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1
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Prescott NA, Biaco T, Mansisidor A, Bram Y, Rendleman J, Faulkner SC, Lemmon AA, Lim C, Tiersky R, Salataj E, Garcia-Martinez L, Borges RL, Morey L, Hamard PJ, Koche RP, Risca VI, Schwartz RE, David Y. A nucleosome switch primes hepatitis B virus infection. Cell 2025; 188:2111-2126.e21. [PMID: 39983728 DOI: 10.1016/j.cell.2025.01.033] [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: 03/29/2023] [Revised: 12/20/2024] [Accepted: 01/24/2025] [Indexed: 02/23/2025]
Abstract
Chronic hepatitis B virus (HBV) infection is an incurable pathogen responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, the Smc5/6 complex. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. By establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA regulates X transcription. We corroborated these findings in situ and further showed that the chromatin-destabilizing molecule CBL137 inhibits full-length X transcription and HBV infection in primary human hepatocytes. Our results shed light on a long-standing paradox and represent a potential therapeutic approach for the treatment of chronic HBV infection.
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Affiliation(s)
- Nicholas A Prescott
- Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA; Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Tracy Biaco
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Pharmacology, Weill Cornell Medicine, New York, NY 10065, USA
| | - Andrés Mansisidor
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University, New York, NY 10065, USA
| | - Yaron Bram
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Justin Rendleman
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University, New York, NY 10065, USA
| | - Sarah C Faulkner
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Abigail A Lemmon
- Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA; Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Christine Lim
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Rachel Tiersky
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Eralda Salataj
- Epigenetics Research Innovation Laboratory, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Liliana Garcia-Martinez
- Sylvester Comprehensive Cancer Center, Biomedical Research Building, Miami, FL 33136, USA; Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Rodrigo L Borges
- Sylvester Comprehensive Cancer Center, Biomedical Research Building, Miami, FL 33136, USA; Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Lluis Morey
- Sylvester Comprehensive Cancer Center, Biomedical Research Building, Miami, FL 33136, USA; Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Pierre-Jacques Hamard
- Epigenetics Research Innovation Laboratory, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Richard P Koche
- Epigenetics Research Innovation Laboratory, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Viviana I Risca
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University, New York, NY 10065, USA.
| | - Robert E Schwartz
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY 10065, USA; Department of Physiology, Biophysics, and System Biology, Weill Cornell Medicine, New York, NY 10065, USA.
| | - Yael David
- Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA; Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Pharmacology, Weill Cornell Medicine, New York, NY 10065, USA; Department of Physiology, Biophysics, and System Biology, Weill Cornell Medicine, New York, NY 10065, USA.
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2
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Lok J, Harris JM, Carey I, Agarwal K, McKeating JA. Assessing the virological response to direct-acting antiviral therapies in the HBV cure programme. Virology 2025; 605:110458. [PMID: 40022943 DOI: 10.1016/j.virol.2025.110458] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Revised: 01/16/2025] [Accepted: 02/20/2025] [Indexed: 03/04/2025]
Abstract
Hepatitis B virus (HBV) is a global health problem with over 250 million people affected worldwide. Nucleos(t)ide analogues remain the standard of care and suppress production of progeny virions; however, they have limited effect on the viral transcriptome and long-term treatment is associated with off-target toxicities. Promising results are emerging from clinical trials and several drug classes have been evaluated, including capsid assembly modulators and RNA interfering agents. Whilst peripheral biomarkers are used to monitor responses and define treatment endpoints, they fail to reflect the full reservoir of infected hepatocytes. Given these limitations, consideration should be given to the merits of sampling liver tissue, especially in the context of clinical trials. In this review article, we will discuss methods for profiling HBV in liver tissue and their value to the HBV cure programme.
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Affiliation(s)
- James Lok
- Institute of Liver Studies, King's College Hospital, London, SE5 9RS, United Kingdom.
| | - James M Harris
- Nuffield Department of Medicine, University of Oxford, OX3 7FZ, United Kingdom
| | - Ivana Carey
- Institute of Liver Studies, King's College Hospital, London, SE5 9RS, United Kingdom
| | - Kosh Agarwal
- Institute of Liver Studies, King's College Hospital, London, SE5 9RS, United Kingdom
| | - Jane A McKeating
- Nuffield Department of Medicine, University of Oxford, OX3 7FZ, United Kingdom; Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, United Kingdom
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3
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Lau DTY, Kim ES, Wang Z, King WC, Kleiner DE, Ghany MG, Hinerman AS, Liu Y, Chung RT, Sterling RK, Cloherty G, Lin SY, Liu HN, Su YH, Guo H. Differential Intrahepatic Integrated HBV DNA Patterns Between HBeAg-Positive and HBeAg-Negative Chronic Hepatitis B. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2025:2025.02.28.25322668. [PMID: 40093236 PMCID: PMC11908316 DOI: 10.1101/2025.02.28.25322668] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Background HBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants. Methods Liver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed. Results Intrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(-) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (-) livers. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(-) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers. Conclusion Serum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(-) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(-) CHB.
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4
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Singh R, Ramakrishna G, Sharma MK, Kumar R, Gupta E, Rastogi A, Tanwar P, Sarin SK, Trehanpati N. Droplet digital PCR technique is ultrasensitive for the quantification of covalently closed circular DNA in the blood of chronic HBV-infected patients. Clin Res Hepatol Gastroenterol 2025; 49:102531. [PMID: 39832728 DOI: 10.1016/j.clinre.2025.102531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Revised: 01/13/2025] [Accepted: 01/17/2025] [Indexed: 01/22/2025]
Abstract
BACKGROUND Covalently closed circular DNA (cccDNA) is a stable, episomal form of HBV DNA. cccDNA is a true marker for the intrahepatic events in controlled CHB infection. Quantifying cccDNA is critical for monitoring disease progression, and efficacy of anti-viral therapies. METHODS To standardize the method, total HBV DNA was isolated from HepAD38 cells and digested with three exonuclease enzymes to remove linear and relaxed circular HBV DNA. Purified cccDNA quantification used ddPCR with specific primers. Treatment-naive chronic hepatitis B virus patients (nCHBV, n=36) with detectable HBV DNA and HBsAg were grouped by HBsAg levels: Group I (HBsAglo < 2000 IU/ml, n=11) and Group II (HBsAghi > 2000 IU/ml, n=25). cccDNA, HBV DNA and HBsAg were quantified in plasma and compared between groups. Correlation with clinical/histopathological features was done. RESULTS Non-digested 3.6^10⁶ tet-ve HepAD38 cells showed 316 copies/µl of total viral DNA. After digesting the linear, integrated, and relaxed circular DNA with triple enzymes, 15 copies/µl of cccDNA were detected. Similarly, after DNA digestion, HBsAglo patients showed a median of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAghi gave a median of 11 copies/µl (IQR 4-16 copies/µl) but with no significant difference between groups (p=0.093). Further, HBsAglo patients with low cccDNA copy numbers showed significantly higher fibrosis grades than HBsAghi (p=0.036). CONCLUSIONS We conclude that employing a combined approach utilizing three exonucleases, cccDNA-specific primers, and ddPCR enables the detection of cccDNA copies even in patients exhibiting low levels of HBsAg and HBV DNA. This integrated method offers additional validation as a surrogate diagnostic tool.
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Affiliation(s)
- Ravinder Singh
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Gayatri Ramakrishna
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Manoj Kumar Sharma
- Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Rahul Kumar
- Laboratory Oncology Unit, Dr. B. R. A. Institute Rotary Cancer Hospital (Cancer Centre), All India Institute of Medical Sciences, New Delhi, India
| | - Ekta Gupta
- Department of Virology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Archana Rastogi
- Department of Pathology, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Pranay Tanwar
- Laboratory Oncology Unit, Dr. B. R. A. Institute Rotary Cancer Hospital (Cancer Centre), All India Institute of Medical Sciences, New Delhi, India
| | - Shiv Kumar Sarin
- Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India.
| | - Nirupama Trehanpati
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India.
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5
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Jacobs R, Singh P, Smith T, Arbuthnot P, Maepa MB. Prospects of viral vector-mediated delivery of sequences encoding anti-HBV designer endonucleases. Gene Ther 2025; 32:8-15. [PMID: 35606493 DOI: 10.1038/s41434-022-00342-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 05/05/2022] [Accepted: 05/06/2022] [Indexed: 11/09/2022]
Abstract
Available treatment for chronic hepatitis B virus (HBV) infection offers modest functional curative efficacy. The viral replicative intermediate comprising covalently closed circular DNA (cccDNA) is responsible for persistent chronic HBV infection. Hence, current efforts have focused on developing therapies that disable cccDNA. Employing gene editing tools has emerged as an attractive strategy, with the end goal of establishing permanently inactivated cccDNA. Although anti-HBV designer nucleases are effective in vivo, none has yet progressed to clinical trial. Lack of safe and efficient delivery systems remains the limiting factor. Several vectors may be used to deliver anti-HBV gene editor-encoding sequences, with viral vectors being at the forefront. Despite the challenges associated with packaging large gene editor-encoding sequences into viral vectors, advancement in the field is overcoming such limitations. Translation of viral vector-mediated gene editing against HBV to clinical application is within reach. This review discusses the prospects of delivering HBV targeted designer nucleases using viral vectors.
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Affiliation(s)
- Ridhwaanah Jacobs
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Tiffany Smith
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
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6
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Li Z, Rahman N, Bi C, Mohallem R, Pattnaik A, Kazemian M, Huang F, Aryal UK, Andrisani O. RNA Helicase DDX5 in Association With IFI16 and the Polycomb Repressive Complex 2 Silences Transcription of the Hepatitis B Virus by Interferon. J Med Virol 2024; 96:e70118. [PMID: 39679735 DOI: 10.1002/jmv.70118] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Revised: 10/29/2024] [Accepted: 11/26/2024] [Indexed: 12/17/2024]
Abstract
RNA helicase DDX5 is a host restriction factor for hepatitis B virus (HBV) biosynthesis. Mass spectrometry (LC-MS/MS) identified significant DDX5-interacting partners, including interferon-inducible protein 16 (IFI16) and RBBP4/7, an auxiliary subunit of polycomb repressive complex 2 (PRC2). DDX5 co-eluted with IFI16, RBBP4/7, and core PRC2 subunits in size exclusion chromatography fractions derived from native nuclear extracts. Native gel electrophoresis of DDX5 immunoprecipitants revealed a 750 kDa DDX5/IFI16/PRC2 complex, validated by nanoscale co-localization via super-resolution microscopy. Prior studies demonstrated that IFI16 suppresses HBV transcription by binding to the interferon-sensitive response element of covalently closed circular DNA (cccDNA), reducing H3 acetylation and increasing H3K27me3 levels by an unknown mechanism. Herein, we demonstrate that ectopic expression of IFI16 inhibited HBV transcription from recombinant rcccDNA, correlating with increased IFI16 binding to rcccDNA, reduced H3 acetylation, and elevated H3K27me3, determined by chromatin immunoprecipitation. Importantly, the inhibitory effect of ectopic IFI16 on HBV transcription was reversed by siRNA-mediated knockdown of DDX5 and EZH2, the methyltransferase subunit of PRC2. This reversal was associated with decreased IFI16 binding to rcccDNA, enhanced H3 acetylation, and reduced H3K27me3. Similarly, endogenous IFI16 induced by interferon-α inhibited HBV rcccDNA transcription in a DDX5- and PRC2-dependent manner. In HBV-infected HepG2-NTCP cells, the antiviral effect of interferon-α was abrogated upon knockdown of DDX5 and EZH2, underscoring the crucial role of the DDX5 complex in IFI16-mediated antiviral response. In conclusion, in response to interferon, DDX5 partners with IFI16 to bind cccDNA, directing PRC2 to epigenetically silence cccDNA chromatin, thereby regulating immune signaling and HBV transcription.
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Affiliation(s)
- Zhili Li
- Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
- Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, USA
| | - Naimur Rahman
- Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
- Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, USA
| | - Cheng Bi
- Department of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA
| | - Rodrigo Mohallem
- Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, USA
- Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA
| | - Aryamav Pattnaik
- Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, USA
- Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA
| | - Majid Kazemian
- Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, USA
- Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA
- Department of Computer Science, Purdue University, West Lafayette, Indiana, USA
| | - Fang Huang
- Department of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA
| | - Uma K Aryal
- Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana, USA
- Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA
| | - Ourania Andrisani
- Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
- Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana, USA
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Ye J, Li F, Hua T, Ma K, Wang J, Zhao Z, Yang Z, Luo C, Jia R, Li Y, Hao M, Wu J, Lu M, Yuan Z, Zhang J, Chen J. Liver mechanosignaling as a natural anti-hepatitis B virus mechanism. Nat Commun 2024; 15:8375. [PMID: 39333106 PMCID: PMC11437074 DOI: 10.1038/s41467-024-52718-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2024] [Accepted: 09/18/2024] [Indexed: 09/29/2024] Open
Abstract
The mechanisms underlying the natural control of hepatitis B virus (HBV) infection have long been an intriguing question. Given the wide physiological range of liver stiffness and the growing attention to the role of mechanical microenvironment in homeostasis and diseases, we investigated how physical matrix cues impact HBV replication. High matrix stiffness significantly inhibited HBV replication and activated YAP in primary hepatocyte culture system, a key molecule in mechanosignaling. YAP activation notably suppressed HBV transcription and antigen expression. Several YAP-induced genes exhibited strong anti-HBV effects. Single-cell analysis of liver tissue from male individuals with active HBV replication revealed a strong significant negative correlation between YAP signature activation and HBV transcript levels. Intraperitoneal administration of YAP small molecule agonist potently controls HBV in male mouse models. These findings unveil a mechanism that involves the mechanical environment of hepatocytes and YAP to clear hepatotropic viral infection in the liver, providing new perspectives for HBV cure studies and antiviral development.
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Affiliation(s)
- Jianyu Ye
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Fahong Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Ting Hua
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Kewei Ma
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
| | - Jinyu Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Zixin Zhao
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
| | - Zhongning Yang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Chen Luo
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Ruohan Jia
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Yaming Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Menghan Hao
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China
| | - Jian Wu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China
| | - Mengji Lu
- Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China.
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China.
| | - Jiming Zhang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China.
- Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
| | - Jieliang Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Huashan Hospital, Shanghai Medical College Fudan University, Shanghai, China.
- Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, Shanghai Institute of Infectious Diseases and Biosecurity, Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Fudan University, Shanghai, China.
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8
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Prescott NA, Mansisidor A, Bram Y, Biaco T, Rendleman J, Faulkner SC, Lemmon AA, Lim C, Hamard PJ, Koche RP, Risca VI, Schwartz RE, David Y. A nucleosome switch primes Hepatitis B Virus infection. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.03.03.531011. [PMID: 38915612 PMCID: PMC11195122 DOI: 10.1101/2023.03.03.531011] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/26/2024]
Abstract
Chronic hepatitis B virus (HBV) infection is an incurable global health threat responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, Smc5/6. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. Establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA drives X transcription. We corroborated these findings in cells and further showed that the chromatin destabilizing molecule CBL137 inhibits X transcription and HBV infection in hepatocytes. Our results shed light on a long-standing paradox and represent a potential new therapeutic avenue for the treatment of chronic HBV infection.
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Affiliation(s)
- Nicholas A. Prescott
- Tri-Institutional PhD Program in Chemical Biology; New York, NY 10065, USA
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
| | - Andrés Mansisidor
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University; New York, NY 10065, USA
- These authors contributed equally
| | - Yaron Bram
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine; New York, NY 10065, USA
- These authors contributed equally
| | - Tracy Biaco
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
- Department of Pharmacology, Weill Cornell Medicine; New York, NY 10065, USA
- These authors contributed equally
| | - Justin Rendleman
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University; New York, NY 10065, USA
| | - Sarah C. Faulkner
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
| | - Abigail A. Lemmon
- Tri-Institutional PhD Program in Chemical Biology; New York, NY 10065, USA
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
| | - Christine Lim
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine; New York, NY 10065, USA
| | - Pierre-Jacques Hamard
- Epigenetics Research Innovation Lab, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
| | - Richard P. Koche
- Epigenetics Research Innovation Lab, Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
| | - Viviana I. Risca
- Laboratory of Genome Architecture and Dynamics, The Rockefeller University; New York, NY 10065, USA
| | - Robert E. Schwartz
- Division of Gastroenterology & Hepatology, Department of Medicine, Weill Cornell Medicine; New York, NY 10065, USA
- Department of Physiology and Biophysics, Weill Cornell Medicine; New York, NY 10065, USA
| | - Yael David
- Tri-Institutional PhD Program in Chemical Biology; New York, NY 10065, USA
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center; New York, NY 10065, USA
- Department of Pharmacology, Weill Cornell Medicine; New York, NY 10065, USA
- Lead Contact
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9
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Rawal P, Tripathi DM, Hemati H, Kumar J, Tyagi P, Sarin SK, Nain V, Kaur S. Targeted HBx gene editing by CRISPR/Cas9 system effectively reduces epithelial to mesenchymal transition and HBV replication in hepatoma cells. Liver Int 2024; 44:614-624. [PMID: 38105495 DOI: 10.1111/liv.15805] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 10/28/2023] [Accepted: 11/12/2023] [Indexed: 12/19/2023]
Abstract
BACKGROUND AND AIMS Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC. METHODOLOGY AND RESULTS We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated. CONCLUSION Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC.
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Affiliation(s)
- Preety Rawal
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
| | - Dinesh Mani Tripathi
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, Delhi, India
| | - Hamed Hemati
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, Delhi, India
| | - Jitendra Kumar
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, Delhi, India
| | - Purnima Tyagi
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, Delhi, India
| | - Shiv Kumar Sarin
- Department of Hepatology, Institute of Liver and Biliary Sciences, Delhi, India
| | - Vikrant Nain
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
| | - Savneet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, Delhi, India
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10
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Ren EC, Zhuo NZ, Goh ZY, Bonne I, Malleret B, Ko HL. cccDNA-Targeted Drug Screen Reveals a Class of Antihistamines as Suppressors of HBV Genome Levels. Biomolecules 2023; 13:1438. [PMID: 37892121 PMCID: PMC10604930 DOI: 10.3390/biom13101438] [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: 07/19/2023] [Revised: 09/08/2023] [Accepted: 09/22/2023] [Indexed: 10/29/2023] Open
Abstract
Chronic infection with hepatitis B virus (HBV) is incurable, as the current therapeutics cannot eliminate its persistent genomic material, cccDNA. Screening systems for cccDNA-targeting therapeutics are unavailable, as low copies of cccDNA in vitro complicate detection. To address this, cccDNA copies were massively increased to levels detectable via automated plate readers. This was achieved via continuous infection in a contact-free co-culture of an HBV generator (clone F881), which stably produced clinically relevant amounts of HBV, and HBV acceptors selected to carry high cccDNA loads. cccDNA-targeted therapeutics were then identified via reduced cccDNA-specific fluorescence, taking differences in the cell numbers and viability into account. Amongst the drugs tested, the H1 antihistamine Bilastine, HBVCP inhibitors and, surprisingly, current HBV therapeutics downregulated the cccDNA significantly, reflecting the assay's accuracy and sensitivity in identifying drugs that induce subtle changes in cccDNA levels, which take years to manifest in vivo. Bilastine was the only therapeutic that did not reduce HBV production from F881, indicating it to be a novel direct suppressor of cccDNA levels. When further assessed, only the structurally similar antihistamines Pitolisant and Nizatidine suppressed cccDNA levels when other H1 antihistamines could not. Taken together, our rapid fluorescence cccDNA-targeted drug screen successfully identified a class of molecules with the potential to treat hepatitis B.
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Affiliation(s)
- Ee Chee Ren
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
| | - Nicole Ziyi Zhuo
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
| | - Zhi Yi Goh
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
| | - Isabelle Bonne
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
- Electron Microscopy Unit, Yong Loo Lin School of Medicine, National University of Singapore, MD1, Tahir Foundation Building, #B1-01, 12 Science Drive 2, Singapore 117549, Singapore
- Immunology Programme, Life Sciences Institute, Center for Life Sciences, National University of Singapore, #05-02, 28 Medical Drive, Singapore 117456, Singapore
| | - Benoît Malleret
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
- Immunology Translational Research Programme, Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Block MD4, Level 3, Singapore 117545, Singapore;
- Electron Microscopy Unit, Yong Loo Lin School of Medicine, National University of Singapore, MD1, Tahir Foundation Building, #B1-01, 12 Science Drive 2, Singapore 117549, Singapore
| | - Hui Ling Ko
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, #03-06, Singapore 138648, Singapore; (N.Z.Z.); (Z.Y.G.); (B.M.)
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11
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Kostyushev D, Brezgin S, Kostyusheva A, Ponomareva N, Bayurova E, Zakirova N, Kondrashova A, Goptar I, Nikiforova A, Sudina A, Babin Y, Gordeychuk I, Lukashev A, Zamyatnin AA, Ivanov A, Chulanov V. Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 32:478-493. [PMID: 37187708 PMCID: PMC10176074 DOI: 10.1016/j.omtn.2023.04.016] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Accepted: 04/17/2023] [Indexed: 05/17/2023]
Abstract
APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4-800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%-99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.
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Affiliation(s)
- Dmitry Kostyushev
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
- Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, 354340 Sochi, Russia
| | - Sergey Brezgin
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
- Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, 354340 Sochi, Russia
| | - Anastasiya Kostyusheva
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
| | - Natalia Ponomareva
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
- Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, 354340 Sochi, Russia
- Department of Pharmaceutical and Toxicological Chemistry, Sechenov First Moscow State Medical University, 119146 Moscow, Russia
| | - Ekaterina Bayurova
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
| | - Natalia Zakirova
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Science, 119991 Moscow, Russia
| | - Alla Kondrashova
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
| | - Irina Goptar
- Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia
| | | | - Anna Sudina
- Federal State Budgetary Institution Centre for Strategic Planning and Management of Biomedical Health Risks of the Federal Medical Biological Agency, 119435 Moscow, Russia
| | - Yurii Babin
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
| | - Ilya Gordeychuk
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
- Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, 127994 Moscow, Russia
- Department of Infectious Diseases, Sechenov First Moscow State Medical University, 119146 Moscow, Russia
| | - Alexander Lukashev
- Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, 119991 Moscow, Russia
| | - Andrey A. Zamyatnin
- Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, 354340 Sochi, Russia
- Institute of Molecular Medicine, Sechenov First Moscow State Medical University, 119991 Moscow, Russia
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
- Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7X, UK
| | - Alexander Ivanov
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Science, 119991 Moscow, Russia
| | - Vladimir Chulanov
- Scientific Center for Genetics and Life Sciences, Division of Biotechnology, Sirius University of Science and Technology, 354340 Sochi, Russia
- Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, 127994 Moscow, Russia
- Department of Infectious Diseases, Sechenov First Moscow State Medical University, 119146 Moscow, Russia
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12
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Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu B, Lütgehetmann M, Guo H, Urban S, Fletcher SP, Protzer U, Levrero M, Zoulim F, Dandri M. Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. Gut 2023; 72:972-983. [PMID: 36707234 PMCID: PMC10086470 DOI: 10.1136/gutjnl-2022-328380] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 01/15/2023] [Indexed: 01/29/2023]
Abstract
OBJECTIVES A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. DESIGN Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. RESULTS Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. CONCLUSIONS We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
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Affiliation(s)
- Lena Allweiss
- I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany
| | - Barbara Testoni
- Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France
- ANRS HBV Cure Task Force, Lyon, France
| | - Mei Yu
- Gilead Sciences, Foster City, California, USA
| | - Julie Lucifora
- Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France
- ANRS HBV Cure Task Force, Lyon, France
- CIRI, Centre International de Recherche en Infectiologie, INSERM U1111, Université Claude Bernard Lyon 1, Lyon, France
| | - Chunkyu Ko
- Institute of Virology, Technical University of Munich, Munchen, Germany
- Infectious Diseases Therapeutic Research Center, Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Korea (the Republic of)
| | - Bingqian Qu
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
- Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Marc Lütgehetmann
- German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany
- Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf Hamburg, Hamburg, Germany
| | - Haitao Guo
- Indiana University School of Medicine, Indianapolis, Indiana, USA
- Department of Microbiology and Molecular Genetics, Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Stephan Urban
- German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | | | - Ulrike Protzer
- German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany
- Institute of Virology, Technical University of Munich, Munchen, Germany
| | - Massimo Levrero
- Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France
- ANRS HBV Cure Task Force, Lyon, France
| | - Fabien Zoulim
- Cancer Research Center of Lyon, INSERM U1052, Lyon University, Hospices de Lyon, Lyon, France
- ANRS HBV Cure Task Force, Lyon, France
| | - Maura Dandri
- I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- German Center for Infection Research, Hamburg-Lübeck-Borstel-Riems, Munich and Heidelberg sites, Germany
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Characterization of Intracellular Precore-Derived Proteins and Their Functions in Hepatitis B Virus-Infected Human Hepatocytes. mBio 2023; 14:e0350122. [PMID: 36715515 PMCID: PMC9973328 DOI: 10.1128/mbio.03501-22] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Hepatitis B virus (HBV) precore protein is not essential for viral replication but is thought to facilitate chronic infection. In addition to the secreted precore products, including the hepatitis B e antigen (HBeAg) and PreC protein, intracellular precore-derived proteins in HBV-infected human hepatocytes remain poorly characterized, and their roles, if any, remain largely unknown. Here, we detected multiple precore derivatives, including the nonprocessed precursor p25 and the processing intermediate p22, in HBV-infected human hepatocytes as well as human hepatoma cells overexpressing the HBV precore protein. Both p25 and p22 showed phosphorylated and unphosphorylated forms, which were located in different intracellular compartments. Interestingly, precore expression was associated with decreases in intracellular HBV core protein (HBc) and secreted DNA-containing virions but was also associated with an increase in secreted empty virions. The decrease in HBc by precore could be attributed to cytosolic p22, which caused HBc degradation, at least in part by the proteasome, and consequently decreased HBV pregenomic RNA packaging and DNA synthesis. In addition, cytosolic p22 formed chimeric capsids with HBc in the cell, which were further secreted in virions. In contrast, the PreC antigen, like HBeAg, was secreted via the endoplasmic reticulum (ER)-Golgi secretory pathway and was thus unable to form capsids in the cell or be secreted in virions. Furthermore, p25, as well as p22, were secreted in virions from HBV-infected human hepatocytes and were detected in the sera of HBV-infected chimpanzees. In summary, we have detected multiple intracellular precore-derived proteins in HBV-infected human hepatocytes and revealed novel precore functions in the viral life cycle. IMPORTANCE Chronic hepatitis B remains a worldwide public health issue. The hepatitis B virus (HBV) precore protein is not essential for HBV replication but may facilitate viral persistence. In this study, we have detected multiple precore protein species in HBV-infected human hepatocytes and studied their functions in the HBV life cycle. We found that the HBV precore proteins decreased intracellular HBV core protein and reduced secretion of complete virions but enhanced secretion of empty virions. Interestingly, the cytosolic precore protein species formed chimeric capsids with the core protein and were secreted in virions. Our results shed new light on the functions of intracellular precore protein species in the HBV life cycle and have implications for the roles of precore proteins in HBV persistence and pathogenesis.
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14
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Batskikh S, Morozov S, Dorofeev A, Borunova Z, Kostyushev D, Brezgin S, Kostyusheva A, Chulanov V. Previous hepatitis B viral infection-an underestimated cause of pancreatic cancer. World J Gastroenterol 2022; 28:4812-4822. [PMID: 36156926 PMCID: PMC9476854 DOI: 10.3748/wjg.v28.i33.4812] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2022] [Revised: 06/15/2022] [Accepted: 08/16/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The etiology of pancreatic cancer remains unclear. This limits the possibility of prevention and effective treatment. Hepatitis B virus (HBV) is responsible for the development of different types of cancer, but its role in pancreatic cancer is still being discussed. AIM To assess the prevalence of previous HBV infection and to identify viral biomarkers in patients with pancreatic ductal adenocarcinoma (PDAC) to support the role of the virus in etiology of this cancer. METHODS The data of 130 hepatitis B surface antigen-negative subjects were available for the final analysis, including 60 patients with PDAC confirmed by cytology or histology and 70 sex- and age-matched controls. All the participants were tested for HBV biomarkers in blood [antibody to hepatitis B core antigen (anti-HBc), antibody to hepatitis B surface antigen (anti-HBs) and HBV DNA], and for those with PDAC, biomarkers in resected pancreatic tissues were tested (HBV DNA, HBV pregenomic RNA and covalently closed circular DNA). We performed immunohistochemistry staining of pancreatic tissues for hepatitis B virus X antigen and Ki-67 protein. Non-parametric statistics were used for the analysis. RESULTS Anti-HBc was detected in 18/60 (30%) patients with PDAC and in 9/70 (13%) participants in the control group (P = 0.029). Accordingly, the odds of PDAC in anti-HBc-positive subjects were higher compared to those with no previous HBV infection (odds ratio: 2.905, 95% confidence interval: 1.191-7.084, standard error 0.455). HBV DNA was detected in 8 cases of PDAC and in 6 of them in the pancreatic tumor tissue samples only (all patients were anti-HBc positive). Blood HBV DNA was negative in all subjects of the control group with positive results of the serum anti-HBc test. Among 9 patients with PDAC, 5 revealed signs of replicative competence of the virus (covalently closed circular DNA with or without pregenomic RNA) in the pancreatic tumor tissue samples. Hepatitis B virus X antigen expression and active cell proliferation was revealed by immunohistochemistry in 4 patients with PDAC in the pancreatic tumor tissue samples. CONCLUSION We found significantly higher risks of PDAC in anti-HBc-positive patients. Detection of viral replication and hepatitis B virus X protein expression in the tumor tissue prove involvement of HBV infection in pancreatic cancer development.
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Affiliation(s)
- Sergey Batskikh
- Department of Hepatology, Moscow Clinical Scientific Center N.A. A.S. Loginov, Moscow 111123, Russia
| | - Sergey Morozov
- Department of Gastroenterology, Hepatology and Nutrition, Federal Research Center of Nutrition, Biotechnology and Food Safety, Moscow 115446, Russia
| | - Alexey Dorofeev
- Department of Scientific and Clinical Laboratory Research, Moscow Clinical Scientific Center N.A. A.S. Loginov, Moscow 111123, Russia
| | - Zanna Borunova
- Department of Scientific and Clinical Laboratory Research, Moscow Clinical Scientific Center N.A. A.S. Loginov, Moscow 111123, Russia
| | - Dmitry Kostyushev
- Laboratory of Genetic Technologies, Sechenov University, Moscow 119435, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, Sochi 354340, Russia
| | - Sergey Brezgin
- Laboratory of Genetic Technologies, Sechenov University, Moscow 119435, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, Sochi 354340, Russia
| | | | - Vladimir Chulanov
- Laboratory of Genetic Technologies, Sechenov University, Moscow 119435, Russia
- Division of Biotechnology, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, Sochi 354340, Russia
- Laboratory of Genetic Technologies and Translational Research, National Medical Research Center for Tuberculosis and Infectious Diseases of Ministry of Health of Russia, Moscow 127994, Russia
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15
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Qu B, Nebioglu F, Leuthold MM, Ni Y, Mutz P, Beneke J, Erfle H, Vondran FW, Bartenschlager R, Urban S. Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen. JHEP Rep 2022; 4:100551. [PMID: 36124123 PMCID: PMC9482114 DOI: 10.1016/j.jhepr.2022.100551] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 07/10/2022] [Accepted: 07/14/2022] [Indexed: 02/07/2023] Open
Abstract
Background & Aims HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants. Methods An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression. Results siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment. Conclusions Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B. Lay summary Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.
Neddylation plays a dual role in HBV expression from viral integrants and episomal cccDNA. Impaired neddylation suppresses production of HBsAg expressed from viral integrants. Neddylation promotes HBsAg generation from viral integrants in an HBx-independent manner. MLN4924 also inhibits the synthesis of viral transcripts from episomal cccDNA.
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16
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Batskikh S, Morozov S, Kostyushev D. Hepatitis B virus markers in hepatitis B surface antigen negative patients with pancreatic cancer: Two case reports. World J Hepatol 2022; 14:1512-1519. [PMID: 36158906 PMCID: PMC9376784 DOI: 10.4254/wjh.v14.i7.1512] [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] [Received: 03/29/2022] [Revised: 05/25/2022] [Accepted: 06/27/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) is a known carcinogen that may be involved in pancreatic cancer development. Detection of HBV biomarkers [especially expression of HBV regulatory X protein (HBx)] within the tumor tissue may provide direct support for this. However, there is still a lack of such reports, particularly in non-endemic regions for HBV infection. Here we present two cases of patients with pancreatic ductal adenocarcinoma, without a history of viral hepatitis, in whom the markers of HBV infection were detected in blood and in the resected pancreatic tissue. CASE SUMMARY The results of examination of two patients with pancreatic cancer, who gave informed consent for participation and publication, were the source for this study. Besides standards of care, special examination to reveal occult HBV infection was performed. This included blood tests for HBsAg, anti-HBc, anti-HBs, HBV DNA, and pancreatic tissue examinations with polymerase chain reaction for HBV DNA, pregenomic HBV RNA (pgRNA HBV), and covalently closed circular DNA HBV (cccDNA) and immunohistochemistry staining for HBxAg and Ki-67. Both subjects were operated on due to pancreatic ductal adenocarcinoma and serum HBsAg was not detected. However, in both of them anti-HBc antibodies were detected in blood, although HBV DNA was not found. Examination of the resected pancreatic tissue gave positive results for HBV DNA, expression of HBx, and active cellular proliferation by Ki-67 index in both cases. However, HBV pgRNA and cccDNA were detected only in case 1. CONCLUSION These cases may reflect potential involvement of HBV infection in the development of pancreatic cancer.
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Affiliation(s)
- Sergey Batskikh
- Department of Hepatology, Moscow Clinical Research Center named after A.S. Loginov, Moscow 111123, Russia
| | - Sergey Morozov
- Department of Gastroenterology, Hepatology and Nutrition, Federal Research Center of Nutrition and Biotechnology, Moscow 115446, Russia.
| | - Dmitry Kostyushev
- Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia
- Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia
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17
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Wei XF, Fan SY, Wang YW, Li S, Long SY, Gan CY, Li J, Sun YX, Guo L, Wang PY, Yang X, Wang JL, Cui J, Zhang WL, Huang AL, Hu JL. Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling. iScience 2022; 25:104416. [PMID: 35663023 PMCID: PMC9156947 DOI: 10.1016/j.isci.2022.104416] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 04/21/2022] [Accepted: 05/12/2022] [Indexed: 11/17/2022] Open
Abstract
The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.
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Affiliation(s)
- Xia-Fei Wei
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, China
| | - Shu-Ying Fan
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Yu-Wei Wang
- Department of Laboratory Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing, China
| | - Shan Li
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Shao-Yuan Long
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Chun-Yang Gan
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Jie Li
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Yu-Xue Sun
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Lin Guo
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Pei-Yun Wang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Xue Yang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Jin-Lan Wang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Jing Cui
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Wen-Lu Zhang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Ai-Long Huang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Jie-Li Hu
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China
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18
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Hong X, Kawasawa YI, Menne S, Hu J. Host cell-dependent late entry step as determinant of hepatitis B virus infection. PLoS Pathog 2022; 18:e1010633. [PMID: 35714170 PMCID: PMC9246237 DOI: 10.1371/journal.ppat.1010633] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Revised: 06/30/2022] [Accepted: 06/01/2022] [Indexed: 12/19/2022] Open
Abstract
Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection. Fundamental studies on, and development of therapies against, chronic hepatitis B virus (HBV) infection, which inflicts hundreds of millions worldwide, are impeded by deficiencies in HBV-susceptible animal models. HBV displays a strict species and cell tropism that are not clearly understood. Here, by studying replication of HBV, and the related woodchuck hepatitis virus, in human and woodchuck hepatic or non-hepatic cells, we found that non-hepatic human cells and some woodchuck hepatic cells could support productive HBV entry after expression of the human cell receptor for HBV. Moreover, by studying the infection of hepatitis D virus, which shares the same entry receptor and initial steps of entry with HBV, we could narrow down a host determinant of HBV infection operating at a late entry/trafficking step(s). Our study thus provides new insights into determinants of HBV host tropism and facilitates the development of HBV-susceptible animal models.
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Affiliation(s)
- Xupeng Hong
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Yuka Imamura Kawasawa
- Department of Pharmacology, Department of Biochemistry and Molecular Biology, Institute for Personalized Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Stephan Menne
- Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, District of Columbia, United States of America
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- * E-mail:
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19
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Zhang X, Tian Y, Xu L, Fan Z, Cao Y, Ma Y, Li H, Ren F. CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection. Hepatol Int 2022; 16:306-315. [PMID: 35298777 PMCID: PMC9013339 DOI: 10.1007/s12072-022-10311-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 02/06/2022] [Indexed: 11/10/2022]
Abstract
Background and aims The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology. Methods We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs). Results Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods. Conclusions We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance. Supplementary Information The online version contains supplementary material available at 10.1007/s12072-022-10311-0.
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Affiliation(s)
- Xiangying Zhang
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China
| | - Yuan Tian
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China
| | - Ling Xu
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China
| | - Zihao Fan
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China
| | - Yaling Cao
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China
| | - Yingmin Ma
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China.
| | - Hao Li
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.
| | - Feng Ren
- Beijing Youan Hospital, Capital Medical University, No. 8, XitouTiao Road, Youwai Street, Fengtai District, Beijing, 100069, China.
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20
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Kamiya N, Sugimoto T, Abe-Chayama H, Akiyama R, Tsuboi Y, Mogami A, Imamura M, Hayes CN, Chayama K. Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA. J Gen Virol 2022; 103. [PMID: 35130138 DOI: 10.1099/jgv.0.001591] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.
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Affiliation(s)
- Naohiro Kamiya
- Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan.,Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Takahiko Sugimoto
- Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Hiromi Abe-Chayama
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Rie Akiyama
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Yasunori Tsuboi
- Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Akira Mogami
- Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - C Nelson Hayes
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Institute of Physical and Chemical Research (RIKEN) Center for Integrative Medical Sciences, Yokohama, Japan
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21
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Wang Z, Chen Y, Deng H, Zhen X, Xiong J, Hu Y. Quantification of intrahepatic cccDNA in HBV associated hepatocellular carcinoma by improved ddPCR method. J Virol Methods 2021; 299:114334. [PMID: 34688781 DOI: 10.1016/j.jviromet.2021.114334] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 10/11/2021] [Accepted: 10/12/2021] [Indexed: 11/26/2022]
Abstract
The quantification of intrahepatic covalently closed circular DNA (cccDNA) is important for assessing the efficiency of anti-HBV therapy. Exonuclease treatment is essential before real-time quantitative PCR (qPCR) or droplet digital PCR (ddPCR) measurement to improve the specificity of cccDNA quantification. In this research, we compared the limit of detection (LOD) of qPCR and ddPCR and evaluated the digestion efficiency of three exonuclease treatments, PSAD, exonuclease III and T5 exonuclease, when measuring cccDNA in cells or clinical samples by ddPCR. We demonstrated that the LOD of ddCPR was 5.9 copies/reaction, which was much lower than that of qPCR (54.9 copies/reaction), indicating that ddPCR is more sensitive than qPCR. Meanwhile, compared to PSAD or Exo III, UNG and T5 exonuclease treatment combined with ddPCR is more effective in detecting intrahepatic cccDNA in clinical samples. Finally, the median intrahepatic cccDNA was 2.6 copies/104 cells in 26 pairs of HCC samples determined by the improved ddPCR method. Therefore, we developed an optimized ddPCR method, which can be used for the absolute quantification of low levels of intrahepatic cccDNA more precisely.
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Affiliation(s)
- Zhuo Wang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China
| | - YanMeng Chen
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China
| | - Haijun Deng
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China
| | - Xiaochuan Zhen
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China
| | - Jing Xiong
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China
| | - Yuan Hu
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, 109# Chongqing Medical University, 1 Yi Xue Yuan Road, Yuzhong District, Chongqing, 400016, China.
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22
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Zhang H, Tu T. Approaches to quantifying Hepatitis B Virus covalently closed circular (ccc)DNA. Clin Mol Hepatol 2021; 28:135-149. [PMID: 34674513 PMCID: PMC9013611 DOI: 10.3350/cmh.2021.0283] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 10/21/2021] [Indexed: 11/19/2022] Open
Abstract
Chronic hepatitis B is a major cause of liver disease worldwide and is currently incurable. Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is a key form of the virus responsible for its persistence and is the transcriptional template for all viral transcripts. The field is focussed on methods to clear HBV cccDNA but this been limited by technical difficulties in its quantification due to: identical sequence to other forms of HBV DNA; low copy number per cell; and high resistance to denaturation by heat, leading to difficulty using polymerase chain reaction or hybridization methods for detection. A number of assays have been developed in order to overcome these hurdles either directly or detecting cccDNA levels indirectly via its transcriptional products. In this review, we summarize the approaches to cccDNA quantification that are currently used, and outline key open questions in the cccDNA biology field which remain to be answered due to the limitations of current methods.
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Affiliation(s)
- Henrik Zhang
- Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia
| | - Thomas Tu
- Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.,Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney at Westmead Hospital, Westmead NSW 2145, Australia
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23
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Ibrahim MK, Abdelhafez TH, Takeuchi JS, Wakae K, Sugiyama M, Tsuge M, Ito M, Watashi K, El Kassas M, Kato T, Murayama A, Suzuki T, Chayama K, Shimotohno K, Muramatsu M, Aly HH, Wakita T. MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter. J Virol 2021; 95:e0076721. [PMID: 33980595 PMCID: PMC8274605 DOI: 10.1128/jvi.00767-21] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 05/06/2021] [Indexed: 01/04/2023] Open
Abstract
Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (NL) reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. IMPORTANCE HBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Therefore, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis and developing anti-HBV strategies. MafF was shown to suppress transcription from the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1β and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.
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Affiliation(s)
- Marwa K. Ibrahim
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Microbial Biotechnology, Division of Genetic Engineering and Biotechnology Research, National Research Centre, Giza, Egypt
| | - Tawfeek H. Abdelhafez
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Microbial Biotechnology, Division of Genetic Engineering and Biotechnology Research, National Research Centre, Giza, Egypt
| | - Junko S. Takeuchi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
- Organization for the Strategic Coordination of Research and Intellectual Properties, Meiji University, Kawasaki, Japan
| | - Kosho Wakae
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Masaya Sugiyama
- Genome Medical Sciences Project, National Center for Global Health and Medicine, Ichikawa, Japan
| | - Masataka Tsuge
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
| | - Masahiko Ito
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Mohamed El Kassas
- Endemic Medicine Department, Faculty of Medicine, Helwan University, Cairo, Egypt
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Kazuaki Chayama
- Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kunitada Shimotohno
- Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hussein H. Aly
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
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24
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Qu B, Brown RJP. Strategies to Inhibit Hepatitis B Virus at the Transcript Level. Viruses 2021; 13:v13071327. [PMID: 34372533 PMCID: PMC8310268 DOI: 10.3390/v13071327] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 07/02/2021] [Accepted: 07/06/2021] [Indexed: 12/11/2022] Open
Abstract
Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.
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Affiliation(s)
- Bingqian Qu
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- European Virus Bioinformatics Center, 07743 Jena, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
| | - Richard J. P. Brown
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
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25
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Zehnder B, Urban S, Tu T. A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA. Bio Protoc 2021; 11:e3986. [PMID: 34124289 DOI: 10.21769/bioprotoc.3986] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 02/13/2021] [Accepted: 02/18/2021] [Indexed: 01/20/2023] Open
Abstract
Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels. Graphic abstract: Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a ).
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Affiliation(s)
- Benno Zehnder
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.,German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany
| | - Thomas Tu
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.,German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany.,Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Westmead, NSW, Australia.,Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Australia
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Hsu CW, Chu YD, Lai MW, Lin CL, Liang KH, Lin YH, Yeh CT. Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression. Cancers (Basel) 2021; 13:cancers13030538. [PMID: 33572617 PMCID: PMC7867012 DOI: 10.3390/cancers13030538] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2020] [Revised: 01/19/2021] [Accepted: 01/27/2021] [Indexed: 02/07/2023] Open
Abstract
New antiviral therapies against hepatitis B virus (HBV) focus on the elimination of covalently closed circular DNA (cccDNA). However, traditional cccDNA-specific quantitative PCR (qPCR) has a narrow effective range, hindering a reliable comparison between the levels of biopsy-derived cccDNAs. Collaterally, the prognostic role of cccDNA in HBV-related hepatocellular carcinoma (HCC) cannot be clearly defined. Here, we developed a peptide nucleic acid (PNA)-clamping qPCR method to provide a wider range of specific cccDNA quantification (up to 5 logs of effective range). Extrachromosomal DNA was extracted from para-neoplastic tissues for cccDNA quantification. In total, 350 HBV-related HCC patients were included for an outcome analysis. Without differential pre-dilution, cccDNA levels in para-neoplastic liver tissues were determined, ranging from < 2 × 103 to 123.0 × 106 copies/gram. The multivariate linear regression analysis showed that cccDNA was independently correlated with the HBV e antigen (p < 0.001) and serum HBV-DNA levels (p = 0.012). The Cox proportional hazard model analysis showed that cccDNA independently predicted overall survival (p = 0.003) and extrahepatic metastasis-free survival (p = 0.001). In virologically suppressed HCC patients, cccDNA still effectively predicted intrahepatic recurrence-free (p = 0.003) and extrahepatic metastasis-free (p = 0.009) survivals. In conclusion, cccDNA independently predicted postoperative extrahepatic metastasis-free survival.
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Affiliation(s)
- Chao-Wei Hsu
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (C.-W.H.); (Y.-D.C.); (M.-W.L.); (Y.-H.L.)
- Department of Hepatogastroenterology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
| | - Yu-De Chu
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (C.-W.H.); (Y.-D.C.); (M.-W.L.); (Y.-H.L.)
| | - Ming-Wei Lai
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (C.-W.H.); (Y.-D.C.); (M.-W.L.); (Y.-H.L.)
- Division of Pediatric Gastroenterology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
| | - Chih-Lang Lin
- Liver Research Unit, Chang Gung Memorial Hospital, Keelung 204, Taiwan;
| | - Kung-Hao Liang
- Medical Research Department, Taipei Veterans General Hospital, Taipei 112, Taiwan;
| | - Yang-Hsiang Lin
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (C.-W.H.); (Y.-D.C.); (M.-W.L.); (Y.-H.L.)
| | - Chau-Ting Yeh
- Liver Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (C.-W.H.); (Y.-D.C.); (M.-W.L.); (Y.-H.L.)
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 333, Taiwan
- Correspondence: ; Tel.: +886-3-3281200 (ext. 8129)
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Caviglia GP, Armandi A, Rosso C, Ribaldone DG, Pellicano R, Fagoonee S. Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis. Diagnostics (Basel) 2021; 11:187. [PMID: 33525443 PMCID: PMC7910971 DOI: 10.3390/diagnostics11020187] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Revised: 01/25/2021] [Accepted: 01/26/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA is the key molecule responsible for viral persistence within infected hepatocytes. The evaluation of HBV cccDNA is crucial for the management of patients with chronic HBV infection and for the personalization of treatment. However, the need for liver biopsy is the principal obstacle for the assessment of intrahepatic HBV cccDNA. In the last decade, several studies have investigated the performance of hepatitis B core-related antigen (HBcrAg) as a surrogate of HBV cccDNA amount in the liver. In this meta-analysis, we collected 14 studies (1271 patients) investigating the correlation between serum HBcrAg and intrahepatic HBV cccDNA. Serum HBcrAg showed a high correlation with intrahepatic HBV cccDNA (r = 0.641, 95% confidence interval (CI) 0.510-0.743, p < 0.001). In a head-to-head comparison, we observed that the performance of HBcrAg was significantly superior to that of hepatitis B surface antigen (r = 0.665 vs. r = 0.475, respectively, p < 0.001). Subgroup analysis showed that the correlation between HBcrAg and intrahepatic HBV cccDNA was high, both in hepatitis B e antigen-positive and -negative patients (r = 0.678, 95% CI 0.403-0.840, p < 0.001, and r = 0.578, 95% CI 0.344-0.744, p < 0.001, respectively). In conclusion, the measurement of serum HBcrAg qualifies as a reliable non-invasive surrogate for the assessment of an intrahepatic HBV cccDNA reservoir.
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Affiliation(s)
- Gian Paolo Caviglia
- Department of Medical Sciences, University of Turin, 10124 Turin, Italy; (A.A.); (C.R.); (D.G.R.)
| | - Angelo Armandi
- Department of Medical Sciences, University of Turin, 10124 Turin, Italy; (A.A.); (C.R.); (D.G.R.)
| | - Chiara Rosso
- Department of Medical Sciences, University of Turin, 10124 Turin, Italy; (A.A.); (C.R.); (D.G.R.)
| | | | - Rinaldo Pellicano
- Unit of Gastroenterology, Città della Salute e della Scienza di Torino-Molinette Hospital, 10126 Turin, Italy;
| | - Sharmila Fagoonee
- Institute of Biostructure and Bioimaging (CNR), Molecular Biotechnology Center, 10126 Turin, Italy
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In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide. Viruses 2021; 13:v13010097. [PMID: 33445753 PMCID: PMC7828204 DOI: 10.3390/v13010097] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Revised: 01/08/2021] [Accepted: 01/08/2021] [Indexed: 02/07/2023] Open
Abstract
An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.
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Huang Q, Zhou B, Cai D, Zong Y, Wu Y, Liu S, Mercier A, Guo H, Hou J, Colonno R, Sun J. Rapid Turnover of Hepatitis B Virus Covalently Closed Circular DNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients. Hepatology 2021; 73:41-52. [PMID: 32189364 PMCID: PMC7898704 DOI: 10.1002/hep.31240] [Citation(s) in RCA: 66] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Revised: 02/07/2020] [Accepted: 03/10/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIMS Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a pivotal role in the establishment and persistence of HBV infection. Understanding the turnover time of preexisting cccDNA pools would be helpful in designing strategies to clear HBV by fully blocking the de novo generation of cccDNA. APPROACH AND RESULTS In this study, we retrospectively monitored the emergence and reversion of the rtM204I/V mutant, a signature lamivudine resistance (LAMR ) mutation serving as a biomarker of cccDNA turnover in liver biopsies and longitudinal serum samples from two clinical trials. Methodologies were optimized to differentially isolate and sequence HBV virion DNA, cccDNA, and HBV RNA from clinical samples. A strong correlation was observed between LAMR composition of cccDNA with that of serum and intrahepatic HBV RNA in paired liver and serum samples (r = 0.96 and 0.90, respectively), suggesting that serum HBV RNA can serve as a surrogate marker of cccDNA genetic composition when liver biopsies are unavailable. LAMR mutations emerged and increased from undetectable to 40%-90% within 16-28 weeks in serum HBV RNA from telbivudine-treated patients experiencing virological breakthrough. Similarly, in lamivudine-resistant patients who switched to interferon therapy, serum HBV-RNA population bearing 100% LAMR mutations fully reversed back to wild type within 24-48 weeks. CONCLUSIONS The genetic composition dynamics of serum HBV RNA and biopsy cccDNA in treated HBV patients indicates that cccDNA turnover occurs relatively rapidly (several months), offering a possibility of HBV cure with finite therapy through completely blocking cccDNA replenishment.
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Affiliation(s)
- Qi Huang
- Assembly Biosciences, Inc.South San FranciscoCA
| | - Bin Zhou
- State Key Laboratory of Organ Failure ResearchGuangdong Provincial Key Laboratory of Viral Hepatitis ResearchDepartment of Infectious DiseasesNanfang HospitalSouthern Medical UniversityGuangzhouChina
- Department of Microbiology and ImmunologyIndiana UniversityIndianapolisIN
| | - Dawei Cai
- Assembly Biosciences, Inc.South San FranciscoCA
| | - Yuhua Zong
- Assembly Biosciences, Inc.South San FranciscoCA
| | - Yaobo Wu
- State Key Laboratory of Organ Failure ResearchGuangdong Provincial Key Laboratory of Viral Hepatitis ResearchDepartment of Infectious DiseasesNanfang HospitalSouthern Medical UniversityGuangzhouChina
| | - Shi Liu
- State Key Laboratory of Organ Failure ResearchGuangdong Provincial Key Laboratory of Viral Hepatitis ResearchDepartment of Infectious DiseasesNanfang HospitalSouthern Medical UniversityGuangzhouChina
| | | | - Haitao Guo
- Department of Microbiology and ImmunologyIndiana UniversityIndianapolisIN
- Cancer Virology ProgramUPMC Hillman Cancer CenterDepartment of Microbiology and Molecular GeneticsUniversity of PittsburghPittsburghPA
| | - Jinlin Hou
- State Key Laboratory of Organ Failure ResearchGuangdong Provincial Key Laboratory of Viral Hepatitis ResearchDepartment of Infectious DiseasesNanfang HospitalSouthern Medical UniversityGuangzhouChina
| | | | - Jian Sun
- State Key Laboratory of Organ Failure ResearchGuangdong Provincial Key Laboratory of Viral Hepatitis ResearchDepartment of Infectious DiseasesNanfang HospitalSouthern Medical UniversityGuangzhouChina
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Bassit L, Ono SK, Schinazi RF. Moving Fast Toward Hepatitis B Virus Elimination. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1322:115-138. [PMID: 34258739 DOI: 10.1007/978-981-16-0267-2_5] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Currently, there are two safe and effective therapeutic strategies for chronic hepatitis B treatment, namely, nucleoside analogs and interferon alpha (pegylated or non-pegylated). These treatments can control viral replication and improve survival; however, they do not eliminate the virus and therefore require long-term continued therapy. In addition, there are significant concerns about virus rebound on discontinuation of therapy and the development of fibrosis and hepatocellular carcinoma despite therapy. Therefore, the search for new, more effective, and safer antiviral agents that can cure hepatitis B virus (HBV) continues. Anti-HBV drug discovery and development is fundamentally impacted by our current understanding of HBV replication, disease physiopathology, and persistence of HBV covalently closed circular DNA (cccDNA). Several HBV replication targets are the basis for novel anti-HBV drug development strategies. Many of them are already in clinical trial phase 1 or 2, while others with promising results are still in preclinical stages. As research intensifies, potential HBV curative therapies and modalities in the pipeline are now on the horizon.
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Affiliation(s)
- Leda Bassit
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, GA, USA
| | - Suzane Kioko Ono
- Department of Gastroenterology, University of Sao Paulo School of Medicine, Sao Paulo, SP, Brazil
| | - Raymond F Schinazi
- Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Children's Healthcare of Atlanta, Atlanta, GA, USA.
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D e novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA. JHEP Rep 2020; 3:100195. [PMID: 33385130 PMCID: PMC7771110 DOI: 10.1016/j.jhepr.2020.100195] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/21/2020] [Revised: 08/24/2020] [Accepted: 09/20/2020] [Indexed: 12/14/2022] Open
Abstract
Background & Aims Chronic HBV infection cannot be cured by current therapeutics owing to their limited ability to reduce covalently closed circular (ccc)DNA levels in the livers of infected individuals. Therefore, greater understanding of the molecular determinants of cccDNA formation and persistence is required. One key issue is the extent to which de novo nucleocapsid-mediated replenishment (reimport) contributes to cccDNA levels in an infected hepatocyte. Methods We engineered an infectious HBV mutant with a genome encoding a stop codon at position T67 in the HBV core open reading frame (ΔHBc HBV). Importantly, ΔHBc HBV virions cannot initiate nucleocapsid synthesis upon infection. Long-term in vitro HBV infection markers were followed for up for 9 weeks in HepG2-NTCP cells (A3 clone) and HBV DNA was quantified using a newly-developed, highly-precise PCR assay (cccDNA inversion quantitative PCR). Results ΔHBc and wild-type (WT) HBV resulted in comparable expression of HBV surface antigen (HBsAg), which could be blocked using the entry inhibitor Myrcludex B, confirming bona fide infection via the receptor sodium taurocholate cotransporting polypeptide (NTCP). In primary human hepatocytes, Huh7-NTCP, HepG2-NTCP, and HepaRG-NTCP cells, comparable copy numbers of cccDNA were formed. cccDNA levels, transcription of viral RNA, and HBsAg secretion remained comparably stable in WT and ΔHBc HBV-infected cells for at least 9 weeks. Conclusions Our results imply that de novo synthesised HBc plays a minor role in transcriptional regulation of cccDNA. Importantly, we show that initially-formed cccDNA is stable in hepatocytes without requiring continuous replenishment in in vitro infection systems and contribution from de novo DNA-containing nucleocapsids is not required. Thus, short-term therapeutic targeting of capsid-reimport is likely an inefficient strategy in eliminating cccDNA in chronically infected hepatocytes. Lay summary The hepatitis B virus can maintain itself in the liver for a patient's lifetime, causing liver injury and cancer. We have clarified exactly how it maintains itself in an infected cell. This now means we have a better idea at how to target the virus and cure a chronic infection.
Covalently closed circular (ccc)DNA is key for maintaining chronic HBV infection. Virus core protein expression is not required for cccDNA formation, stability, or transcription within 9 weeks of in vitro infection. Our results suggest that targeting HBV core with short-term treatment is inefficient in clearing intrahepatic cccDNA. Viral entry inhibitors or capsid inhibitors could prevent breakthrough of novel HBV variants.
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Key Words
- ALT, alanine aminotransferase
- Antivirals
- Bulevirtide
- CIs, capsid inhibitors
- Capsid inhibitors
- Core protein
- Covalently closed circular DNA
- DHBV, duck hepatitis B virus
- HBV DNA integration
- HBV persistence
- HBV, hepatitis B virus
- HBcAg
- HBsAg, hepatitis B virus surface antigen
- Hepcludex
- Myrcludex B
- NC, naked capsids
- NTCP, sodium taurocholate cotransporting polypeptide
- NUCs, nucleos(t)ide analogues
- ORF, open reading frame
- PEG, polyethylene glycol
- PHH, primary human hepatocytes
- SN, supernatant
- VP, virions
- WT, wild-type
- cccDNA, covalently closed circular DNA
- dpi, days post inoculation
- mge, multiplicity of genomic equivalent
- pgRNA, pregenomic RNA
- rcDNA, relaxed circular DNA
- vge, viral genome equivalents
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Tu T, Zehnder B, Qu B, Ni Y, Main N, Allweiss L, Dandri M, Shackel N, George J, Urban S. A novel method to precisely quantify hepatitis B virus covalently closed circular (ccc)DNA formation and maintenance. Antiviral Res 2020; 181:104865. [PMID: 32726641 DOI: 10.1016/j.antiviral.2020.104865] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/21/2020] [Accepted: 06/23/2020] [Indexed: 01/05/2023]
Abstract
Hepatitis B virus (HBV) is the major cause of virus-associated liver disease. Persistent HBV infection is maintained by its episomal genome (covalently closed circular DNA, cccDNA), which acts as a template for viral transcripts. The formation of cccDNA is poorly characterised due to limited ability to quantify it accurately in the presence of replicative intermediates. Here, we describe a novel cccDNA quantification assay (cccDNA inversion quantitative PCR, cinqPCR), which uses restriction enzymes to invert a DNA sequence close to the gap region of Genotype D HBV strains, including the isolate widely used in experimental studies. Importantly, cinqPCR allows simultaneous normalisation to cellular DNA in a single reaction, provides absolute copy numbers without requiring a standard curve, and has high precision, sensitivity, and specificity for cccDNA compared to previous assays. We first established that cinqPCR gives values consistent with classical approaches in both in vitro and in vivo (humanised mice) HBV infections. We then used cinqPCR to find that cccDNA is formed within 12 h post-inoculation (hpi). cccDNA formation slowed by 28 hpi despite de novo synthesis of HBV DNA, indicating inefficient conversion of new viral genomes to cccDNA within infected cells. Finally, we show that cinqPCR can be used as a 96-well screening assay. Thus, we have developed an ideal method for testing current and future anti-cccDNA therapeutics with high precision and sensitivity.
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Affiliation(s)
- Thomas Tu
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany; Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Westmead, NSW, Australia.
| | - Benno Zehnder
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Bingqian Qu
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Yi Ni
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Nathan Main
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia; Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia
| | - Lena Allweiss
- Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Maura Dandri
- Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZIF, Hamburg-Lübeck-Borstel Partner Site, Germany
| | - Nicholas Shackel
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia; Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia; South Western Sydney Clinical School, University of New South Wales, Australia
| | - Jacob George
- Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Westmead, NSW, Australia
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany; German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany
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Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation. Nat Microbiol 2020; 5:715-726. [PMID: 32152586 PMCID: PMC7190442 DOI: 10.1038/s41564-020-0678-0] [Citation(s) in RCA: 85] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2019] [Accepted: 01/30/2020] [Indexed: 12/14/2022]
Abstract
Chronic hepatitis B virus (HBV) infection results in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA (rcDNA) delivered by the virions to hepatocytes. A complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV rcDNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast and human extract screenings, we identified five core components of lagging strand synthesis as essential for cccDNA formation: PCNA, the replication factor C (RFC) complex, DNA polymerase δ (POLδ), FEN-1, and DNA ligase 1 (LIG1). We reconstituted cccDNA formation with purified human homologs, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with DNA polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Altogether, our findings define key components in HBV cccDNA formation.
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34
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Prescott NA, Bram Y, Schwartz RE, David Y. Targeting Hepatitis B Virus Covalently Closed Circular DNA and Hepatitis B Virus X Protein: Recent Advances and New Approaches. ACS Infect Dis 2019; 5:1657-1667. [PMID: 31525994 DOI: 10.1021/acsinfecdis.9b00249] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Chronic Hepatitis B virus (HBV) infection remains a worldwide concern and public health problem. Two key aspects of the HBV life cycle are essential for viral replication and thus the development of chronic infections: the establishment of the viral minichromosome, covalently closed circular (ccc) DNA, within the nucleus of infected hepatocytes and the expression of the regulatory Hepatitis B virus X protein (HBx). Interestingly, nuclear HBx redirects host epigenetic machinery to activate cccDNA transcription. In this Perspective, we provide an overview of recent advances in understanding the regulation of cccDNA and the mechanistic and functional roles of HBx. We also describe the progress toward targeting both cccDNA and HBx for therapeutic purposes. Finally, we outline standing questions in the field and propose complementary chemical biology approaches to address them.
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Affiliation(s)
- Nicholas A. Prescott
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States
- Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States
| | - Yaron Bram
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, United States
| | - Robert E. Schwartz
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, United States
- Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, United States
| | - Yael David
- Chemical Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States
- Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States
- Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, United States
- Department of Pharmacology, Weill Cornell Medicine, 1300 York Avenue, New York, New York 10065, United States
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35
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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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Tong S, Pan J, Tang J. Study on the structure optimization and anti-hepatitis B virus activity of novel human La protein inhibitor HBSC11. J Med Virol 2019; 91:1818-1829. [PMID: 31241178 PMCID: PMC6771476 DOI: 10.1002/jmv.25528] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2019] [Accepted: 06/17/2019] [Indexed: 12/23/2022]
Abstract
In our previous study, Methyl pyrazolo[1,5‐a] pyridine‐2‐carboxylate (HBSC11) was shown to combine with La protein, which conferred anti‐hepatitis B virus (HBV) effects. The purpose of this study was to optimize, synthesize, and evaluate the anti‐HBV activity of HBSC11. The methyl group of HBSC11 was substituted with hydrophobic, hydrophilic, and tricyclic groups to generate novel HBV inhibitors with desirable potency. On in vitro evaluation, several derivatives exhibited good anti‐HBV activity compared with control. In particular, compound 5a reduced the level of HBV antigen by approximately 50%, which was similar to the activity of entecavir. In a mouse model, 5a showed 98.9% inhibition rate for HBV DNA, 57.4% for HBsAg, and 46.4% for HBeAg; the corresponding rates in the control group were 90.8, 3.8, and 9.8%, respectively. In addition, prediction of binding modes and physicochemical properties showed that 5a formed hydrogen bonds with La protein and conformed well to the Lipinski's rule of five. Our results suggest that 5a is a potential new anti‐HBV drug.
La protein protects HBV RNA from destruction by combining with HBV RNA and covers up the RNA cleavage site. HBSC11 (Methyl pyrazolo[1,5‐a] pyridine‐2‐carboxylate) is a novel La protein inhibitor which we identified as previous. 10 derivatives (3a‐3f, 5a‐5d) were obtained by 2 sections‐scaffold and kept the active site form leading compound HBSC11. Candidate compound 5a exhibited potent anti‐HBV activity with safety concentration and satisfied physicochemical properties.
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Affiliation(s)
- Shuangmei Tong
- Department of Pharmacy, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China
| | - Jiaqian Pan
- Department of Pharmacy, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China
| | - Jing Tang
- Department of Pharmacy, The Obstetrics & Gynecology Hospital of Fudan University, Shanghai, China
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An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes. PLoS One 2019; 14:e0216139. [PMID: 31188831 PMCID: PMC6561549 DOI: 10.1371/journal.pone.0216139] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Accepted: 04/15/2019] [Indexed: 01/04/2023] Open
Abstract
Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.
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Nkongolo S, Nußbaum L, Lempp FA, Wodrich H, Urban S, Ni Y. The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA. Antiviral Res 2019; 168:146-155. [PMID: 31018112 DOI: 10.1016/j.antiviral.2019.04.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Revised: 04/14/2019] [Accepted: 04/17/2019] [Indexed: 01/01/2023]
Abstract
Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning.
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Affiliation(s)
- Shirin Nkongolo
- University Hospital Heidelberg (Germany), Center for Infectious Diseases, Molecular Virology, Germany; German Center for Infection Research (DZIF), Partner Site Heidelberg, TTU Hepatitis, Germany.
| | - Lea Nußbaum
- University Hospital Heidelberg (Germany), Center for Infectious Diseases, Molecular Virology, Germany.
| | - Florian A Lempp
- University Hospital Heidelberg (Germany), Center for Infectious Diseases, Molecular Virology, Germany; German Center for Infection Research (DZIF), Partner Site Heidelberg, TTU Hepatitis, Germany.
| | - Harald Wodrich
- Laboratoire de Microbiologie Fondamentale et Pathogénicité, University of Bordeaux, France.
| | - Stephan Urban
- University Hospital Heidelberg (Germany), Center for Infectious Diseases, Molecular Virology, Germany; German Center for Infection Research (DZIF), Partner Site Heidelberg, TTU Hepatitis, Germany.
| | - Yi Ni
- University Hospital Heidelberg (Germany), Center for Infectious Diseases, Molecular Virology, Germany; German Center for Infection Research (DZIF), Partner Site Heidelberg, TTU Hepatitis, Germany.
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Qu B, Urban S. Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR. Bio Protoc 2019; 9:e3202. [PMID: 33654998 DOI: 10.21769/bioprotoc.3202] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2018] [Revised: 03/14/2019] [Accepted: 03/15/2019] [Indexed: 01/27/2023] Open
Abstract
Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate "cccDNA copies per (infected) cell".
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Affiliation(s)
- Bingqian Qu
- Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), University Hospital Heidelberg, Heidelberg, Germany
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, Centre for Integrative Infectious Disease Research (CIID), University Hospital Heidelberg, Heidelberg, Germany.,German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
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Caviglia GP, Olivero A, Smedile A. Reply to: "Over-gap PCR amplification to identify presence of replication-competent HBV DNA from integrated HBV DNA: An updated occult HBV infection definition". J Hepatol 2019; 70:559-560. [PMID: 30470481 DOI: 10.1016/j.jhep.2018.10.029] [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] [Received: 10/24/2018] [Accepted: 10/25/2018] [Indexed: 12/04/2022]
Affiliation(s)
- Gian Paolo Caviglia
- Department of Medical Sciences, University of Turin, and Gastroenterology Division of Città della Salute e della Scienza of Turin, University Hospital, Turin, Italy.
| | - Antonella Olivero
- Department of Medical Sciences, University of Turin, and Gastroenterology Division of Città della Salute e della Scienza of Turin, University Hospital, Turin, Italy
| | - Antonina Smedile
- Department of Medical Sciences, University of Turin, and Gastroenterology Division of Città della Salute e della Scienza of Turin, University Hospital, Turin, Italy
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