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1
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Liu J, Wang L, Zhang X, Wang S, Qin Q. Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process. Virulence 2024; 15:2301244. [PMID: 38230744 PMCID: PMC10795790 DOI: 10.1080/21505594.2023.2301244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Accepted: 12/28/2023] [Indexed: 01/18/2024] Open
Abstract
Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 μm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.
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Affiliation(s)
- Jiaxin Liu
- Biosafety Laboratory, Guangdong Second Provincial General Hospital, Guangzhou, China
- Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Liqun Wang
- Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Xinyue Zhang
- Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Shaowen Wang
- Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Qiwei Qin
- Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China
- Guangdong Laboratory for Lingnan Modern Agriculture, College of Marine Sciences, South China Agricultural University, Guangzhou, China
- Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
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2
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Uchida T, Imamura M, Hayes CN, Suehiro Y, Teraoka Y, Ohya K, Aikata H, Abe-Chayama H, Ishida Y, Tateno C, Hara Y, Hino K, Okamoto T, Matsuura Y, Aizaki H, Wake K, Kohara M, Liang TJ, Oka S, Chayama K. HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice. Hepatology 2023; 78:929-942. [PMID: 36896966 PMCID: PMC11519831 DOI: 10.1097/hep.0000000000000335] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 01/25/2023] [Indexed: 03/11/2023]
Abstract
BACKGROUND AND AIMS Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.
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Affiliation(s)
- Takuro Uchida
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - C. Nelson Hayes
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Yosuke Suehiro
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Yuji Teraoka
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kazuki Ohya
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Aikata
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Research Center for Hepatology and Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- Center for Medical Specialist Graduate Education and Research, Hiroshima, Japan
| | - Yuji Ishida
- Research Center for Hepatology and Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- PhoenixBio Co., Ltd., Higashihiroshima, Japan
| | - Chise Tateno
- Research Center for Hepatology and Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- PhoenixBio Co., Ltd., Higashihiroshima, Japan
| | - Yuichi Hara
- Department of Hepatology and Pancreatology, Kawasaki Medical School, Kurashiki, Japan
| | - Keisuke Hino
- Department of Hepatology and Pancreatology, Kawasaki Medical School, Kurashiki, Japan
| | - Toru Okamoto
- Institute for Advanced Co-creation Studies, Research Institute for Microbial Diseases Osaka University, Osaka, Japan
- Center for Infectious Disease Education and Research, Osaka University, Osaka, Japan
| | - Yoshiharu Matsuura
- Center for Infectious Disease Education and Research, Osaka University, Osaka, Japan
- Department of Virus Control, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kenjiro Wake
- Liver Research Unit, Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - T. Jake Liang
- Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Shiro Oka
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kazuaki Chayama
- Research Center for Hepatology and Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
- Hiroshima Institute of Life Sciences, Hiroshima, Japan
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3
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Piewbang C, Dankaona W, Poonsin P, Yostawonkul J, Lacharoje S, Sirivisoot S, Kasantikul T, Tummaruk P, Techangamsuwan S. Domestic cat hepadnavirus associated with hepatopathy in cats: A retrospective study. Vet Med (Auckl) 2022; 36:1648-1659. [PMID: 36054642 PMCID: PMC9511090 DOI: 10.1111/jvim.16525] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Accepted: 08/10/2022] [Indexed: 11/28/2022]
Abstract
Background Whether domestic cat hepadnavirus (DCH) infection is associated with clinical disease remains to be determined. Objectives To determine the relationship between DCH detection, hematology, serum bichemistry and liver histology in DCH‐positive cats. Animals One thousand twenty‐two cats in Thailand without concurrent diseases and not undergoing treatments adversely affecting the liver. Methods Retrospective cross‐sectional study. Samples derived from cats with concurrent virus detection were excluded. DCH detection was determined in blood and fresh‐frozen liver by quantitative polymerase chain reaction (qPCR) and further investigated in liver sections showing histological parenchymal disorders (HPD) and normal liver (HNL) using in situ hybridization (ISH). Proliferative/apoptotic activities were determined using immunohistochemistry and ISH panels. Biochemical variables and risk factors for DCH infection were investigated. Results Six hundred sixty‐one (557 blood and 119 liver samples) cats were included. DCH was detected in 18.50% (103/557), 13.85% (9/65), and 3.70% (2/54) of the blood, HPD, and HNL groups, respectively. Cats with DCH revealed abnormally high activity of aspartate aminotransferase (AST) (P = .001) and alanine aminotransferase (ALT) (P < .001). Among DCH‐positive HPD case 2/9 an 7/9 were acute and chronic hepatitis, of which 4/7 had hepatitis. Log viral copy number (LVCN) was positively correlated with ALT (P < .001), triglyceride (P < .001), and gamma‐glutamyl transpeptidase (GGT) (P = .022). The LVCN also had a positive association with degree of hepatitis (P < .05). There was hepatocyte proliferation activity in DHC positive cats. Conclusion and Clinical Importance Domestic cat hepadnavirus infection was associated with high serum activity of liver enzymes and chronic lymphoplasmacytic hepatitis (LPH).
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Affiliation(s)
- Chutchai Piewbang
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
- Animal Virome and Diagnostic Development Research Group, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Wichan Dankaona
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
- Animal Virome and Diagnostic Development Research Group, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Panida Poonsin
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
- Animal Virome and Diagnostic Development Research Group, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Jakarwan Yostawonkul
- National Nanotechnology Center (NANOTEC) National Science and Technology Development Agency (NSTDA) Pathumthani Thailand
| | - Sitthichok Lacharoje
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
- Animal Virome and Diagnostic Development Research Group, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Sirintra Sirivisoot
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Tanit Kasantikul
- Clemson Veterinary Diagnostic Center Clemson University Columbia South Carolina USA
| | - Padet Tummaruk
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
| | - Somporn Techangamsuwan
- Department of Pathology, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
- Animal Virome and Diagnostic Development Research Group, Faculty of Veterinary Science Chulalongkorn University Bangkok Thailand
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4
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Sun H, Chang L, Yan Y, Wang L. Hepatitis B virus pre-S region: Clinical implications and applications. Rev Med Virol 2020; 31. [PMID: 33314434 DOI: 10.1002/rmv.2201] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 11/22/2020] [Accepted: 11/29/2020] [Indexed: 12/12/2022]
Abstract
Hepatitis B virus (HBV) infection is a major threat to global public health, which can result in many acute and chronic liver diseases. HBV, a member of the family Hepadnaviridae, is a small enveloped DNA virus containing a circular genome of 3.2 kb. Located upstream of the S-open-reading frame of the HBV genome is the pre-S region, which is vital to the viral life cycle. The pre-S region has high variability and many mutations in the pre-S region are associated with several liver diseases, such as fulminant hepatitis (FH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). In addition, the pre-S region has been applied in the development of several pre-S-based materials and systems to prevent or treat HBV infection. In conclusion, the pre-S region plays an essential role in the occurrence, diagnosis, and treatment of HBV-related liver diseases, which may provide a novel perspective for the study of HBV infection and relevant diseases.
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Affiliation(s)
- Huizhen Sun
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
- Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, PR China
| | - Le Chang
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
| | - Ying Yan
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
| | - Lunan Wang
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
- Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, PR China
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5
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Huang Y, Zhang Y, Liu Z, Liu C, Zheng J, Qin Q, Huang X. Autophagy Participates in Lysosomal Vacuolation-Mediated Cell Death in RGNNV-Infected Cells. Front Microbiol 2020; 11:790. [PMID: 32425913 PMCID: PMC7212415 DOI: 10.3389/fmicb.2020.00790] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2020] [Accepted: 04/02/2020] [Indexed: 01/14/2023] Open
Abstract
Nervous necrosis virus (NNV) is the etiological agent of viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER), which results in heavy economic losses to the aquaculture industry worldwide. Dramatic cytoplasmic vacuoles were observed during NNV infection both in vitro and in vivo; however, the origin and mechanism of cytoplasmic vacuolization remains unknown. In this report, we found that the cytoplasmic vacuole morphology became fused and enlarged during infection with red spotted grouper nervous necrosis virus (RGNNV), which was accompanied by increased cell death. Notably, Lyso-Tracker, but not Mito-Tracker or ER-Tracker, was accumulated in the vacuoles, and abnormal lysosome swelling was observed in RGNNV-infected cells, suggesting that the cytoplasmic vacuoles originated from lysosomal organelles. Cytoplasmic vacuolization and cell death in RGNNV-infected cells was completely blocked by the vacuolar H+-ATPase inhibitor (bafilomycin A1), and was significantly weakened by chloroquine (CQ), a lysosomotropic agent that induces the acidification of the lysosomes. This suggests that lysosome acidification was essential for vacuole formation. Significant inhibitory effects on vacuolization and cell death were also observed in the RGNNV-infected cells following treatment with nigericin and monensin (ionophores that uncouple the proton gradient present in lysosomes). This indicated that lysosome function was tightly associated with RGNNV infection-induced cell death. In addition, vacuoles were found to be partially co-localized with GFP-LC3II punctate dots during RGNNV infection. Moreover, the severity of vacuolization and cell death were both significantly decreased after treatment with the autophagy inhibitor, 3-MA, suggesting that autophagy was involved in lysosomal vacuolization and cell death evoked by RGNNV infection. Thus, our results demonstrate that autophagy participates in lysosomal vacuolation-mediated cell death during RGNNV infection, and provides new insight into our understanding of the potential mechanisms underlying nodavirus pathogenesis in vitro.
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Affiliation(s)
- Youhua Huang
- College of Marine Sciences, South China Agricultural University, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
| | - Ya Zhang
- College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Zetian Liu
- College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Chuanhe Liu
- Instrumental Analysis & Research Center, South China Agricultural University, Guangzhou, China
| | - Jiaying Zheng
- College of Marine Sciences, South China Agricultural University, Guangzhou, China
| | - Qiwei Qin
- College of Marine Sciences, South China Agricultural University, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China.,Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
| | - Xiaohong Huang
- College of Marine Sciences, South China Agricultural University, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
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6
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Nie YZ, Zheng YW, Miyakawa K, Murata S, Zhang RR, Sekine K, Ueno Y, Takebe T, Wakita T, Ryo A, Taniguchi H. Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells. EBioMedicine 2018; 35:114-123. [PMID: 30120080 PMCID: PMC6156717 DOI: 10.1016/j.ebiom.2018.08.014] [Citation(s) in RCA: 127] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Revised: 07/23/2018] [Accepted: 08/06/2018] [Indexed: 02/07/2023] Open
Abstract
Therapies against hepatitis B virus (HBV) have improved in recent decades; however, the development of individualized treatments has been limited by the lack of individualized infection models. In this study, we used human induced pluripotent stem cell (hiPSC) to generate a functional liver organoid (LO) that inherited the genetic background of the donor, and evaluated its application in modeling HBV infection and exploring virus-host interactions. To establish a functional hiPSC-LO, we cultured hiPSC-derived endodermal, mesenchymal, and endothelial cells with a chemically defined medium in a three-dimensional microwell culture system. Based on cell-cell interactions, these cells could organize themselves and gradually differentiate into a functional organoid, which exhibited stronger hepatic functions than hiPSC derived hepatic like cell (HLC). Moreover, the functional LO demonstrated more susceptibility to HBV infection than hiPSC-HLC, and could maintain HBV propagation and produce infectious virus for a prolonged duration. Furthermore, we found that virus infection could cause hepatic dysfunction of hiPSC-LOs, with down-regulation of hepatic gene expression, induced release of early acute liver failure markers, and altered hepatic ultrastructure. Therefore, our study demonstrated that HBV infection in hiPSC-LOs could recapitulate virus life cycle and virus induced hepatic dysfunction, suggesting that hiPSC-LOs may provide a promising individualized infection model for the development of individualized treatment for hepatitis.
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Affiliation(s)
- Yun-Zhong Nie
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Yun-Wen Zheng
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan; Department of Advanced Gastroenterological Surgical Science and Technology, University of Tsukuba, Tsukuba-shi, Ibaraki 305-8575, Japan; Research Center of Stem Cells and Regenerative Medicine, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, China,.
| | - Kei Miyakawa
- Department of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Soichiro Murata
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Ran-Ran Zhang
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Keisuke Sekine
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Yasuharu Ueno
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Takanori Takebe
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, 162-8640 Tokyo, Japan
| | - Akihide Ryo
- Department of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan
| | - Hideki Taniguchi
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan; Advanced Medical Research Center, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa 236-0004, Japan.
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7
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Shubin AV, Demidyuk IV, Komissarov AA, Rafieva LM, Kostrov SV. Cytoplasmic vacuolization in cell death and survival. Oncotarget 2018; 7:55863-55889. [PMID: 27331412 PMCID: PMC5342458 DOI: 10.18632/oncotarget.10150] [Citation(s) in RCA: 248] [Impact Index Per Article: 35.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2016] [Accepted: 06/06/2016] [Indexed: 12/15/2022] Open
Abstract
Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival.
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Affiliation(s)
- Andrey V Shubin
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia.,Laboratory of Chemical Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia.,Laboratory of Biologically Active Nanostructures, N.F. Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia
| | - Ilya V Demidyuk
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia
| | - Alexey A Komissarov
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia
| | - Lola M Rafieva
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia
| | - Sergey V Kostrov
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia
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8
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Li G, Zhu Y, Shao D, Chang H, Zhang X, Zhou D, Gao Y, Lan K, Deng Q. Recombinant covalently closed circular DNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model. Hepatology 2018; 67:56-70. [PMID: 28749559 DOI: 10.1002/hep.29406] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Revised: 06/01/2017] [Accepted: 07/24/2017] [Indexed: 12/30/2022]
Abstract
UNLABELLED Covalently closed circular DNA of hepatitis B virus (HBV) is critical for viral persistence in vivo. We recently reported a technique involving recombinant covalently closed circular DNA (rcccDNA) of HBV by site-specific DNA recombination. Using hydrodynamic injection, rcccDNA induces a temporarily prolonged HBV antigenemia in immunocompetent mice, similar to acute resolving HBV infection. In this study, we simulated the pathophysiological impact of chronic hepatitis to reproduce rcccDNA persistence in mouse models. We showed that rcccDNA achieved long-lasting persistence in the presence of a compromised immune response or when transcriptional activity was repressed. To closely mimic chronic hepatitis, we used a replication-defective recombinant adenoviral vector to deliver rcccDNA to the liver, which led to prominent HBV persistence throughout the experiment duration (>62 weeks) in transgenic mice expressing Cre recombinase under the albumin promoter. A sustained necroinflammatory response and fibrosis were identified in mouse livers, with dysplastic lesions commonly seen during the late stage of viral persistence, analogous to the progressive pathology of clinical chronic hepatitis. CONCLUSION rcccDNA was intrinsically stable in vivo, enabling long-term persistence in the context of chronic hepatitis, and viral persistence, in turn, may promote progression of chronic liver disease; our study also presented a surrogate model of HBV cccDNA persistence in mice that could advance our understanding of the pathogenesis of chronic hepatitis B. (Hepatology 2018;67:56-70).
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Affiliation(s)
- Gaiyun Li
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China.,Key Laboratory of Medical Molecular Virology (MOE & MOH), School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Yuanfei Zhu
- Key Laboratory of Medical Molecular Virology (MOE & MOH), School of Basic Medical Sciences, Fudan University, Shanghai, China.,Department of Hepatopathy, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Dianhui Shao
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China
| | - Hao Chang
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China.,Key Laboratory of Medical Molecular Virology (MOE & MOH), School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Xiaoming Zhang
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China
| | - Dongming Zhou
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China
| | - Yueqiu Gao
- Department of Hepatopathy, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Ke Lan
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
| | - Qiang Deng
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai, China.,Key Laboratory of Medical Molecular Virology (MOE & MOH), School of Basic Medical Sciences, Fudan University, Shanghai, China
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9
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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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10
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Sato A, Ishii T, Sano F, Yamada T, Takahashi H, Matsumoto N. Severe de novo Hepatitis B Recovered from Late-Onset Liver Insufficiency with Prolonged Ascites and Hypoalbuminemia due to Hepatitis B Virus Genotype Bj with Precore Mutation. Case Rep Gastroenterol 2016; 10:553-559. [PMID: 27920641 PMCID: PMC5121562 DOI: 10.1159/000450543] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/02/2016] [Accepted: 08/31/2016] [Indexed: 12/16/2022] Open
Abstract
De novo hepatitis B is associated with a high risk of hepatic failure often resulting in fatal fulminant hepatitis even when nucleotide analogues are administered. A 77-year-old female developed de novo hepatitis B after R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) treatment for diffuse large B-cell lymphoma. Hepatitis B virus (HBV) isolated from the patient was of genotype Bj, with a precore mutation (G1896A) exhibiting an extremely high viral load at the onset of hepatitis. She showed markedly high levels of transaminase with mild jaundice on admission and rapid decrease of prothrombin activity after admission. Although acute liver failure was averted by the administration of entecavir and corticosteroid pulse therapy, liver volume decreased to 860 ml, and marked hypoalbuminemia accompanying massive ascites occurred 2 months after the onset of hepatitis and persisted for 3 months with high levels of HBV DNA and mild abnormal alanine aminotransferase levels. Frequent infusions of albumin solution, nutrition support, and alleviation therapy showed limited effect. However, overall improvement along with HBV DNA reduction was observed after increasing the dose of entecavir and completion of prednisolone that was administered with a minimum dose for adrenal insufficiency. An immediate and sufficient suppression of virus replication with potent antiviral therapy is critical, particularly in patients infected with HBV precore mutation (G1896A) and/or Bj genotype, which may have a high viral replication and direct hepatocellular damage.
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Affiliation(s)
- Akira Sato
- Division of Gastroenterology, Department of Internal Medicine, St. Marianna University School of Medicine Yokohama City Seibu Hospital, Yokohama, Japan
| | - Toshiya Ishii
- Division of Gastroenterology, Department of Internal Medicine, St. Marianna University School of Medicine Yokohama City Seibu Hospital, Yokohama, Japan
| | - Fumiaki Sano
- Division of Hematology and Oncology, Department of Internal Medicine, St. Marianna University School of Medicine Yokohama City Seibu Hospital, Yokohama, Japan
| | - Takayuki Yamada
- Department of Radiology, St. Marianna University School of Medicine Yokohama City Seibu Hospital, Yokohama, Japan
| | - Hideaki Takahashi
- Division of Gastroenterology, Department of Internal Medicine, St. Marianna University School of Medicine Yokohama City Seibu Hospital, Yokohama, Japan
| | - Nobuyuki Matsumoto
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Japan
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Shubin AV, Demidyuk IV, Lunina NA, Komissarov AA, Roschina MP, Leonova OG, Kostrov SV. Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death. BMC Cell Biol 2015; 16:4. [PMID: 25886889 PMCID: PMC4355371 DOI: 10.1186/s12860-015-0050-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2014] [Accepted: 01/26/2015] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND 3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus. RESULTS Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis. CONCLUSIONS 3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.
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Affiliation(s)
- Andrey V Shubin
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
| | - Ilya V Demidyuk
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
| | - Nataliya A Lunina
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
| | - Alexey A Komissarov
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
| | - Marina P Roschina
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
| | - Olga G Leonova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119992, Russia.
| | - Sergey V Kostrov
- Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Science, Moscow, 123182, Russia.
- National Research Center "Kurchatov Institute", Moscow, 123182, Russia.
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12
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Hu Z, Li M, Liu J, Yu L, Xue Y, Chen Y. Detection of Hepatitis B Virus Large Surface Protein Using a Time-Resolved Immunofluorometric Assay. J Clin Lab Anal 2014; 29:498-504. [PMID: 25277704 DOI: 10.1002/jcla.21800] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2013] [Revised: 06/17/2014] [Accepted: 08/07/2014] [Indexed: 01/28/2023] Open
Abstract
BACKGROUND To establish a novel method based on time-resolved immunofluorometric assay (TR-IFMA) with higher sensitivity and a broader detection range for detecting serum hepatitis B virus large surface protein (L protein). METHODS The precision, sensitivity, specificity, coefficient of recovery, and stability of the assay were evaluated and comparison with the classical enzyme-linked immunosorbent assay (ELISA) was also executed. RESULTS The precision, specificity, and sensitivity of the TR-IFMA were clearly better than ELISA. Particularly, the sensitivity was 0.1 ng/ml; moreover, the specificity was 100%, 96%, 92.5%, 96.9%, 97.8%, and 100% in the sera of healthy blood donors, systemic lupus erythematosus (SLE) patients, rheumatoid arthritis (RA) patients, hepatitis C virus (HCV) patients, cytomegalovirus (CMV) infection patients, and pregnant patients, respectively. Meanwhile, we observed that the established TR-IFMA kit has a wider acceptable linear range of 0.63-10,367 ng/ml rather than the regular commercial ELISA kit having range of only 10.12-1095.9 ng/ml. Subsequently, correlation coefficient between the TR-IFMA and ELISA was 0.8009. The intra- and interassay precision rates were less than 5% for three different concentrations. The average recovery rate for L protein was 101.17%. In sum, the established assay kit performed better in terms of stability than the commercial ELISA kit. CONCLUSION The TR-IFMA that we developed for L protein presented a higher sensitivity and wider detecting range than regular commercial ELISA. Therefore, this TR-IFMA has promising value both in the screening of HBV and monitoring of antiviral therapy.
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Affiliation(s)
- Zhigang Hu
- Department of Laboratory Medicine, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Mei Li
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Jie Liu
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Lei Yu
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Yifeng Xue
- Department of Laboratory Medicine, Affiliated Wuxi people's Hospital of Nanjing Medical University, Wuxi, China
| | - Yu Chen
- Department of Laboratory Medicine, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
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13
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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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14
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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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15
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Na B, Huang Z, Wang Q, Qi Z, Tian Y, Lu CC, Yu J, Hanes MA, Kakar S, Huang EJ, Ou JHJ, Liu L, Yen TSB. Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury. PLoS One 2011; 6:e26240. [PMID: 22022578 PMCID: PMC3192172 DOI: 10.1371/journal.pone.0026240] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2011] [Accepted: 09/22/2011] [Indexed: 12/12/2022] Open
Abstract
Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.
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Affiliation(s)
- Bing Na
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
| | - Zhiming Huang
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
| | - Qian Wang
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
| | - Zhongxia Qi
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, United States of America
| | - Yongjun Tian
- Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, California, United States of America
| | - Cheng-Chan Lu
- Department of Pathology, National Cheng Kung University College of Medicine, Tainan, Taiwan
| | - Jingwei Yu
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, United States of America
| | - Martha A. Hanes
- Department of Laboratory Animal Resources, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Sanjay Kakar
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
- Department of Pathology, University of California, San Francisco, San Francisco, California, United States of America
| | - Eric J. Huang
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
- Department of Pathology, University of California, San Francisco, San Francisco, California, United States of America
| | - J.-H. James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, California, United States of America
| | - Limin Liu
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
| | - T. S. Benedict Yen
- Pathology Service, Veterans Administration Medical Center, San Francisco, California, United States of America
- Department of Pathology, University of California, San Francisco, San Francisco, California, United States of America
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16
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Wang NY, Zhang D, Zhao W, Li BA, Lin CQ. Hepatitis B virus large surface protein in serum as a candidate biomarker for evaluating hepatitis B virus infection. Clin Biochem 2011; 44:1199-204. [DOI: 10.1016/j.clinbiochem.2011.07.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2011] [Revised: 07/03/2011] [Accepted: 07/05/2011] [Indexed: 01/12/2023]
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17
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Giannousis IP, Manolakopoulos SG, Hadziyannis E, Georgiou A, Papatheodoridis GV. Changes of serum levels of keratin-18 fragments in hepatitis B e antigen-negative chronic hepatitis B patients under oral antiviral therapy. Antivir Ther 2011; 16:505-14. [DOI: 10.3851/imp1775] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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18
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Pan JS, Zhou F, Xie CX, Cai JY, Chen JM, Zhang ZP, Dong J, Xu HZ, Shi HX, Ren JL. Aldolase A-HBsAg interaction and its effect on ultraviolet radiation induced apoptosis in 293FT cells. J Gastroenterol Hepatol 2010; 25:1702-1709. [PMID: 20880182 DOI: 10.1111/j.1440-1746.2010.06237.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
BACKGROUND AND AIM Hepatitis B virus (HBV) infection poses great challenges to humans, claiming one million lives annually worldwide. Solid data have related HBV to hepatocellular carcinoma. METHODS In the present research, we verified the interaction between surface protein (HBs) encoded by HBV and aldolase A (ALDA) using yeast two-hybrid, mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal. RESULTS Anti-ALDA antibody precipitated Gal4-HBs fusion protein in the presence of HBs. Anti-HBs antibody precipitated p65ΔN-ALDA only in the presence of ALDA. Small HBs could be pulled down by GST-ALDA. Cells transfected with pCMV-AD-ALDA showed a protection from ultraviolet radiation-induced apoptosis (21.3% ± 1.3% for ALDA, 35.4% ± 2.1% for control, P < 0.05). CONCLUSIONS An interaction does exist between ALDA and HBs. The S region within HBs is sufficient for binding ALDA. In addition, ALDA conferred protection to ultraviolet radiation-induced apoptosis, and this effect was enhanced by the interaction between HBs and ALDA.
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Affiliation(s)
- Jin-Shui Pan
- Division of Gastroenterology, Zhongshan Hospital Xiamen University, and Gastroenterology Institute of Xiamen University, and Gastroenterology Center of Xiamen, Xiamen, Fujian Province, China
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Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen. J Virol 2010; 84:5860-7. [PMID: 20357083 DOI: 10.1128/jvi.02594-09] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.
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20
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Zhao C, Zhang W, Tian X, Fang C, Lu H, Yuan Z, Yang P, Wen Y. Proteomic analysis of cell lines expressing small hepatitis B surface antigen revealed decreased glucose-regulated protein 78 kDa expression in association with higher susceptibility to apoptosis. J Med Virol 2010; 82:14-22. [PMID: 19950238 DOI: 10.1002/jmv.21654] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Accumulating evidence suggests a key role of hepatocyte apoptosis in the pathogenesis of viral hepatitis B. It was found in this study that stable expression of small hepatitis B surface antigen (SHBs) in HepG2 and Huh7 cells increased susceptibility to apoptosis. Proteomic analysis of SHBs expressing HepG2 cells revealed 43 down-regulated and 38 up-regulated proteins. Some have been implicated in apoptosis, including glucose-regulated protein 78 kDa (GRP78), heterogeneous nuclear ribonucleoprotein H3 (hnRNP H), Rho GDP dissociation inhibitor (GDI), cystatin B, far upstream element-binding protein (FUSEbp), and TNF receptor-associated protein 1 (TRAP1). Differential expression of GRP78 and several other proteins was confirmed by Western blot analysis. Replenishing GRP78 improved cellular resistance to apoptosis, whereas reduction of GRP78 by siRNA increased susceptibility even in the absence of SHBs. Taken together, these results suggest that HBsAg plays a pro-apoptotic role through down-regulation of GRP78.
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Affiliation(s)
- Chao Zhao
- Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Institute of Biomedical Sciences, Fudan University, Shanghai, China
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21
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Levrero M, Belloni L. HBV Signaling. SIGNALING PATHWAYS IN LIVER DISEASES 2010:465-481. [DOI: 10.1007/978-3-642-00150-5_31] [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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Inoue J, Ueno Y, Nagasaki F, Wakui Y, Kondo Y, Fukushima K, Niitsuma H, Shimosegawa T. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 2009; 395:202-209. [PMID: 19850315 DOI: 10.1016/j.virol.2009.09.028] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2009] [Revised: 06/22/2009] [Accepted: 09/23/2009] [Indexed: 12/17/2022]
Abstract
A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.
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Affiliation(s)
- Jun Inoue
- Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai 980-8574, Japan.
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23
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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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Inoue J. Factors involved in the development of fulminant hepatitis B: Are the mutations of hepatitis B virus implicated? Hepatol Res 2009; 39:1053-5. [PMID: 19878349 DOI: 10.1111/j.1872-034x.2009.00609.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Jun Inoue
- Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan
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Huang Q, Wang L, Bai S, Lin W, Chen W, Lin J, Lin X. Global proteome analysis of hepatitis B virus expressing human hepatoblastoma cell line HepG2. J Med Virol 2009; 81:1539-50. [PMID: 19626621 DOI: 10.1002/jmv.21593] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
In countries where hepatitis B virus (HBV) is endemic, a high incidence of hepatocellular carcinoma (HCC) occur in HBV carriers and the prolonged replication and expression of HBV proteins in the liver is considered an important risk factor for progression to malignancy. However, the mechanism of pathogenesis of HBV-associated carcinoma remains elusive. In this study, the human hepatoblastoma HepG2 cell line harboring 1.2 x unit-length of the HBV genome was generated and subjected to a proteomic approach analyzing the global protein expression profiles of HepG2 cells with and without HBV replication and protein expression. By using fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), followed by MALDI-TOF-MS and database searching, a total of 50 differentially expressed proteins were identified, including some cell cycle-related proteins. These cycle-related proteins may lead to accumulation of HepG2-HBV cells in the G2/M phase, and an increase in the proportion of HepG2 cells with tripolar or multipolar spindles. This study described the proteomic alterations in HepG2 cells HBV-harboring, which may provide new insights into the underlying molecular mechanisms involved in HBV replication and pathogenesis.
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Affiliation(s)
- Qingling Huang
- Key Laboratory of Infection and Oncology, Research Center of Molecular Medicine, Fujian Medical University, Fuzhou, China
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Studach LL, Rakotomalala L, Wang WH, Hullinger RL, Cairo S, Buendia MA, Andrisani OM. Polo-like kinase 1 inhibition suppresses hepatitis B virus X protein-induced transformation in an in vitro model of liver cancer progression. Hepatology 2009; 50:414-23. [PMID: 19472310 PMCID: PMC2788305 DOI: 10.1002/hep.22996] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
UNLABELLED Chronic hepatitis B virus (HBV) infection is linked to development of hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis acting as a weak oncogene or a cofactor in hepatocarcinogenesis. pX induces DNA re-replication, DNA damage, and partial polyploidy in a poorly differentiated, immortalized hepatocyte cell line. In this study we employed sorted, pX-induced polyploid cells to investigate their growth and oncogenic transformation potential over the course of 70 cell doublings. Immediately after live cell-sorting, nearly 40% of pX-induced polyploid cells undergo apoptosis, whereas the surviving cells exhibit proliferation sensitive to p53. After 40 cell generations the pX-expressing polyploid cultures exhibit loss of p53 function and become growth factor- and anchorage-independent, indicative of oncogenic transformation. The pX-induced polyploid cultures in the course of 70 cell generations undergo progressively increasing DNA damage, propagate damaged DNA to daughter cells, and display increased expression of a cluster of proliferation genes shown to be elevated in human HCC, including HBV-HCC. One of these genes is the mitotic kinase Polo-like kinase 1 (Plk1). Oncogenic transformation is suppressed in the absence of pX expression, and significantly, by inhibition of Plk1. These results identify Plk1 as crucial in pX-mediated oncogenic transformation. CONCLUSION Partial polyploidy induced by pX is not immediately associated with oncogenic transformation. Continued DNA damage for 40 cell generations is reproducibly associated with loss of p53 function, enhanced expression of Plk1, and oncogenic transformation. Because Plk1 expression is also elevated in HBV-HCC tumors, this in vitro cellular model simulates liver cancer progression and pathogenesis in chronic HBV patients. Inhibition of Plk1 activity suppresses pX-mediated oncogenic transformation, identifying Plk1 as a promising therapeutic target for HBV-mediated HCC.
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Affiliation(s)
- Leo L. Studach
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA
| | - Lova Rakotomalala
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA
| | - Wen-Horng Wang
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA
| | - Ronald L. Hullinger
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA
| | - Stefano Cairo
- Oncogenesis and Molecular Virology Unit, Institut Pasteur, Paris Cedex 15, France
| | - Marie-Annick Buendia
- Oncogenesis and Molecular Virology Unit, Institut Pasteur, Paris Cedex 15, France
| | - Ourania M. Andrisani
- Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA
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Ding C, Wei H, Sun R, Zhang J, Tian Z. Hepatocytes proteomic alteration and seroproteome analysis of HBV-transgenic mice. Proteomics 2009; 9:87-105. [PMID: 19053081 DOI: 10.1002/pmic.200701053] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Hepatitis B is the most common and serious liver disease, especially in developing countries. Although HBV pathogenesis has been extensively investigated, the proteomic alteration of hepatocytes during HBV chronic infection is still unclear. Using the purified hepatocytes, we compared the protein profiles by 2-DE and LC-MS between HBV-transgenic (Tg) and corresponding background mice. Twenty-seven altered proteins were identified in hepatocytes from HBV-Tg mice, among which 13 proteins were involved in mitochondrion metabolism pathway including tricarboxylic acid (TCA) cycle and oxidative response; four proteins (SELENBP, SCP2, RGN and PRDX1) were also dramatically changed in liver samples from HBV-infected patients. Important genes (gpx, sod, ogg et al.) correlated to oxidative damage were up-regulated in the liver of HBV-Tg mice. Reactive oxygen species production was significantly increased while ATP production was decreased in liver mitochondria from HBV-Tg mice. Moreover, hepatocytes of HBV-Tg mice were more sensitive to hydrogen peroxide-induced cell death than that of wild-type control. Using 2-D Western blotting analysis, eight hepatocyte proteins were found to react with sera of HBV-Tg mice but not with that of background mice. Interestingly, two (Etfa and Dmgdh) of the eight reactive proteins were overexpressed in HBV-Tg mice. We believe this study is the first proteomic and seroproteome analysis of HBV-infected mammalian hepatocyte and provides insightful links between HBV infection and HBV-induced liver diseases.
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Affiliation(s)
- Chen Ding
- Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China
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Osawa Y, Seki E, Brenner DA. Apoptosis in Liver Injury and Liver Diseases. ESSENTIALS OF APOPTOSIS 2009:547-564. [DOI: 10.1007/978-1-60327-381-7_24] [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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Abstract
Hepatitis B viruses are small enveloped DNA viruses referred to as Hepadnaviridae that cause transient or persistent (chronic) infections of the liver. This family is divided into two genera, orthohepadnavirus and avihepadnavirus, which infect mammals or birds as natural hosts, respectively. They possess a narrow host range determined by the initial steps of viral attachment and entry. Hepatitis B virus is the focus of biomedical research owing to its medical significance. Approximately 2 billion people have serological evidence of hepatitis B, and of these approximately 350 million people have chronic infections (World Health Organisation, Fact Sheet WHO/204, October 2000). Depending on viral and host factors, the outcomes of infection with hepatitis B virus vary between acute hepatitis, mild or severe chronic hepatitis or cirrhosis. Chronic infections are associated with an increased risk for the development of hepatocellular carcinoma.
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Affiliation(s)
- Hans-Jürgen Netter
- Monash University, Department of Microbiology, Clayton Campus, Victoria 3800, Australia
| | - Shau-Feng Chang
- Industrial Technology Research Institute, Biomedical Engineering Laboratories, 300 Hsinchu, Taiwan
| | - Michael Bruns
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, 20251 Hamburg, Germany
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30
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Abstract
The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which, collectively, represent a large fraction of the total protein output of a mammalian cell. Therefore, the protein flux through the ER must be carefully monitored for abnormalities, including the buildup of misfolded proteins. Mammalian cells have evolved an intricate set of signaling pathways from the ER to the cytosol and nucleus, to allow the cell to respond to the presence of misfolded proteins within the ER. These pathways, known collectively as the unfolded protein response, are important for normal cellular homeostasis and organismal development and may also play key roles in the pathogenesis of many diseases. This review provides background information on the unfolded protein response and discusses a selection of diseases whose pathogenesis involves ER stress.
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Affiliation(s)
- Jonathan H. Lin
- Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
- Department of Pathology, University of California, San Francisco, California 94143
| | - Peter Walter
- Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
- Howard Hughes Medical Institute, Chevy Chase, Maryland 2081
| | - T.S. Benedict Yen
- Department of Pathology, University of California, San Francisco, California 94143
- Pathology Service, San Francisco Veterans Affairs Medical Center, San Francisco, California 94121
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31
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Voronina SG, Sherwood MW, Gerasimenko OV, Petersen OH, Tepikin AV. Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles. Am J Physiol Gastrointest Liver Physiol 2007; 293:G1333-8. [PMID: 17717043 DOI: 10.1152/ajpgi.00275.2007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.
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Affiliation(s)
- Svetlana G Voronina
- The Physiological Laboratory, The Univ. of Liverpool, Crown St., Liverpool L69 3BX UK
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32
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Dougherty AM, Guo H, Westby G, Liu Y, Simsek E, Guo JT, Mehta A, Norton P, Gu B, Block T, Cuconati A. A substituted tetrahydro-tetrazolo-pyrimidine is a specific and novel inhibitor of hepatitis B virus surface antigen secretion. Antimicrob Agents Chemother 2007; 51:4427-37. [PMID: 17875990 PMCID: PMC2167973 DOI: 10.1128/aac.00541-07] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals are thought to play a role in suppressing the HBV-specific immune response. Current therapeutics are not directed at reducing this viral antigenemia; thus, our group has focused on identifying inhibitors of HBsAg secretion. By using the HBV-expressing cell line HepG2.2.15, high-throughput screening of an 80,288-compound synthetic small-molecule library identified HBF-0259, an aromatically substituted tetrahydro-tetrazolo-(1, 5-a)-pyrimidine. Following resynthesis, HBF-0259 had a 50% effective concentration of approximately 1.5 microM in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 microM. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 microM; the selective index was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing viral particles, while the secretion of alpha-1-acid glycoprotein and alpha-1-antitrypsin was unaffected. The HBV e antigen, which is not a constituent of HBV particles, was also unaffected, suggesting that the secretion of particles bearing HBV structural glycoproteins is targeted directly. Inhibitory activity was also confirmed by transfection of HBsAg, indicating that the action of the compound is independent of those of other viral proteins. HBF-0259 had no effect on HBV DNA synthesis, demonstrating that inhibition is independent of viral genomic replication. Finally, HBF-0259 had little or no effect on the cell-to-cell spread of two unrelated viruses, suggesting that it is a specific inhibitor of secretion of HBsAg. Possible mechanisms of action and the implications for its development are discussed.
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Affiliation(s)
- Anne Marie Dougherty
- Institute for Hepatitis and Virus Research, Hepatitis B Foundation, 3805 Old Easton Road, Doylestown, PA 18902, USA
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Perlmutter DH, Brodsky JL, Balistreri WF, Trapnell BC. Molecular pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: a meeting review. Hepatology 2007; 45:1313-23. [PMID: 17464974 DOI: 10.1002/hep.21628] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
In recent years, we have witnessed several important paradigm shifts in understanding the molecular basis of liver disease in alpha-1-antitrypsin (AT) deficiency. These shifts have become possible as a result of a number of advances in research on the cell biology of aggregation-prone mutant proteins and in research on the pathobiological mechanisms of liver disease in general. Late-breaking research in these areas was the subject of an AASLD/Alpha-1 Foundation Single Topic Conference in Atlanta, Georgia, on January 26 to 28, 2006. The conference was titled "Alpha-1-Antitrypsin Deficiency and Other Liver Diseases Caused by Aggregated Proteins." Investigators from all over the world, representing a broad array of scientific disciplines and perspectives, discussed the pathobiology of AT deficiency, mechanisms of cell injury in diseases associated with aggregation-prone proteins, pathways by which cells respond to protein aggregation and mislocalization, and mechanisms of liver injury in general and in diseases related to AT deficiency. A session of the meeting was devoted to novel therapeutic strategies being developed for AT deficiency as well as to strategies either in development or already being applied to the class of diseases associated with mutant proteins.
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Affiliation(s)
- David H Perlmutter
- Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA.
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Abstract
Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma. Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. In this review, we will discuss the impact of virus-host interactions for the pathogenesis of HBV infection and liver disease. These interactions include the relevance of naturally occurring viral variants for clinical disease, the role of virus-induced apoptosis for HBV-induced liver cell injury and the impact of antiviral immune responses for outcome of infection.
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Affiliation(s)
- Thomas F Baumert
- Department of Medicine I, Schlosspark Klinik, Teaching Hospital of the Charite, Humboldt University, Heubnerweg 2, D-14059 Berlin, Germany
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35
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Phromjai J, Aiba N, Suzuki M, Sato H, Takahara T, Kondo S, Shiraki K. Infection and direct injury in human hepatocyte explants and a hepatoblastoma cell line due to hepatiticomimetic (non-hepatitis) viruses. J Med Virol 2007; 79:413-25. [PMID: 17311334 DOI: 10.1002/jmv.20783] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Hepatitis is caused by hepatitis viruses, but hepatitis or hepatocellular enzyme abnormalities is sometimes associated with infection by the hepatiticomimetic viruses. The direct and indirect effects of infection with hepatiticomimetic viruses were examined in two human hepatocyte systems. Poliovirus, adenovirus, and herpes simplex virus (HSV) induced cytopathology in Hep G2 cells. Measles virus caused no change in hepatocytes. Poliovirus infection did not affect cellular protein synthesis, and the peak of hepatocellular enzyme release coincided with the peak of virus release. The increase in adenovirus protein synthesis correlated with the decrease of transferrin synthesis, and enzyme release was not prominent. HSV induced viral protein synthesis with enhanced processing and inhibition of synthesis of alpha1-antitrypsin. The peak of enzyme release was later than the peak of virus release. In primary hepatocytes, poliovirus, adenovirus, and induced extensive cytopathology and enzyme release, and VZV caused cytopathology and significant but minute enzyme release. The ratio of lactate dehydrogenase to aspartate aminotransferase release was larger in poliovirus infection in both hepatocytes than in HSV or VZV infection. Although poliovirus and adenovirus are released by cytolysis and HSV and VZV are secreted by exocytosis of cytoplasmic vacuoles, enzyme release was independent of the type of virus release. Adenovirus showed strong cytotoxicity but did not modify the membrane nor cause enzyme release. Enzyme release was associated with modification of the surface membrane due to apoptosis with poliovirus and necrosis with HSV. Consequently hepatocellular injury by viral infection did not reflect the amount or pattern of hepatocellular enzyme release.
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Wen ZL, Tan DM, Cheng J, Yang YF, Liu GZ, Liu HB. Comparison of effect on hepatocellular apoptosis between core proteins of hepatitis B virus genotype B and C. Shijie Huaren Xiaohua Zazhi 2006; 14:3228-3232. [DOI: 10.11569/wcjd.v14.i33.3228] [Citation(s) in RCA: 0] [Impact Index Per Article: 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 compare the proapoptosis effect of hepatitis B virus (HBV) genotype B and C core proteins on liver cells, and explore the pathogenesis of HBV infection with different genotypes.
METHODS: Four serum samples infected by HBV (2 for type B and C, respectively) were used to amplify HBV core fragment using polymerase chain reaction (PCR). After recombination, cloning, and subcloning, four eucaryotic plasmids with different genotypes and clinical phenotypes were obtained. Then the plasmids were transfected into hepatocellular carcinoma cell line HepG2, and MTT assay and flow cytometry were used to detect the proliferation and apoptosis of HepG2 cells.
RESULTS: All the four recombinant eucaryotic plasmids were constructed successfully, and HBV core protein expression was confirmed by internal reference for transfection. The proliferation of HepG2 cells was not significantly different between the four plasmids, but the apoptosis rate in C-type (C2) group was markedly higher than that in B-type (B1) group (8.8% ± 2.0%vs 6.4% ± 0.8%, P < 0.05).
CONCLUSION: C-type HBV can induce more severe cell apoptosis than B-genotype HBV.
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37
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Yang YM, Wang HX, Wang GL, Cui YL, Liang P. Comparison between hepatitis B virus large protein and DNA and its association with alanine aminotransferase level. Shijie Huaren Xiaohua Zazhi 2006; 14:2733-2736. [DOI: 10.11569/wcjd.v14.i27.2733] [Citation(s) in RCA: 0] [Impact Index Per Article: 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 explore the clinical significance of hepatitis B virus large protein (HBV-LP) in the diagnosis of viral replication.
METHODS: Serum samples were collected from 177 patients with HBV infection. HBV-LP, HBV preS1 and HBV markers were examined using enzyme linked immunosorbent assay (ELISA). HBV DNA was quantitatively detected by real-time polymerase chain reaction. The level of alanine aminotransferase was obtained by an automated biochemistry analyzer.
RESULTS: No significant difference was found between the detectable rates of HBV DNA and HBV-LP in patient with the same HBV markers. The positive rates of HBV-LP and HBV DNA in HBeAg-positive patients were higher than those of HBeAg negative ones (95.45% vs 44.36%, P < 0.05; 93.18% vs 41.35%, P < 0.05). There was significant difference between the detectable rates of HBV DNA and HBV preS1 in patients with the same HBV markers (P < 0.05). The average level of ALT in HBV-LP-positive patients was higher than that of HBV-LP-negative ones.
CONCLUSION: There is a perfect correlation between the positive rate of HBV-LP and HBV DNA, and HBV-LP is a reliable serological marker that can reflect the replication of HBV as well as liver function.
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Paik YH, Brenner DA. Immunosuppression, hepatitis B virus variants: Synergistic role in hepatic fibrogenesis. Gastroenterology 2006; 131:957-60. [PMID: 16952565 DOI: 10.1053/j.gastro.2006.07.029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
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Märschenz S, Endres AS, Brinckmann A, Heise T, Kristiansen G, Nürnberg P, Krüger DH, Günther S, Meisel H. Functional analysis of complex hepatitis B virus variants associated with development of liver cirrhosis. Gastroenterology 2006; 131:765-80. [PMID: 16952546 DOI: 10.1053/j.gastro.2006.07.008] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2005] [Accepted: 06/08/2006] [Indexed: 12/31/2022]
Abstract
BACKGROUND & AIMS Development of cirrhosis in renal transplant recipients with chronic hepatitis B is associated with the accumulation of complex hepatitis B virus (HBV) variants carrying deletions in the C gene and/or preS region and deletions/insertions in the core promoter. Here, we characterized for the first time the phenotype of these complex HBV variants. METHODS Representative full-length genomes of the HBV variants that were isolated and cloned from serum and liver of an immunosuppressed renal transplant recipient before and during end-stage liver disease were transfected into the human hepatoma cell line HuH7 and functionally analyzed. RESULTS The variant genomes showed considerably reduced levels of precore and surface messenger RNA (mRNA) and of the major spliced pregenomic RNA, an increased level of pregenomic RNA, and a partial or complete defect in hepatitis B e antigen, core, and surface protein expression/secretion. Very low amounts of variant core protein with internal deletion were detectable. Reduced hepatitis B surface antigen secretion of some variants correlated with aberrant localization of surface proteins in endoplasmic reticulum. Despite the defects in viral protein expression, enhanced replication and enrichment in competition to wild-type HBV were observed. Enhanced reverse transcription and possibly increased levels of pregenomic RNA seem to be responsible for this effect. CONCLUSIONS Development of cirrhosis is associated with accumulation of complex variants, which exhibit a drastically altered phenotype combining enhanced replication with defects in protein expression. This phenotype appears to be based on the major mutations in the core promoter and C gene but is considerably influenced by additional mutations throughout the genome.
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Affiliation(s)
- Stefanie Märschenz
- Institut für Virologie (Helmut-Ruska-Haus), Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany
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Tan TL, Feng Z, Lu YW, Chan V, Chen WN. Adhesion contact kinetics of HepG2 cells during Hepatitis B virus replication: Involvement of SH3-binding motif in HBX. Biochim Biophys Acta Mol Basis Dis 2006; 1762:755-66. [PMID: 16935477 DOI: 10.1016/j.bbadis.2006.06.016] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2006] [Revised: 05/26/2006] [Accepted: 06/12/2006] [Indexed: 11/18/2022]
Abstract
It has been shown that Hepatitis B virus (HBV) replication directly alters the expression of key cytoskeleton-associated proteins which play key roles in mechanochemical signal transduction. Nevertheless, little is known on the correlation between HBV replication and the subsequent adhesion mechanism of HBV-replicating cells. In this study, it is demonstrated that the lag time of adhesion contact evolution of HepG2 cells with HBV replication is significantly increased by two times compared to that of normal HepG2 cell on collagen coated substrate. During the initial 20 min of cell seeding, only diffuse forms of vinculin was detected in HBV replicating cells while vinculin-associated focal complexes were found in normal and control cells. Similar delay in cell adhesion in HBV-replicating cells was observed in cells transfected with HBX, the smallest HBV protein, suggesting its involvement in this cellular process. In addition, a proline rich region found in many SH3 binding proteins was identified in HBX. HBX was found to interact with the focal adhesion protein, vinexin-beta, through the SH3 binding. Furthermore, HepG2 cells with HBV replication showed evidence of cell rounding up, possibly resulting from cytoskeletal reorganizations associated with interaction between HBX and vinexin-beta. Taken together, our results suggest that HBX is involved in the cytoskeletal reorganization in response to HBV replication.
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Affiliation(s)
- Tuan Lin Tan
- School of Biological Sciences, Nanyang Technological University, Singapore 637551
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Le AP, Ju BH, Wang W, Zhang WJ. Clinical significance of serum hepatitis B virus large surface protein in diagnosis and treatment of patients with hepatitis B. Shijie Huaren Xiaohua Zazhi 2006; 14:1578-1581. [DOI: 10.11569/wcjd.v14.i16.1578] [Citation(s) in RCA: 0] [Impact Index Per Article: 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 discuss the clinical significance of hepatitis B virus large surface protein (HBV-LP) detection in the diagnosis and treatment of patients with hepatitis B.
METHODS: Enzyme-linked immunosorbent assay was used to measure the levels of serum HBV-LP, HBV preS1, HBV preS2 and HBV markers, and fluorescence quantitative-polymerase chain reaction (FQ-PCR) was used to detect HBV DNA in 162 patients with hepatitis B as well as 47 normal controls.
RESULTS: In serum samples of the patient with HBV infection, the level of HBV-LP had significant correlation with that of HBV DNA, HBeAg, HBVpreS2 and HBVpreS1 antigen (c2 = 9.22, 11.89, 60.35, 99.87; all P < 0.01). The contents of serum HBV-LP was positively correlated with HBV DNA copies (rs = 0.64, P < 0.001). The level of serum HBV-LP was also significantly different between the groups with different HBV DNA copies (P < 0.01). The level and positive rate of serum HBV-LP were significantly different between HBV DNA-positive and negative patients (Z = 5.85, P < 0.001), and they were in the same situation for HBeAg, HBVpreS2 and HBVpreS1 antigen (Z = 8.70, 8.44, 8.84; all P < 0.001). The serum HBV-LP was more sensitive than HBV DNA, HBVpreS2 and HBVpreS1 antigen in HBeAg-negative patients (76.8%). Serum HBV-LP was significantly different between the patients with HBV infection and normal controls (P < 0.01).
CONCLUSION: HBV-LP may serve as a reliable marker in the reflection of HBV replication at protein level, and it is valuable to monitor HBV replication and prognosis of the disease, especially in HBeAg-negative HBV infected patients.
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Liu ZY, Huang YB, Wei HS, Song SJ, Feng X, Liu YN, Cheng J. Effects of HBeAg and HBsAg on cytokine excretion and angiotensin-converting enzyme activity in HepG 2 cell line. Shijie Huaren Xiaohua Zazhi 2006; 14:904-907. [DOI: 10.11569/wcjd.v14.i9.904] [Citation(s) in RCA: 0] [Impact Index Per Article: 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 evaluate the effects of hepatitis B virus surface and e antigens (HBeAg and HBsAg) on the activity of angiotensin-converting enzyme (ACE) and cytokines excretion in hepatocellular carcinoma cell line HepG2.
METHODS: The eukaryotic expression plasmids, including pcDNA3.1(-)-HBsAg, pcDNA3.1(-)-HBeAg, and empty plasmid pcDNA3.1(-), were transfected into HepG2 cells, and the levels of cytokines, such as IL-6 and IL-8, and the activity of ACE were determined in the supernatants 48 h after transfection by flow cytometry and biochemical methods, respectively.
RESULTS: The expression of HBeAg and HBsAg were detected in the supernatants, but there was no significant difference of ACE activity between the cells tranfected with pcDNA3.1(-)-HBsAg, pcDNA3.1(-)-HBeAg, and empty plasmid pcDNA3.1(-). The levels of IL-6 and IL-8 in the supernatants had no significant differences between the cells transfected with pcDNA3.1(-)-HBsAg, pcDNA3.1(-)-HBeAg and the control cells.
CONCLUSION: HBeAg and HBsAg has no significant effects on ACE activity and IL-6, IL-8 secretion in HepG2 cells.
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Meuleman P, Libbrecht L, Wieland S, De Vos R, Habib N, Kramvis A, Roskams T, Leroux-Roels G. Immune suppression uncovers endogenous cytopathic effects of the hepatitis B virus. J Virol 2006; 80:2797-807. [PMID: 16501088 PMCID: PMC1395427 DOI: 10.1128/jvi.80.6.2797-2807.2006] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2005] [Accepted: 12/27/2005] [Indexed: 02/07/2023] Open
Abstract
It is generally accepted that the host's immune response rather than the virus itself is causing the hepatocellular damage seen in acute and chronic hepatitis B virus (HBV) infections. However, in situations of severe immune suppression, chronic HBV patients may develop a considerable degree of liver disease. To examine whether HBV has direct cytopathic effects in severely immune compromised hosts, we have infected severe combined immune deficient mice (uPA-SCID), harboring human liver cells, with HBV. Serologic analysis of the plasma of HBV-infected animals revealed the presence of extremely high amounts of viral genomes and proteins. Histological analysis of the livers of uPA-SCID chimeras infected with HBV for more than 2 months showed that the majority of human hepatocytes had a ground-glass appearance, stained intensely for viral proteins, and showed signs of considerable damage and cell death. This histopathologic pattern closely resembles the picture observed in the livers of immunosuppressed HBV patients. These lesions were not observed in animals infected with HBV for less than 1 month. Ultrastructural analysis of long-term-infected hepatocytes showed a highly increased presence of cylindrical HBsAg structures, core particles, and Dane particles compared to short-term-infected hepatocytes. These long-term-infected hepatocytes also contained elevated amounts of HBV cccDNA. In conclusion, HBV causes dramatic intracellular changes and hepatocellular damage in the human hepatocytes that reside in a severely immune deficient mouse. These lesions show much resemblance to the ones encountered in immunosuppressed chronic HBV patients. Our observations indicate that HBV may be directly cytopathic in conditions of severe immune suppression.
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Affiliation(s)
- Philip Meuleman
- Center for Vaccinology, Ghent University and Hospital, Building A, First Floor, De Pintelaan 185, 9000 Ghent, Belgium
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Jiang S, Xie Q, Zhang W, Zhou XQ, Jin YX. Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro. World J Gastroenterol 2005; 11:4094-7. [PMID: 15996037 PMCID: PMC4502108 DOI: 10.3748/wjg.v11.i26.4094] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [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 prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis.
METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea).
RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%.
CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.
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Affiliation(s)
- Shan Jiang
- Department of Infectious Disease, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
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Martirosian G, Jóźwiak J, Radosz-Komoniewska H. Vacuolization of target cells: response to microbial toxins. World J Microbiol Biotechnol 2005. [DOI: 10.1007/s11274-004-5520-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Abstract
The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral capsid replicating via reverse transcription. The crystal structure of the capsid is known. It has a diameter of 36 nm and is formed by one protein species (C protein). The viral envelope contains three different coterminal proteins (S, M, and L proteins) spanning the membrane several times. These proteins are not only released from infected cells as components of the viral envelope but in 10,000-fold excess as subviral lipoprotein particles with a diameter of 22 nm containing no capsid. Assembly of the capsid occurs in the cytosol and results in packaging of a 3.5 kb RNA molecule together with viral and cellular factors. This newly formed capsid cannot be enveloped. Rather, synthesis of the viral DNA genome in the lumen of the capsid by reverse transcription is required to induce a budding competent state. Envelopment then takes place at an intracellular membrane of the pre-Golgi compartment. The S and the L protein, but not the M protein, is required for this process. The L protein forms two different transmembrane topologies. The isoform exposing the N-terminal part at the cytosolic side of the membrane is essential for budding. In this domain, a 22 amino acid (aa) long linear stretch has been mapped genetically to play a vital role in the morphogenetic process. This domain probably mediates the contact to the capsid. A second matrix domain was mapped to the cytosolic loop of the S protein. A similar genetic approach identified two small areas on the capsid surface, which might interact with the envelope proteins during envelopment.
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Affiliation(s)
- Volker Bruss
- Department of Virology, University of Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.
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Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes. Hepatology 2005; 41:247-56. [PMID: 15660384 DOI: 10.1002/hep.20553] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus-cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase-3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations.
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48
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Lambert C, Prange R. Development and characterization of a 293 cell line with regulatable expression of the hepatitis B virus large envelope protein. J Virol Methods 2004; 121:181-90. [PMID: 15381355 DOI: 10.1016/j.jviromet.2004.06.018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2004] [Revised: 06/16/2004] [Accepted: 06/21/2004] [Indexed: 11/23/2022]
Abstract
During the life cycle of hepatitis B virus (HBV) the large L envelope protein plays a pivotal role that is related to its peculiar dual transmembrane topology. To study the complex structure and diverse functions of L under regulated conditions of production, a human 293 cell line stably expressing L under the control of the ecdysone-inducible promoter was generated. Cells demonstrated stringent dose- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer. Temporal control of L expression allowed to trace (i) its posttranslational reorientation resulting in the mixed topology; (ii) its spatial redistribution from the endoplasmic reticulum to Golgi-like structures; and (iii) its intracellular retention in a membrane-associated configuration. On regulated overproduction, L blocked the secretion of HBV small envelope polypeptides without impairing the cell secretory apparatus. Despite the continuous high-level storage of L within the 293 cell line, no cytopathic effects could be detected. This is in contrast to ground-glass hepatocytes of chronic HBV carriers and HBV transgenic mice and may imply that the intracellular storage of L is particularly damaging to the liver cell.
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Affiliation(s)
- Carsten Lambert
- Department of Medical Microbiology and Hygiene, University of Mainz, Augustusplatz, 55101 Mainz, Germany
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Akay S, Karasu Z, Akyildiz M, Tokat Y. Adefovir treatment in posttransplant hepatitis B virus infection resistant to lamivudine plus hepatitis B virus immunoglobulin. Transplant Proc 2004; 36:2768-70. [PMID: 15621144 DOI: 10.1016/j.transproceed.2004.09.062] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Failure of prophylaxis for hepatitis B virus (HBV) recurrence in liver transplant patients with HBV immunoglobulin (HBIG) or lamivudine or both can be associated with rapid development of liver failure. Some of these patients develop a devastating clinicopathological state characterized by jaundice and rapidly progressive liver failure or fibrosing cholestatic hepatitis. We present two liver transplant recipients who experienced HBV recurrence while they were under lamivudine and HBIG prophylaxis. One of them had finding of severe HBV infection; the other, fibrosing cholestatic hepatitis. After commencing adefovir dipivoxil both patients showed improvements in clinical status and laboratory data. At month 4 of treatment, HBV DNA values became negative and liver function tests almost normalized. In addition, in one case showed HBs ag/anti-HBs seroconversion. When failure of prophylaxis with lamivudine and HBIG occurs, adefovir dipivoxil should be considered to be a safe and effective choice for recurrent HBV infections in liver transplant patients.
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Affiliation(s)
- S Akay
- Department of Gastroenterology, Ege University, Izmir 35040, Turkey.
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Ji D, Cheng J, Lu YY, Dong J, Guo J, Liu Y. Autonomous activation of hepatitis B virus large surface protein. Shijie Huaren Xiaohua Zazhi 2004; 12:2321-2324. [DOI: 10.11569/wcjd.v12.i10.2321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct the yeast expression vector of hepatitis B virus large surface protein (LHBs), and to study its autonomous activation.
METHODS: The Matchmaker GAL4 two-hybrid technique was used. The LHBs, pre-S1, pre-S2 and SHBs genes were amplified by polymerase chain reaction (PCR) with respective primers. The amplified PCR fragments were then subcloned into the EcoR I/BamH I sites (5'ands) of pGBKT7 vector to obtain the expression vectors including pGBKT7(-)-LHBs, pGBKT7(-)-preS1, pGBKT7(-)-preS2 and pGBKT7(-)-SHBs. This vectors were identifed by PCR and digestion of EcoR I/BamH I. After the constructed vectors were transformed into yeast AH109, the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing x-a-gal for testing their autonomous activation.
RESULTS: The yeast expression vectors were constructed. The yeast cells transformed with pGBKT7-LHB and pGBKT7-preS1 vectors could grow well on both of the media. However, cells transformed with pGBKT7-preS2 and pGBKT7-SHBs vectors could only grow on the SD/-Trp medium.
CONCLUSION: The LHBs functions as a transcriptional transactivator, and serves as the functional GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). The autonomous activation of LHBs roots in its pre-S1 domain.
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