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1
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Xu R, Hu P, Li Y, Tian A, Li J, Zhu C. Advances in HBV infection and replication systems in vitro. Virol J 2021; 18:105. [PMID: 34051803 PMCID: PMC8164799 DOI: 10.1186/s12985-021-01580-6] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 05/18/2021] [Indexed: 12/17/2022] Open
Abstract
Background Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family that has limited tissue and species specificity. Due to the persistence of HBV covalently closed circular DNA (cccDNA) in host cells after HBV infection, current antiviral drugs cannot eradicate HBV. Therefore, the development of an active cell culture system supporting HBV infection has become the key to studying HBV and developing effective therapeutic drugs. Main body This review summarizes the significant research achievements in HBV cell culture systems in vitro, including embryonic hepatocytes and primary hepatocytes, which support the virus infection process most similar to that in the body and various liver tumor cells. The discovery of the bile-acid pump sodium-taurocholate co-transporting polypeptide (NTCP) as the receptor of HBV has advanced our understanding of HBV biology. Subsequently, various liver cancer cells overexpressing NTCP that support HBV infection have been established, opening a new door for studying HBV infection. The fact that induced pluripotent stem cells that differentiate into hepatocyte-like cells support HBV infection provides a novel idea for the establishment of an HBV cell culture system. Conclusion Because of the host and tissue specificity of HBV, a suitable in vitro HBV infection system is critical for the study of HBV pathogenesis. Nevertheless, recent advances regarding HBV infection in vitro offer hope for better studying the biological characteristics of HBV, the pathogenesis of hepatitis B, the screening of anti-HBV drugs and the mechanism of carcinogenesis.
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Affiliation(s)
- Ruirui Xu
- Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China
| | - Pingping Hu
- Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China
| | - Yuwen Li
- Department of Pediatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China
| | - Anran Tian
- Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China
| | - Jun Li
- Department of Infectious Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, Jiangsu, China
| | - Chuanlong Zhu
- Department of Tropical Diseases, The Second Affiliated Hospital of Hainan Medical University, Haikou, 570311, Hainan, China.
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Goh ZY, Ren EC, Ko HL. Intracellular interferon signalling pathways as potential regulators of covalently closed circular DNA in the treatment of chronic hepatitis B. World J Gastroenterol 2021; 27:1369-1391. [PMID: 33911462 PMCID: PMC8047536 DOI: 10.3748/wjg.v27.i14.1369] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Revised: 02/23/2021] [Accepted: 03/17/2021] [Indexed: 02/06/2023] Open
Abstract
Infection with the hepatitis B virus (HBV) is still a major global health threat as 250 million people worldwide continue to be chronically infected with the virus. While patients may be treated with nucleoside/nucleotide analogues, this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA (cccDNA) genome. As a result, HBV infection cannot be cured, and the virus reactivates when conditions are favorable. Interferons (IFNs) are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells. They have been shown to induce cccDNA clearance, but their use in the treatment of HBV infection is limited as HBV-targeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling. Thus, to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV, instead of direct IFN administration, novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed. This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions. These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications. In addition, the mechanisms that HBV employs to disrupt IFN signalling will be discussed. Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections. Together, these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.
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Affiliation(s)
- Zhi Yi Goh
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Integrative Sciences and Engineering Programme, NUS Graduate School, National University of Singapore, Singapore 119077, Singapore
| | - Ee Chee Ren
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore
| | - Hui Ling Ko
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
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3
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Amir F, Siddiqui ZI, Farooqui SR, Anwer A, Khan S, Azmi MI, Mehmankhah M, Dohare R, Khan LA, Kazim SN. Impact of length of replication competent genome of hepatitis B virus over the differential antigenic secretion. J Cell Biochem 2019; 120:17858-17871. [PMID: 31310366 DOI: 10.1002/jcb.29054] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 04/12/2019] [Accepted: 04/18/2019] [Indexed: 12/18/2022]
Abstract
Hepatitis B virus (HBV) genome consists of circular partially double stranded DNA of 3.2 kb size which gets converted into covalently closed circular DNA (cccDNA) during its life cycle. It then acts as a template for formation of pregenomicRNA (pgRNA) of 3.5 kb. Absence of appropriate animal models prompted a need to establish a better in vitro culture system to uncover the propagation and survival mechanisms of the virus. There is scarcity of data to represent the significance of varying length of replication competent viral genome on the secretion of viral secretory proteins/antigens and in turn on the overall effects on the accomplishment of the viral life cycle. The present study was undertaken to ascertain a suitable replication competent construct in which the viral life cycle of HBV with varying clinical relevance can be studied efficiently. Two constructs (pHBV 1.3 and pHBV 1X) of different sizes were used to transfect hepatoma cells and consequently the secretory antigens were monitored. In vector free approach (pHBV 1X), 3.2 kb viral DNA is directly transfected in the culture system whereas in vector mediated approach more than full length of viral genome is cloned in a vector (pHBV 1.3X) and transfected to obtain a 3.5 kb pgRNA intermediate. HBV secretes two important antigens; HBsAg and HBeAg. HBsAg is a hallmark of infection and is the first to be secreted in the blood stream whereas HBeAg is a secretory protein and remains associated with the viral replication. The construct pHBV 1.3X referring to as more than full length, by virtue of being capable of undergoing transcription without the synthesis of cccDNA intermediate (unlike the clinical situation where an intermediate step of cccDNA synthesis is an essential component to initiate the viral life cycle) appears to be better system for studying viral life cycle in in vitro culture system. The reasons could be assigned to the fact that as low as 100 ng of viral DNA was shown to quantify the replicative phenotypes with this construct. The better efficiency of this construct at prima facie, appears to be mediated through the significantly higher levels of pgRNA transcript during the viral life cycle.
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Affiliation(s)
- Fatima Amir
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.,Department of Biosciences, Jamia Millia Islamia, New Delhi, India
| | - Zaheenul Islam Siddiqui
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Sabihur Rahman Farooqui
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.,Department of Biotechnology, Jamia Millia Islamia, New Delhi, India
| | - Ayesha Anwer
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Saniya Khan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Md Iqbal Azmi
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.,Multidisciplinary Centre for Advanced Research and Studies, Jamia Millia Islamia, New Delhi, India
| | - Mahboubeh Mehmankhah
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | - Ravins Dohare
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
| | | | - Syed Naqui Kazim
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
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4
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Meier-Stephenson V, Bremner WTR, Dalton CS, van Marle G, Coffin CS, Patel TR. Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutations. Viruses 2018; 10:E603. [PMID: 30388827 PMCID: PMC6265984 DOI: 10.3390/v10110603] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2018] [Revised: 10/27/2018] [Accepted: 10/31/2018] [Indexed: 02/06/2023] Open
Abstract
Over 250 million people are infected chronically with hepatitis B virus (HBV), the leading cause of liver cancer worldwide. HBV persists, due, in part, to its compact, stable minichromosome, the covalently-closed, circular DNA (cccDNA), which resides in the hepatocytes' nuclei. Current therapies target downstream replication products, however, a true virological cure will require targeting the cccDNA. Finding targets on such a small, compact genome is challenging. For HBV, to remain replication-competent, it needs to maintain nucleotide fidelity in key regions, such as the promoter regions, to ensure that it can continue to utilize the necessary host proteins. HBVdb (HBV database) is a repository of HBV sequences spanning all genotypes (A⁻H) amplified from clinical samples, and hence implying an extensive collection of replication-competent viruses. Here, we analyzed the HBV sequences from HBVdb using bioinformatics tools to comprehensively assess the HBV core and X promoter regions amongst the nearly 70,000 HBV sequences for highly-conserved nucleotides and variant frequencies. Notably, there is a high degree of nucleotide conservation within specific segments of these promoter regions highlighting their importance in potential host protein-viral interactions and thus the virus' viability. Such findings may have key implications for designing antivirals to target these areas.
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Affiliation(s)
- Vanessa Meier-Stephenson
- Department of Microbiology, Immunology and Infectious Diseases, Cumming, School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
- Alberta RNA Research & Training Institute, Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
| | - William T R Bremner
- Department of Ecosystem & Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
| | - Chimone S Dalton
- Department of Ecosystem & Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
| | - Guido van Marle
- Department of Microbiology, Immunology and Infectious Diseases, Cumming, School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
| | - Carla S Coffin
- Department of Microbiology, Immunology and Infectious Diseases, Cumming, School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
- Liver Unit, Division of Gastroenterology and Hepatology, Department of Medicine, Calgary, AB T2N 4Z6, Canada.
| | - Trushar R Patel
- Department of Microbiology, Immunology and Infectious Diseases, Cumming, School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada.
- Alberta RNA Research & Training Institute, Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada.
- DiscoveryLab, Faculty of Medicine & Dentistry, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2H7, Canada.
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Influential Factors of Hepatitis B Virus cccDNA in Peripheral Blood Mononuclear Cells Among HBsAg-Positive Pregnant Females Neonates. HEPATITIS MONTHLY 2018. [DOI: 10.5812/hepatmon.55064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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6
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Nishizawa T, Hoshino T, Naganuma A, Kobayashi T, Nagashima S, Takahashi M, Takagi H, Okamoto H. Enhanced pregenomic RNA levels and lowered precore mRNA transcription efficiency in a genotype A hepatitis B virus genome with C1766T and T1768A mutations obtained from a fulminant hepatitis patient. J Gen Virol 2016; 97:2643-2656. [PMID: 27473751 DOI: 10.1099/jgv.0.000566] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The viral factors associated with the development of fulminant hepatitis B are not fully understood. We recently found four unique mutations [G to A at nucleotide 1742 (G1742A), C1766T, T1768A and T1809C] in the basal core promoter (BCP) region of a genotype A hepatitis B virus (HBV) strain (FH) obtained from a 53-year-old man with fatal fulminant hepatitis. To elucidate the association of the mutations of the FH genome with the disease, we constructed a 1.3-fold FH genome and its five variants by replacing one or two mutated nucleotides with wild-type nucleotide(s) via site-directed mutagenesis, and transfected human hepatoma cells (HepG2/C3A) with the constructs. There were no discernible differences between FH and two variants (FH_A1742G and FH_C1809T) with regard to viral replication and protein expression. However, in comparison to three other variants (FH_T1766C, FH_A1768T and FH_T1766C/A1768T) with wild-type nucleotide(s) at 1766 and/or 1768, the FH genome exhibited a 2.5-5-fold enhancement of viral replication by heightened pregenomic RNA synthesis and a 1.5-2.5-fold reduction in the hepatitis B e antigen (HBeAg) synthesis by the downregulation of the precore mRNA level. An immunofluorescence analysis revealed the increased and predominant cytoplasmic localization of the core protein in the FH genome. The present study demonstrates that the C1766T/T1768A mutations in the BCP region of genotype A HBV enhance viral replication, downregulate HBeAg expression and are responsible for the predominant localization of the core protein in the cytoplasm, which are likely associated with the development of fulminant hepatitis.
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Affiliation(s)
- Tsutomu Nishizawa
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
| | - Takashi Hoshino
- Department of Gastroenterology, National Hospital Organization Takasaki General Medical Center, Gunma 370-0829, Japan
| | - Atsushi Naganuma
- Department of Gastroenterology, National Hospital Organization Takasaki General Medical Center, Gunma 370-0829, Japan
| | - Tominari Kobayashi
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
| | - Shigeo Nagashima
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
| | - Masaharu Takahashi
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
| | - Hitoshi Takagi
- Department of Gastroenterology, National Hospital Organization Takasaki General Medical Center, Gunma 370-0829, Japan.,Department of Gastroenterology and Hepatology, Kusunoki Hospital, Gunma 375-0024, Japan
| | - Hiroaki Okamoto
- Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
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Simsek E, Lu X, Ouzounov S, Block TM, Mehta AS. α-Glucosidase Inhibitors Have a Prolonged Antiviral Effect against Hepatitis B Virus through the Sustained Inhibition of the Large and Middle Envelope Glycoproteins. ACTA ACUST UNITED AC 2016; 17:259-67. [PMID: 17176630 DOI: 10.1177/095632020601700503] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Previous work has shown that the secretion of enveloped hepatitis B virus (HBV) DNA and the HBV middle envelope protein (MHBs) are sensitive to glucosidase inhibition. Here, it is shown that HBV DNA secretion remains depressed after the removal of the glucosidase inhibitor and long after glucosidase function returns to normal. For example, glycoprocessing and the secretion of α-1 anti-trypsin returned to normal within 3 h of the removal of the glucosidase inhibitor. In contrast, the secretion of HBV did not return to normal for more than 7 days after the removal of the inhibitor. Consistent with the inhibition of HBV virion secretion, the levels of HBV L and HBV M proteins were also reduced by treatment with the glucosidase inhibitor and remained reduced for 7 days after compound withdrawal. The implications of the prolonged antiviral effect against HBV and the use of glucosidase inhibitors as antiviral agents are discussed.
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Affiliation(s)
- Ender Simsek
- Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University College of Medicine, Philadelphia, PA, USA
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8
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A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation. Antimicrob Agents Chemother 2015; 59:7061-72. [PMID: 26349829 DOI: 10.1128/aac.01558-15] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Accepted: 09/02/2015] [Indexed: 12/18/2022] Open
Abstract
Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection.
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9
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Cavallone D, Moriconi F, Colombatto P, Oliveri F, Bonino F, Brunetto MR. Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line. J Virol Methods 2013; 189:110-7. [DOI: 10.1016/j.jviromet.2013.01.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2012] [Revised: 01/04/2013] [Accepted: 01/28/2013] [Indexed: 12/12/2022]
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Wang M, Qiu N, Lu S, Xiu D, Yu J, Wang XT, Lu F, Li T, Liu X, Zhuang H. Serum hepatitis B surface antigen is correlated with intrahepatic total HBV DNA and cccDNA in treatment-naïve patients with chronic hepatitis B but not in patients with HBV related hepatocellular carcinoma. J Med Virol 2012; 85:219-27. [PMID: 23168998 DOI: 10.1002/jmv.23461] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/09/2012] [Indexed: 01/08/2023]
Abstract
The aim of the study was to investigate correlations between intrahepatic hepatitis B virus total DNA, covalently closed circular DNA (cccDNA), and serum HBsAg in treatment-naïve chronic hepatitis B and HBV related hepatocellular carcinoma (HCC). Liver tissues were taken from 42 HBV related HCC and 36 patients with chronic hepatitis B. A fraction of DNA extracted from liver tissue was digested with a plasmid-safe ATP-dependent DNase and used for HBV cccDNA detection. The remaining DNA was used for the detection of HBV total DNA and β-globin, the latter of which is a housekeeping gene and quantified for normalization by real-time PCR. Quantitation of serum HBsAg was performed by a chemiluminescence assay. Serum HBsAg had positive correlations with serum HBV DNA (r = 0.636, P < 0.001), intrahepatic HBV total DNA (r = 0.519, P = 0.001) and cccDNA (r = 0.733, P < 0.001) in 36 treatment-naïve chronic hepatitis B, while HBsAg correlated poorly with DNA (r = 0.224, P = 0.210), intrahepatic total DNA and cccDNA in the tumor (r = 0.351, P = 0.031; r = 0.164, P = 0.324, respectively) and non-tumor (r = 0.237, P = 0.152; r = 0.072, P = 0.667, respectively) liver tissues of 42 HCC. HBV cccDNA and total DNA were significantly higher in liver tissue from chronic hepatitis B than in tumor and non-tumor of HCC (P < 0.001). Serum HBsAg and HBV DNA were also higher in chronic hepatitis B than in HCC (P < 0.001). It was concluded that levels of serum HBsAg and intrahepatic cccDNA and total DNA were significantly higher in chronic hepatitis B than in HCC, and significant correlations among them were observed in treatment-naïve chronic hepatitis B but not in HCC.
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Affiliation(s)
- Meirong Wang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
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Shi H, Lu L, Zhang NP, Zhang SC, Shen XZ. Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment. World J Gastroenterol 2012; 18:3617-22. [PMID: 22826629 PMCID: PMC3400866 DOI: 10.3748/wjg.v18.i27.3617] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2012] [Revised: 04/18/2012] [Accepted: 04/20/2012] [Indexed: 02/06/2023] Open
Abstract
AIM To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells. METHODS HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 10(5) HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS Compared to lamivudine alone (22.63% ± 0.12%), both sequential (51.50% ± 0.17%, P = 0.034) and cytokine treatment (49.66% ± 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% ± 0.02% vs 82.48% ± 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 ± 0.04 (control), 3.09 ± 0.08 (lamivudine), 2.55 ± 0.13 (cytokine), 2.32 ± 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 ± 0.01 (control), 3.08 ± 0.08 (lamivudine), 2.57 ± 0.15 (cytokine), 2.34 ± 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells (58.03% ± 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002]. CONCLUSION Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load.
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12
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Specific expression of human interferon-gamma controls hepatitis B virus replication in vitro in secreting hepatitis B surface antigen hepatocytes. J Virol Methods 2012; 180:84-90. [DOI: 10.1016/j.jviromet.2011.12.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2011] [Revised: 11/04/2011] [Accepted: 12/30/2011] [Indexed: 12/31/2022]
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13
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Chong CL, Chen ML, Wu YC, Tsai KN, Huang CC, Hu CP, Jeng KS, Chou YC, Chang C. Dynamics of HBV cccDNA expression and transcription in different cell growth phase. J Biomed Sci 2011; 18:96. [PMID: 22208719 PMCID: PMC3262020 DOI: 10.1186/1423-0127-18-96] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Accepted: 12/30/2011] [Indexed: 01/04/2023] Open
Abstract
Background The covalently closed-circular DNA (cccDNA) of hepatitis B virus (HBV) is associated with viral persistence in HBV-infected hepatocytes. However, the regulation of cccDNA and its transcription in the host cells at different growth stages is not well understood. Methods We took advantages of a stably HBV-producing cell line, 1.3ES2, and examine the dynamic changes of HBV cccDNA, viral transcripts, and viral replication intermediates in different cellular growth stages. Results In this study, we showed that cccDNA increased suddenly in the initial proliferation phase of cell growth, probably attributable to its nuclear replenishment by intracellular nucleocapsids. The amount of cccDNA then decreased dramatically in the cells during their exponential proliferation similar to the loss of extrachromosomal plasmid DNA during cell division, after which it accumulated gradually while the host cells grew to confluency. We found that cccDNA was reduced in dividing cells and could be removed when proliferating cells were subjected to long term of lamivudine (3TC) treatment. The amounts of viral replicative intermediates were rapidly reduced in these proliferating cells and were significantly increased after cells reaching confluency. The expression levels of viral transcripts were increased in parallel with the elevated expression of hepatic transcription factors (HNF4α, CEBPα, PPARα, etc.) during cell growth confluency. The HBV transcripts were transcribed from both integrated viral genome and cccDNA, however the transcriptional abilities of cccDNA was less efficient then that from integrated viral genome in all cell growth stages. We also noted increases in the accumulation of intracellular viral particles and the secretion of mature virions as the cells reached confluency and ceased to grow. Conclusions Based on the dynamics of HBV replication, we propose that HBV replication is modulated differently in the different stages of cell growth, and can be divided into three phases (initial proliferation phase, exponential proliferation phase and growth confluency phase) according to the cell growth curve. The regulation of cccDNA in different cell growth phase and its importance regarding HBV replication are discussed.
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Affiliation(s)
- Chin-Liew Chong
- Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
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14
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Abstract
The pathogenesis of hepatitis B virus (HBV) is complex and it appears that molecular variants play a role in this process. HBV undergoes numerous rounds of error prone production within an infected host. The resulting quasispecies are heterogeneous and in the absence of archaeological records of past infection, the evolution of HBV can only be inferred indirectly from its epidemiology and by genetic analysis. This review gathered the controversies about the HBV origin and factors influencing its quasispecies. Also, it provided some evidence on how HBV genotypes correlated with human history and patterns of migration. It is our belief that this topic deserves further attention and thus it is likely that more critical research work will be performed to elucidate the unknown mechanisms and processes in this area.
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Affiliation(s)
- S M Jazayeri
- Hepatitis B Molecular Laboratory, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
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15
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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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16
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Starkey JL, Chiari EF, Isom HC. Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro. J Gen Virol 2009; 90:115-26. [PMID: 19088280 PMCID: PMC2659548 DOI: 10.1099/vir.0.004408-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.
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Affiliation(s)
- Jason L. Starkey
- Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State University College of Medicine, 500 University Drive, H069, Hershey, PA 17033
| | - Estelle F. Chiari
- Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State University College of Medicine, 500 University Drive, H069, Hershey, PA 17033
| | - Harriet C. Isom
- Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State University College of Medicine, 500 University Drive, H069, Hershey, PA 17033
- Department of Pathology, Milton S. Hershey Medical Center, The Penn State University College of Medicine, 500 University Drive, H069, Hershey, PA 17033
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17
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Yao X, Han Q, Song J, Liang C, Wakita T, Yang R, Chen X. Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase. Mol Biotechnol 2008; 40:186-94. [DOI: 10.1007/s12033-008-9075-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2008] [Accepted: 05/21/2008] [Indexed: 02/05/2023]
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18
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Heipertz RA, Miller TG, Kelley CM, Delaney WE, Locarnini SA, Isom HC. In vitro study of the effects of precore and lamivudine-resistant mutations on hepatitis B virus replication. J Virol 2007; 81:3068-76. [PMID: 17215289 PMCID: PMC1866076 DOI: 10.1128/jvi.02341-06] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Understanding the consequences of mutation in the hepatitis B virus (HBV) genome on HBV replication is critical for treating chronic HBV infection. In this study, HBV replication in HepG2 cells initiated by transduction with precore (PC), rtM204I, and wild-type (wt) HBV recombinant baculoviruses was compared. The pattern and magnitude of HBV replication initiated by the PC HBV recombinant baculovirus were similar to those observed for wt HBV throughout the time course examined. In contrast, when the rtM204I mutation was introduced into wt HBV, by day 10 postinfection the levels of intra- and extracellular HBV DNA were markedly reduced compared to those for wt HBV. Although the rtM204I mutation reduced the production of HBV replicative intermediates, no effect on the level of covalently closed circular DNA or HBV transcripts was observed at late time points. Coinfection studies with different ratios of wt and rtM204I baculoviruses showed that the rtM204I variant did not produce a product that inhibited HBV replication. However, the combination of the wt and rtM204I baculoviruses yielded HBV DNA levels at late time points that were greater than those for the wt alone, suggesting that wt polymerase may function in trans to boost rtM204I replication. We concluded that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro.
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Affiliation(s)
- Richard A Heipertz
- Milton S. Hershey Medical Center, The Penn State College of Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033, USA
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19
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Bourne EJ, Dienstag JL, Lopez VA, Sander TJ, Longlet JM, Hall JG, Kwiatkowski RW, Wright T, Lai CL, Condreay LD. Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy. J Viral Hepat 2007; 14:55-63. [PMID: 17212645 DOI: 10.1111/j.1365-2893.2006.00775.x] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.
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Affiliation(s)
- E J Bourne
- Discovery Virology Department, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
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20
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Condreay JP, Ames RS, Hassan NJ, Kost TA, Merrihew RV, Mossakowska DE, Pountney DJ, Romanos MA. Baculoviruses and mammalian cell-based assays for drug screening. Adv Virus Res 2006; 68:255-86. [PMID: 16997014 DOI: 10.1016/s0065-3527(06)68007-x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
- J Patrick Condreay
- Department of Gene Expression and Protein Biochemistry GlaxoSmithKline Discovery Research, Research Triangle Park, North Carolina 27709, USA
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21
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Abstract
Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for < or = 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-alpha and pegylated IFN-alpha2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural beta-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with < or = 9 log10 decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log10, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.
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Affiliation(s)
- Chee-Kin Hui
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China
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22
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Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 2005; 23:567-75. [PMID: 15877075 PMCID: PMC3610534 DOI: 10.1038/nbt1095] [Citation(s) in RCA: 700] [Impact Index Per Article: 35.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
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23
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Guo HY, Tan DM, Xu XW. Predictive value of HBV cccDNA in PBMC of response to lamivudine therapy in patients with chronic hepatitis B. Shijie Huaren Xiaohua Zazhi 2005; 13:1202-1205. [DOI: 10.11569/wcjd.v13.i10.1202] [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 assess the value of hepatitis B virus (HBV) covalently closed circle DNA (cccDNA) in peripheral blood mononuclear cells (PBMC) for the prediction of the sustained response to lamivudine therapy in patients with chronic hepatitis B.
METHODS: Seventeen chronic hepatitis B patients with complete or partial response (serum HBV DNA turned negative, and ALT was normalized with or without HBeAg/anti-HBe seroconversion) were selected. HBV cccDNA in PBMC was detected at the end of lamivudine treatment, and 1 year after treatment.
RESULTS: Nine out of 17 patients were positive for HBV cccDNA in their PBMC at the end of lamivudine treatment. Virological relapse were found during 1-year follow-up in all of these 9 patients; whereas only 1 relapse was observed among the 8 patients without HBV cccDNA.
CONCLUSION: HBV cccDNA in PBMC at the end of treatment is of good predictive value for the sustained response to lamivudine treatment in chronic hepatitis B patients.
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24
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Sallie R. Replicative homeostasis: a fundamental mechanism mediating selective viral replication and escape mutation. Virol J 2005; 2:10. [PMID: 15707489 PMCID: PMC552327 DOI: 10.1186/1743-422x-2-10] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2005] [Accepted: 02/11/2005] [Indexed: 01/12/2023] Open
Abstract
Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation - Replicative Homeostasis - is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression.
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25
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Chou YC, Jeng KS, Chen ML, Liu HH, Liu TL, Chen YL, Liu YC, Hu CP, Chang C. Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method. J Virol 2005; 79:1813-23. [PMID: 15650205 PMCID: PMC544084 DOI: 10.1128/jvi.79.3.1813-1823.2005] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Virus persistence in chronic hepatitis B patients is due to the sustaining level of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. In this study, we used a modified 1.3-fold hepatitis B virus (HBV) genome, with a BclI genetic marker embedded in the redundancy region, to examine the transcriptional activity of cccDNA and the effect of the HBx protein on transcriptional regulation. After harvesting total RNA from transfected cells or stable lines, we specifically identified and monitored the transcripts from cccDNA by using reverse transcription-PCR (RT-PCR) combined with the restriction enzyme digestion method. In this approach, we have found that (i) RT-PCR combined with detection of the BclI marker is a highly specific method for distinguishing cccDNA-derived transcripts from the original integrated viral genome, (ii) the transcriptional ability of cccDNA was less efficient than that from the integrated viral genome, and (iii) the transcriptional activity of cccDNA was significantly regulated by the HBx protein, a potential transcription activator. In conclusion, we provided a tool with which to elucidate the transcriptional regulation of cccDNA and clarified the transcriptional regulation mechanism of HBx on cccDNA. The results obtained may be helpful in the development of a clinical intervention for patients with chronic HBV infections.
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Affiliation(s)
- Yu-Chi Chou
- Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
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26
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Sallie R. Replicative homeostasis: a mechanism of viral persistence. Med Hypotheses 2005; 63:515-23. [PMID: 15288380 DOI: 10.1016/j.mehy.2004.02.050] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2004] [Accepted: 02/21/2004] [Indexed: 01/12/2023]
Abstract
Acute viral infection is characterised by high-level replication before prompt decline of viraemia and, commonly, viral clearance. This kinetic pattern is generally held to be due to immune control. However, infection with some viruses, notably hepatitis C (HCV), hepatitis B (HBV) and the human immunodeficiency virus (HIV), often results in chronic stable low-level spontaneously fluctuating viraemia, kinetics that are difficult to rationalize on this basis. The persistence of HCV, an RNA virus, is especially problematic and its stability, occurring despite rapid, genomic mutation is highly paradoxical. This paper outlines the hypothesis, and evidence, that viruses autoregulate replication and mutation and describes a mechanism--replicative homeostasis--explaining viral stability. Replicative homeostasis results in stable, but reactive, replicative equilibria that drive quasispecies expansion and immune escape and explain all observed viral behaviours and host responses. This paradigm implies new approaches to antiviral therapy and is broadly relevant to modulation of gene expression.
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Affiliation(s)
- Richard Sallie
- St John of God Hospital, Western Gastroenterology, Suite 35, 95 Monash Avenue, Nedlands, Perth, WA 6009, Australia.
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27
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Gilbert L, Välilehto O, Kirjavainen S, Tikka PJ, Mellett M, Käpylä P, Oker-Blom C, Vuento M. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells. FEBS Lett 2004; 579:385-92. [PMID: 15642348 DOI: 10.1016/j.febslet.2004.11.101] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2004] [Revised: 11/02/2004] [Accepted: 11/16/2004] [Indexed: 11/25/2022]
Abstract
A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.
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Affiliation(s)
- Leona Gilbert
- Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FIN-40014, Finland.
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28
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Clay WC, Condreay JP, Moore LB, Weaver SL, Watson MA, Kost TA, Lorenz JJ. Recombinant baculoviruses used to study estrogen receptor function in human osteosarcoma cells. Assay Drug Dev Technol 2004; 1:801-10. [PMID: 15090226 DOI: 10.1089/154065803772613435] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.
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Affiliation(s)
- William C Clay
- Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline Discovery Research, Research Triangle Park, NC 27709-3398, USA.
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29
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Jun-Bin S, Zhi C, Wei-Qin N, Jun F. A quantitative method to detect HBV cccDNA by chimeric primer and real-time polymerase chain reaction. J Virol Methods 2003; 112:45-52. [PMID: 12951212 DOI: 10.1016/s0166-0934(03)00190-3] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), is a continuous double chain ring, while other DNA forms contain a gap on each chain at the position of direct repeat 1 sequence (DR1) and direct repeat 2 sequence (DR2), respectively. At present it is still difficult to detect with high sensitivity and specificity and quantify the HBV cccDNA pool in the nucleus of the hepatocyte. A chimeric primer was designated in which segment A near 3' end is complementary to HBV plus strand just before the DR2 region gap, and segment B near 5' end is consensus to part of the human immunodefficient virus genomic sequence, without homogenetic relationship to HBV genome. Promoted by taq DNA polymerase, a single nucleotide strand is elongated from chimeric primer generated by HBV cccDNA. In contrast, other HBV DNA forms do not produce a single nucleotide strand due to the cessation of elongation reaction at the DR2 gap. The newly formed single nucleotide strand is subsequently amplified by a new polymerase chain reaction system (PCR), in which a primer, identical to chimeric primer segments B, is used to ensure specific amplification, avoiding other HBV DNA format inference. In addition, a taqman probe was used in the PCR system to report the detection signal, and for constructing a standard curve between cycle threshold (Ct) value and the template quantity. This technique proved to be effective for rapid and sensitive detection and quantitation of HBV cccDNA with high specificity and efficacy.
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Affiliation(s)
- Shao Jun-Bin
- Institute of Infectious Diseases (National Key Laboratory of Infectious Diseases), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
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30
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Di Stefano G, Kratz F, Lanza M, Fiume L. Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier. Dig Liver Dis 2003; 35:428-33. [PMID: 12868680 DOI: 10.1016/s1590-8658(03)00212-3] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND The hepatocyte receptor for asialoglycoproteins, which binds and internalises galactosyl terminating peptides, was found to be expressed also on the cells of the majority of hepatocarcinomas. AIMS To verify whether doxorubicin coupling to lactosaminated albumin, a galactosyl terminating neoglycoprotein, produces selective drug accumulation in hepatocytes with reduced concentrations in extra-hepatic tissues, thus facilitating the use of the drug in hepatocarcinoma treatment. METHODS Doxorubicin concentrations were measured in organs of mice injected with the free or coupled drug. RESULTS In mice injected with the coupled drug, the ratios between doxorubicin concentrations in liver and those in heart, intestine, spleen and kidney were 8-14 times higher than in animals that received the same dose of the free drug. CONCLUSIONS Due to the very efficient liver targeting of doxorubicin, the lactosaminated human albumin-doxorubicin conjugate appears to have the potential of improving the chemotherapy of hepatocellular carcinomas through the asialoglycoprotein receptor.
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Affiliation(s)
- G Di Stefano
- Department of Experimental Pathology, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy
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31
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Abdelhamed AM, Kelley CM, Miller TG, Furman PA, Cable EE, Isom HC. Comparison of anti-hepatitis B virus activities of lamivudine and clevudine by a quantitative assay. Antimicrob Agents Chemother 2003; 47:324-36. [PMID: 12499209 PMCID: PMC148955 DOI: 10.1128/aac.47.1.324-336.2003] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC(50)) and EC(90) values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.
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Affiliation(s)
- Ayman M Abdelhamed
- Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, Pennsylvania 17033, USA
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