Taiwan.
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32
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Liang X, Qu Z, Zhang Z, Du J, Liu Y, Cui M, Liu H, Gao L, Han L, Liu S, Cao L, Zhao P, Sun W. Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL. Biochem Biophys Res Commun 2008; 369:456-63. [DOI: 10.1016/j.bbrc.2008.02.035] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2008] [Accepted: 02/09/2008] [Indexed: 10/22/2022]
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Abstract
Notwithstanding the medical importance of the HBV infection, our understanding of how this pathogen enters hepatocytes is incomplete. This reflects a long-lasting dependence of in vitro infection studies solely on primary human hepatocytes, which are difficult to obtain and maintain in a susceptible state. The establishment of a polarizable HBV-susceptible human hepatoma cell line (HepaRG) and the utilization of Tupaia belangeri hepatocytes (PTHs) resolved this issue. Since then, important insight into viral and cellular determinants participating in HBV binding and infection have been achieved. We now know that the large viral surface protein (L) plays a pivotal role in HBV entry. It mediates diverse functions, commencing binding of virions to heparan sulfate proteoglycans at the hepatocytes surface as a prerequisite for entry. Subsequently, (a) highly specific event(s) involving the myristoylated N-terminal preS1 subdomain of L, as well as the cytosolic and antigenic loops of the S-domain, initiates a series of less well understood steps, resulting in a pH independent, reduction-sensitive fusion of the viral membrane with a cellular membrane. One of these steps is highly sensitive to synthetic N-acylated preS1 lipopeptides and can be blocked in vitro and in vivo at picomolar concentrations. This opens novel therapeutic options addressing virus entry. Future approaches aiming at the elucidation of HBV hepatotropism, the identification of (a) specific receptor(s), the clarification of the endocytic entry pathway and imaging of fluorescently-labeled virions will allow us to decipher more precisely the HBV entry pathway in the near future. Furthermore, clinical efficacy studies with HBV–preS-derived lipopeptides will tell us whether entry inhibition is a passable way to defend acute and chronic HBV and hepatitis delta virus infections.
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Affiliation(s)
- Stephan Urban
- University of Heidelberg, Department of Molecular Virology, Otto-Meyerhof-Zentrum (OMZ), Im Neuenheimer Feld 350, 69120 Heidelberg, Germany
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McLaughlin-Drubin ME, Munger K. Viruses associated with human cancer. BIOCHIMICA ET BIOPHYSICA ACTA 2008; 1782:127-50. [PMID: 18201576 PMCID: PMC2267909 DOI: 10.1016/j.bbadis.2007.12.005] [Citation(s) in RCA: 241] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2007] [Revised: 12/13/2007] [Accepted: 12/18/2007] [Indexed: 02/07/2023]
Abstract
It is estimated that viral infections contribute to 15-20% of all human cancers. As obligatory intracellular parasites, viruses encode proteins that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. These cellular processes are governed by complex and redundant regulatory networks and are surveyed by sentinel mechanisms that ensure that aberrant cells are removed from the proliferative pool. Given that the genome size of a virus is highly restricted to ensure packaging within an infectious structure, viruses must target cellular regulatory nodes with limited redundancy and need to inactivate surveillance mechanisms that would normally recognize and extinguish such abnormal cells. In many cases, key proteins in these same regulatory networks are subject to mutation in non-virally associated diseases and cancers. Oncogenic viruses have thus served as important experimental models to identify and molecularly investigate such cellular networks. These include the discovery of oncogenes and tumor suppressors, identification of regulatory networks that are critical for maintenance of genomic integrity, and processes that govern immune surveillance.
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Affiliation(s)
- Margaret E McLaughlin-Drubin
- The Channing Laboratory, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, 8th Floor, 181 Longwood Avenue, Boston, MA 02115, USA.
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Liu H, Luan F, Ju Y, Shen H, Gao L, Wang X, Liu S, Zhang L, Sun W, Ma C. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase. Biochem Biophys Res Commun 2007; 355:379-84. [PMID: 17307151 DOI: 10.1016/j.bbrc.2007.01.160] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2007] [Accepted: 01/30/2007] [Indexed: 12/12/2022]
Abstract
The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.
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Affiliation(s)
- Hua Liu
- Institute of Immunology, Shandong University School of Medicine, #44 Wenhua Xi Road, Jinan 250012, PR China
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Wang HC, Huang W, Lai MD, Su IJ. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci 2006; 97:683-8. [PMID: 16863502 PMCID: PMC11158693 DOI: 10.1111/j.1349-7006.2006.00235.x] [Citation(s) in RCA: 223] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Although hepatitis B virus (HBV) has been documented to cause hepatocellular carcinoma (HCC), the exact role of HBV in the development of HCC remains enigmatic. Several hypotheses have been proposed to explain the potential mechanism, including insertional mutagenesis of HBV genomes and transcriptional activators of HBV gene products such as hepatitis B x protein (HBx) and truncated middle S mutants. In the past few years, we have identified two types of large HBV surface antigens (LHBs) with deletions at the pre-S1 (DeltaS1-LHBs) and pre-S2 (DeltaS2-LHBs) regions in ground glass hepatocytes. The pre-S mutant LHBs are retained in the endoplasmic reticulum (ER) and escape from immune attack. The pre-S mutants, particularly DeltaS2-LHBs, are increasingly prevalent in patients with hepatitis B e antigen (HBeAg)-positive chronic HBV infection, ranging from 6% before the 3rd decade to 35% in the 6th decade. In HCC patients, the two pre-S mutants were detected in 60% of HCC patients, in the serum and in HCC tissues. Pre-S mutant LHBs can initiate ER stress to induce oxidative DNA damage and genomic instability. Furthermore, pre-S mutant LHBs can upregulate cyclooxygenase-2 and cyclin A to induce cell cycle progression and proliferation of hepatocytes. In transgenic mice, the pre-S mutants can induce dysplasia of hepatocytes and development of HCC. In a nested control study, the presence of pre-S mutants carried a high risk of developing HCC in HBV carriers. In summary, the findings we describe in this review suggest a potential role for HBV pre-S mutants in HBV-related hepatocarcinogenesis, providing a model of viral carcinogenesis associated with ER stress.
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Affiliation(s)
- Hui-Ching Wang
- Division of Clinical Research, National Health Research Institutes, National Cheng Kung University College of Medicine, Tainan 704, Taiwan
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Merle P. [Epidemiology, natural history and pathogenesis of hepatocellular carcinoma]. Cancer Radiother 2005; 9:452-7. [PMID: 16226912 DOI: 10.1016/j.canrad.2005.09.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/05/2005] [Indexed: 10/25/2022]
Abstract
Hepatocellular carcinoma is one of the most prevalent tumors worldwide and its incidence is increasing due to hepatitis C virus infection. Other etiologic factors are hepatitis B virus infection, alcoholic liver disease and hemochromatosis. This tumor mainly develops in cirrhotic livers that are true precancerous states. Although mechanisms of hepatocarcinogenesis remain badly known, some signaling pathways are frequently deregulated: inactivation of the p53 tumor suppressor factor in 25% of HCC, activation of the Wnt signaling and the telomerase immortalization enzyme in most of tumors. Hepatitis viruses play a direct oncogenic role by interaction between viral proteins and cellular ones, which control cell homeostasis, or by integration of hepatitis B virus genome into the host genome. Furthermore, hepatitis viruses play an indirect oncogenic role by chronic inflammation and hepatocyte regeneration related to viral hepatopathy. In a near future, a better understanding of virus-specific oncogenic mechanisms should allow us to set up innovative preventive and curative therapeutic strategies.
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Affiliation(s)
- P Merle
- Service d'hépatogastroentérologie, hôpital de l'Hôtel-Dieu, Inserm U271, 69003 Lyon, France.
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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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Wang HC, Chang WT, Chang WW, Wu HC, Huang W, Lei HY, Lai MD, Fausto N, Su IJ. Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes. Hepatology 2005; 41:761-70. [PMID: 15726643 DOI: 10.1002/hep.20615] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Naturally occurring mutants with a deletion in the pre-S2 region of the large surface protein (Delta S2-LHBs) are prevalent in serum and livers of patients with chronic hepatitis B virus (HBV) infection associated with cirrhosis. The Delta S2-LHBs protein is retained in the endoplasmic reticulum (ER) and may induce ER stress. One interesting observation is the consistently clustered distribution of hepatocytes expressing Delta S2-LHBs. In this study, complementary DNA microarray analysis identified cyclin A and several groups of genes as being significantly upregulated by Delta S2-LHBs in the HuH-7 cell line. This observation was confirmed in liver tissues. The induction of cyclin A expression may occur via the specific transactivator function of Delta S2-LHBs independent of ER stress. In the presence of Delta S2-LHBs, hepatocytes sustained cyclin A expression and cell cycle progression under ER stress and displayed increased BrdU incorporation with multinuclear formation. Furthermore, Delta S2-LHBs could enhance anchorage-independent cell growth in a nontransformed human hepatocyte line and induced nodular proliferation of hepatocytes in transgenic mice. In conclusion, these in vitro and in vivo data support a role for Delta S2-LHBs in the hepatocyte hyperplasia and a likely role in the process of HBV-related tumorigenesis.
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Affiliation(s)
- Hui-Ching Wang
- Division of Clinical Research, National Health Research Institutes, Tainan, Taiwan
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Lu X, Lee M, Tran T, Block T. High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus. Int J Med Sci 2005; 2:30-35. [PMID: 15968337 PMCID: PMC1142222 DOI: 10.7150/ijms.2.30] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2004] [Accepted: 01/01/2005] [Indexed: 01/08/2023] Open
Abstract
The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.
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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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N/A, 成 军, 刘 妍, 杨 倩, 纪 冬, 王 春. N/A. Shijie Huaren Xiaohua Zazhi 2004; 12:160-162. [DOI: 10.11569/wcjd.v12.i1.160] [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: 03/05/2023] Open
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Lu YY, Liu Y, Cheng J, Ling YD, Chen TY, Shao Q, Wang L, Zhang LX. Genes trans-regulated by a novel hepatitis B virus preS2 antigen binding protein S2-29 by cDNA microarray. Shijie Huaren Xiaohua Zazhi 2004; 12:58-61. [DOI: 10.11569/wcjd.v12.i1.58] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the biological functions of a novel hepatitis B virus preS2 antigen binding protein S2-29, and to analyze the gene expression profiles of HepG2 cell transfected with S2-29 gene.
METHODS: S2-29 gene was screened and identified by using yeast two-hybrid system 3 and coimmunoprecipita-tion technique. Full-length encoding frame S2-29 and its amino acid sequences were identified by using bioinformatics method and the recombined eukaryotic expression plasmid pcDNA3.1(-)-S2-29 was constructed and transfected into HepG2 cells. Total mRNA was isolated from the HepG2 cells transfected with pcDNA3.1(-) and pcDNA3.1(-)-S2-29, respectively. cDNA microarray was employed for detecting and analysing of mRNA from the HepG2 cells.
RESULTS: S2-29 cDNA sequence was obtained and identified by yeast two-hybrid screening and the bioinformatics analysis. Among 1 152 genes, there were 10 differences, of which 9 genes were upregulated and 1 gene were downregulated in HepG2 cells transfected with S2-29 protein expression plasmid. These genes differentially down-regulated by S2-29 protein included eukaryotic translation elongation factor 2, MAP-kinase activating death domain, glutathione peroxidase 5, gelsolin-like capping protein (actin filament), NDRG family member 2, prosaposin, SUMO-1 activating enzyme subunit 1, insulin receptor and a novel protein.
CONCLUSION: Microarray technique is successfully used to screen the genes trans-regulated by S2-29, which brings some new clues for studying the trans-regulation and biological function of S2-29.
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Dong J, Cheng J, Yang Q. Identification and characterization of high variable regions in hepatitis B virus genome of adr and adw serotypes. Shijie Huaren Xiaohua Zazhi 2004; 12:42-46. [DOI: 10.11569/wcjd.v12.i1.42] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To identify a hypervariable region or a hyperconservative region in hepatitis B virus (HBV) genome and to study the pre-X region and the pre-pre-S region.
METHODS: Full-length of HBV genomes deposited in GenBank were searched and compared for their identity and homology. According to their sequences, the HBV genomes of adr or adw serotypes were selected. The Vector 6.0 software was used to compare the identity and difference among the strains of HBV genomes.
RESULTS: Twenty-eight strains of HBV genome in GenBank belong to adr serotype HBV genome, and 22 strains belong to adw serotype. After being compared, the total identity rates were 76.6% and 73.9%, respectively. There might be a hypervariable region and a hyperconservative region in adr serotype HBV genome, with the regional identity rate of 54.5% and 92.1%, respectively. There might be a hyperconservative region in adw serotype HBV genome, with the regional identity rate of 85.0%. Region coding pre-pre-S gene was popular in the strains. Pre-X region might be a serotype-specific gene. A2608 C/T and/or C/A2733 T replacement mutation resulted in disability of coding pre-X gene.
CONCLUSION: There may be a hyperconservative region in HBV genome. Pre-pre-S gene and pre-X gene are worthy of further study.
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Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. THE AMERICAN JOURNAL OF PATHOLOGY 2003; 163:2441-9. [PMID: 14633616 PMCID: PMC1892360 DOI: 10.1016/s0002-9440(10)63599-7] [Citation(s) in RCA: 187] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Ground glass hepatocyte (GGH) represents a histological hallmark of chronic hepatitis B virus infection and contains surface antigens in the endoplasmic reticulum (ER). Several types of GGHs are recognized at different hepatitis B virus replicative stages. The recent identification of pre-S mutants from GGHs encourages us to investigate whether different GGHs may harbor specific mutants and exhibit differential biological activities. In this study, we applied laser capture microdissection to isolate specific GGHs from a total of 50 samples on eight resected liver specimens. The surface genes in two major types of GGHs were analyzed. Type I GGHs expressed an inclusion-like pattern of hepatitis B surface antigens and harbored mutants with deletions over pre-S1 region, whereas type II GGHs, distributed in clusters and emerged at late replicative phase, contained mutants with deletions over pre-S2 region that defines a cytotoxic T lymphocyte (CTL) immune epitope, and may represent an immune escape mutant. Transfection of pre-S mutants in Huh7 revealed decreased syntheses of middle and small S proteins with accumulation of large surface antigen in ER, which in turn led to the activation of ER stress response with differential activities for different mutants. This study therefore demonstrates that different GGHs may contain specific mutants and exhibit differential biological activities.
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Affiliation(s)
- Hui-Ching Wang
- Graduate Institutes of Basic Medical Sciences, Molecular Medicine, and Immunology and Microbiology, National Cheng Kung University College of Medicine, Tainan, Taiwan
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Abstract
AIM To identify the pre-X gene in HBV strains of Chinese patients.
METHODS The whole HBV genome was amplified by long-distance and accurate polymerase chain reaction (LA-PCR) method from the serum of 2 patients with chronic HBV infection, and then the PCR products were cloned into pGEM Teasy vectors. Five clones of HBV genome were sequenced. Sequences of our finding were compared with other HBV genome sequence deposited in GenBank.
RESULTS Pre-X region was found in the HBV genome of these 5 clones, just upstream to the X gene. The pre-X gene is 168 bp long, and can be translated with X gene in frame. The amino acids sequence of pre-X gene was as the following: MGLGYWPSPPAWNLCGSSADPYCGTPSSLFCS QPVWSETYRNRQLCCPLSQIHLLS.
CONCLUSION Pre-X region may be a serotype-specific coding gene.
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Affiliation(s)
- Jing Dong
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China
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Abstract
AIM To investigate the new open reading frame (ORF) in hepatitis B virus (HBV) genome.
METHODS The whole HBV genome was amplified by long-distance and accurate polymerase chain reaction (LA-PCR) method from the serum of 2 patients with chronic HBV infection, and then the PCR products were ligased into pGEM Teasy vectors. Five clones of HBV genome were sequenced. Sequences of our finding were compared with other HBV genome sequence deposited in GenBank.
RESULTS A new ORF was found in HBV genome, just before Pre-S1 region. It was defined as pre-pre-S region. The pre-pre-S ORF was deduced to be translated with Pre-S1, Pre-S2 and S gene in frame. Its amino acids sequence was as the following: MQLIITSKLGIIYILCGRLVFYIREKLHAV PHFVGHHILGNKSYS. There was a TA-rich region before the ATG of pre-pre-S ORF, indicating a possible promoter for its transcription.
CONCLUSION There is a new ORF pre-pre-S located upstream to pre-S1 region.
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Affiliation(s)
- Jing Dong
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China
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Lu YY, Cheng TY, Cheng J, Liang YD, Wang L, Liu Y, Li K, Zhang J, Shao Q, Zhang LX. Screening and identification of a novel gene coding for hepatitis B virus pre-S2 antigen interacting protein S2-29. Shijie Huaren Xiaohua Zazhi 2003; 11:1114-1117. [DOI: 10.11569/wcjd.v11.i8.1114] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM The Pre-S2 region of hepatitis B virus (HBV) has been reported to have complex biological functions. It has human polymerized albumin receptor (PAR) activity, which correlates with viral replication, and it can induce neutralization antibody. As an important part of truncated middle surface proteins (MHBs), the Pre-S2 domain binds PKC alpha/beta and triggers a PKC-dependent activation of the c-Raf-1/MAP2-kinase signal transduction cascade, resulting in activation of transcription factors such as AP-1 and NF-kB. To investigate the biological function of hepatitis B virus (HBV) Pre-S2 protein, we used yeast two-hybrid technique to screen proteins interacting with HBV Pre-S2 antigen in hepatocytes.
METHODS The HBV Pre-S2 gene was amplified by polymerase chain reaction (PCR) and cloned into yeast expression vector PGBKT7 to construct HBV Pre-S2 bait plasmid. The bait plasmid was transformed into yeast AH109 and mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After being extracted and sequenced, genes were analyzed by bioinformatics. The complete sequence of new gene S2-29 was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7, then translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro.
RESULTS Twenty-six colonies were obtained, among them two colonies were new genes with unknown function and no homeobox genes were found in Genbank by blast. The complete sequence of new gene S2-29 could be amplified from the mRNA of HepG2 cell and the interaction between HBV Pre-S2 antigen and S2-29 was further confirmed by coimmunoprecipitation technique.
CONCLUSION Genes of HBV Pre-S2 interacting proteins were successfully screened. A novel gene S2-29 was cloned and could express in HepG2 cell. The HBV Pre-S2 antigen could interact with S2-29, which brings new clues for studying the biological functions of HBV Pre-S2 and the pathogenesis of HBV infection.
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Affiliation(s)
- Yin-Ying Lu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Tian-Yan Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jun Cheng
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yao-Dong Liang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Lin Wang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Yan Liu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ke Li
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Jian Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Qing Shao
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
| | - Ling-Xia Zhang
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China
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