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Bell JT, Zhang X. The hepatitis B virus surface antigen: An evolved perfection and its unresolved mysteries. Virology 2025; 608:110527. [PMID: 40220401 DOI: 10.1016/j.virol.2025.110527] [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: 12/07/2024] [Revised: 03/24/2025] [Accepted: 04/04/2025] [Indexed: 04/14/2025]
Abstract
The Hepatitis B Virus has long afflicted the human race, with a widespread impact on the global health system and profound medical implications for those who are chronically infected. Despite its relatively recent discovery, over the last 50 years great advancements have been made towards the characterisation of this complex etiological agent. The virus itself has a highly evolved genome which encodes for seven viral proteins, three of which (the surface antigens) were consequential in the initial discovery and isolation of the virus. These surface antigens are ubiquitously important throughout the viral lifecycle, from capsid envelopment through to receptor-mediated invasion into the hepatocytes. The hepatitis B surface antigens (in particular, the large protein) adopt complex topological folds and tertiary structures, and it is this topological intricacy which facilitates the diverse roles the three surface antigens play in HBV maturation and infection. Here, the biochemical and topological attributes of the three surface antigens are reviewed in detail, with particular focus on their relevance to the establishment of infection. Further research is still required to elucidate the coordinates of the antigen loop and the dynamic topological changes of key motifs during entry and viral morphogenesis; these in turn may provide new leads for therapeutics which may potentiate a functional cure for chronic hepatitis B.
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Affiliation(s)
- Jack Thomas Bell
- Faculty of Science and Technology, University of Canberra, ACT, Australia
| | - Xiaonan Zhang
- Faculty of Science and Technology, University of Canberra, ACT, Australia.
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D'Anna S, Salpini R, Degasperi E, Piermatteo L, Facchetti F, Sambarino D, Torre G, Borghi M, Anolli MP, Monico S, Svicher V, Lampertico P. HBsAg Isoforms as Innovative Biomarkers in Predicting Virological Response to Bulevirtide in Patients With Chronic Hepatitis D. Liver Int 2025; 45:e70094. [PMID: 40290073 DOI: 10.1111/liv.70094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2024] [Revised: 02/17/2025] [Accepted: 04/02/2025] [Indexed: 04/30/2025]
Abstract
BACKGROUND AND AIMS HDV exploits HBV surface-protein (HBsAg) for entering into hepatocytes. HBsAg consists of 3 isoforms: Large- (L-HBs, predominantly present in virions and mediating binding to NTCP-receptor), Middle- (M-HBs) and Small-HBsAg (S-HBs). Here, we investigated the kinetics of HBs isoforms under bulevirtide treatment (BLV). METHODS 67 consecutive patients with HDV-related compensated cirrhosis starting BLV 2 mg/day were enrolled. L-HBs, M-HBs and S-HBs were quantified by ad-hoc ELISAs in baseline and week 48 (W48) samples. RESULTS At baseline, median (IQR) HDV-RNA was 5.1 (4.3-5.7) log IU/mL while median (IQR) S-HBs, M-HBs and L-HBs levels were 3801 (1401-7462), 743 (211-1710) and 5 (1-13) ng/mL. At W48, virological responses (VR) were observed in 72% (48/67) of patients, while 25.4% (17/67) achieved undetectable HDV-RNA (11/17 with ALT-normalisation). A decline of S-HBs, M-HBs and L-HBs levels was observed in 51%, 63% and 31% of patients (median [IQR] decline: 961 [461-1985], 258 [68-626] and 4 [2-12] ng/mL). Notably, patients with undetectable HDV-RNA at W48 had baseline L-HBs and S-HBs levels lower than patients not achieving this end-point (1 [0.3-7] vs. 6 [2-13] ng/mL, p = 0.04 and 1570 [369-5185] vs. 4015 [1646-8687] ng/mL, p = 0.002). By AUROC, patients with baseline L-HBs < 3 ng/mL or S-HBs < 3400 ng/mL were more likely to achieve HDV-RNA undetectability at W48 (39.3% vs. 15.8%, p = 0.04 and 38.7% vs. 13.9%, p = 0.03). Furthermore, the combination of pre-treatment L-HBs < 3 ng/mL + HDV-RNA < 5logIU/mL and S-HBs < 3400 ng/mL + HDV-RNA < 5logIU/mL was the best predictor for achieving undetectable HDV-RNA at W48 (56.3% vs. 15.7%, p = 0.002 and 60% vs. 11%, p < 0.001). CONCLUSIONS Quantification of L-HBs and of S-HBs, along with HDV-RNA, may reflect the burden of circulating infectious virions in HBV/HDV co-infection, providing a promising tool to identify patients more likely to respond to BLV.
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Affiliation(s)
- Stefano D'Anna
- Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - Romina Salpini
- Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - Elisabetta Degasperi
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | | | - Floriana Facchetti
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Dana Sambarino
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Giulia Torre
- Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - Marta Borghi
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Maria Paola Anolli
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Sara Monico
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | | | - Pietro Lampertico
- Division of Gastroenterology and Hepatology, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
- CRC "A. M. and A. Migliavacca" Center for Liver Disease, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy
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Hong X, Schneider WM, Rice CM. Hepatitis B Virus Nucleocapsid Assembly. J Mol Biol 2025:169182. [PMID: 40316009 DOI: 10.1016/j.jmb.2025.169182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2025] [Revised: 04/13/2025] [Accepted: 04/28/2025] [Indexed: 05/04/2025]
Abstract
Hepatitis B virus (HBV), the prototypical member of the Hepadnaviridae family, is a DNA virus that replicates its genome through reverse transcription of a pregenomic RNA (pgRNA) precursor. The selective packaging of pgRNA and viral polymerase (Pol) into assembling capsids formed by the viral core protein-a process known as nucleocapsid assembly-is an essential step in the HBV lifecycle. Advances in cellular and cell-free systems have provided significant insights into the mechanisms underlying capsid assembly, Pol binding to pgRNA, Pol-pgRNA packaging, and initiation of genome replication. However, the absence of a cell-free system capable of reconstituting selective HBV Pol-pgRNA packaging into fully assembled capsids leaves fundamental questions about nucleocapsid assembly unanswered. This review summarizes the current knowledge of HBV nucleocapsid assembly, focusing on the interplay between Pol-pgRNA interactions, capsid formation, and regulation by host factors. It also highlights the contribution of cellular and cell-free systems to these discoveries and underscores the need for new approaches that reconstitute the complete HBV nucleocapsid assembly process. With the growing interest in developing nucleocapsid assembly inhibitors, some of which are currently in clinical trials, targeting Pol-pgRNA interactions and nucleocapsid assembly represents a promising therapeutic strategy for curing chronic hepatitis B.
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Affiliation(s)
- Xupeng Hong
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.
| | - William M Schneider
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA
| | - Charles M Rice
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA
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Seigneuret F, Eymieux S, Sarabia-Vega V, Lemoine R, Burlaud-Gaillard J, Raynal P, Hourioux C, Sureau C, Roingeard P, de Rocquigny H. The HBV large envelope protein initiates virion assembly by recruiting capsids at membrane rich domains related to late endosome. Cell Mol Life Sci 2025; 82:128. [PMID: 40128454 PMCID: PMC11933560 DOI: 10.1007/s00018-025-05574-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 12/06/2024] [Accepted: 01/02/2025] [Indexed: 03/26/2025]
Abstract
A crucial step of HBV (Hepatitis B Virus) virion morphogenesis is the envelopment of the nucleocapsid by the viral envelope proteins, which is triggered by an interaction between the HBV core protein and the large HBV envelope protein. To document this protein-protein interaction, we co-expressed core and large HBV envelope (LHBs) in Huh-7 cells and subjected the cells to microscopy examination by Fluorescence Resonance Energy Transfer (FRET) and Transmission Electron Microscopy (TEM). Our results show that the sole expression of the core protein leads to assembly of capsids that remain individually isolated within the whole cell, but particularly within the nucleus. In the presence of LHBs, capsids were observed as large clusters in a membrane rich region peripheral to the nucleus. In this context, core-LHBs complex co-localize with markers of the late endosome/multivesicular bodies, this co-localization being driven by LHBs. These results thus show that LHBs binds to the core proteins when preassembled into capsid, at membranes of the late endosome, where the inner capsid and the outer envelope meet to assemble a virion.
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Affiliation(s)
- Florian Seigneuret
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Sébastien Eymieux
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Vanessa Sarabia-Vega
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Roxane Lemoine
- Département Cytométrie et Single-Cell Immunobiologie, Plateforme Scientifique et Technique - Analyse des Systèmes Biologiques (PST-ASB), Université de Tours, 10 Boulevard Tonnellé, 37032, Tours Cedex 1, France
| | - Julien Burlaud-Gaillard
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Pierre Raynal
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Christophe Hourioux
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Camille Sureau
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Philippe Roingeard
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Hugues de Rocquigny
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France.
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Chuang YC, Ou JHJ. Hepatitis B virus entry, assembly, and egress. Microbiol Mol Biol Rev 2024; 88:e0001424. [PMID: 39440957 DOI: 10.1128/mmbr.00014-24] [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] [Indexed: 10/25/2024] Open
Abstract
SUMMARYHepatitis B virus (HBV) is an important human pathogen that chronically infects approximately 250 million people in the world, resulting in ~1 million deaths annually. This virus is a hepatotropic virus and can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. The entry of HBV into hepatocytes is initiated by the interaction of its envelope proteins with its receptors. This is followed by the delivery of the viral nucleocapsid to the nucleus for the release of its genomic DNA and the transcription of viral RNAs. The assembly of the viral capsid particles may then take place in the nucleus or the cytoplasm and may involve cellular membranes. This is followed by the egress of the virus from infected cells. In recent years, significant research progresses had been made toward understanding the entry, the assembly, and the egress of HBV particles. In this review, we discuss the molecular pathways of these processes and compare them with those used by hepatitis delta virus and hepatitis C virus , two other hepatotropic viruses that are also enveloped. The understanding of these processes will help us to understand how HBV replicates and causes diseases, which will help to improve the treatments for HBV patients.
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Affiliation(s)
- Yu-Chen Chuang
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
| | - J-H James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
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Zhang Y, Yuan X, Wang J, Han M, Lu H, Wang Y, Liu S, Yang S, Xing HC, Cheng J. TRPV4 promotes HBV replication and capsid assembly via methylation modification of H3K4 and HBc ubiquitin. J Med Virol 2024; 96:e29510. [PMID: 38573018 DOI: 10.1002/jmv.29510] [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: 09/19/2023] [Revised: 02/21/2024] [Accepted: 02/25/2024] [Indexed: 04/05/2024]
Abstract
Hepatitis B virus (HBV) infection poses a significant burden on global public health. Unfortunately, current treatments cannot fully alleviate this burden as they have limited effect on the transcriptional activity of the tenacious covalently closed circular DNA (cccDNA) responsible for viral persistence. Consequently, the HBV life cycle should be further investigated to develop new anti-HBV pharmaceutical targets. Our previous study discovered that the host gene TMEM203 hinders HBV replication by participating in calcium ion regulation. The involvement of intracellular calcium in HBV replication has also been confirmed. In this study, we found that transient receptor potential vanilloid 4 (TRPV4) notably enhances HBV reproduction by investigating the effects of several calcium ion-related molecules on HBV replication. The in-depth study showed that TRPV4 promotes hepatitis B core/capsid protein (HBc) protein stability through the ubiquitination pathway and then promotes the nucleocapsid assembly. HBc binds to cccDNA and reduces the nucleosome spacing of the cccDNA-histones complex, which may regulate HBV transcription by altering the nucleosome arrangement of the HBV genome. Moreover, our results showed that TRPV4 promotes cccDNA-dependent transcription by accelerating the methylation modification of H3K4. In conclusion, TRPV4 could interact with HBV core protein and regulate HBV during transcription and replication. These data suggest that TRPV4 exerts multifaceted HBV-related synergistic factors and may serve as a therapeutic target for CHB.
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Affiliation(s)
- Yu Zhang
- Peking University Ditan Teaching Hospital, Beijing, China
- Department of Hepatology, Beijing Ditan Hospital of Capital Medical University, Beijing, China
| | - Xiaoxue Yuan
- Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Jun Wang
- Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Beijing Key Laboratory of Emerging Infectious Diseases, Peking University Ditan Teaching Hospital, Beijing, China
| | - Ming Han
- Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Hongping Lu
- Beijing Pan-Asia Tongze Institute of Biomedicine Co, Ltd, Beijing, China
| | - Yun Wang
- Department of Hepatology, Beijing Ditan Hospital of Capital Medical University, Beijing, China
- Beijing Key Laboratory of Emerging Infectious Diseases, The First Section of Liver Disease, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Shunai Liu
- Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Song Yang
- Department of Hepatology, Beijing Ditan Hospital of Capital Medical University, Beijing, China
| | - Hui-Chun Xing
- Department of Hepatology, Beijing Ditan Hospital of Capital Medical University, Beijing, China
| | - Jun Cheng
- Peking University Ditan Teaching Hospital, Beijing, China
- Department of Hepatology, Beijing Ditan Hospital of Capital Medical University, Beijing, China
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7
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Wang Y, Li Q, Li C, Wang C, Wang S, Yuan W, Yu D, Zhang K, Shi B, Chen X, Liu T, Yuan Z, Tong S, Nassal M, Wen YM, Wang YX. Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion. Gut 2024; 73:668-681. [PMID: 37973365 DOI: 10.1136/gutjnl-2023-330537] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 11/01/2023] [Indexed: 11/19/2023]
Abstract
OBJECTIVES Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.
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Affiliation(s)
- Yang Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Qiqi Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Cheng Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Cong Wang
- Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai, China
| | - Shijie Wang
- Deparment of Infectious Diseases, Changzheng Hospital, Navy Medical University, Shanghai, China
| | - Wenjie Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Demin Yu
- Department of Infectious Diseases, Institute of Infectious and Respiratory Diseases, Sino-French Research Center for Life Science and Genomics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
| | - Ke Zhang
- SCG Cell Therapy Pte Ltd, Singapore
| | - Bisheng Shi
- Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai, China
| | - Xiaomei Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Tiantian Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Shuping Tong
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Michael Nassal
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
| | - Yu-Mei Wen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Yong-Xiang Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontier Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
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Li J, Lin Y, Wang X, Lu M. Interconnection of cellular autophagy and endosomal vesicle trafficking and its role in hepatitis B virus replication and release. Virol Sin 2024; 39:24-30. [PMID: 38211880 PMCID: PMC10877419 DOI: 10.1016/j.virs.2024.01.001] [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: 09/07/2023] [Accepted: 01/06/2024] [Indexed: 01/13/2024] Open
Abstract
Hepatitis B virus (HBV) produces and releases various particle types, including complete virions, subviral particles with envelope proteins, and naked capsids. Recent studies demonstrate that HBV exploits distinct intracellular membrane trafficking pathways, including the endosomal vesicle trafficking and autophagy pathway, to assemble and release viral and subviral particles. Herein, we summarize the findings about the distinct roles of autophagy and endosomal membrane trafficking and the interaction of both pathways in HBV replication, assembly, and release.
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Affiliation(s)
- Jia Li
- Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, 45122, Germany
| | - Yong Lin
- Key Laboratory of Molecular Biology of Infectious Diseases (Chinese Ministry of Education), Chongqing Medical University, Chongqing, 400016, China
| | - Xueyu Wang
- The Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310000, China
| | - Mengji Lu
- Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, 45122, Germany.
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9
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Lazarevic I, Banko A, Miljanovic D, Cupic M. Hepatitis B Surface Antigen Isoforms: Their Clinical Implications, Utilisation in Diagnosis, Prevention and New Antiviral Strategies. Pathogens 2024; 13:46. [PMID: 38251353 PMCID: PMC10818932 DOI: 10.3390/pathogens13010046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2023] [Revised: 12/27/2023] [Accepted: 01/02/2024] [Indexed: 01/23/2024] Open
Abstract
The hepatitis B surface antigen (HBsAg) is a multifunctional glycoprotein composed of large (LHB), middle (MHB), and small (SHB) subunits. HBsAg isoforms have numerous biological functions during HBV infection-from initial and specific viral attachment to the hepatocytes to initiating chronic infection with their immunomodulatory properties. The genetic variability of HBsAg isoforms may play a role in several HBV-related liver phases and clinical manifestations, from occult hepatitis and viral reactivation upon immunosuppression to fulminant hepatitis and hepatocellular carcinoma (HCC). Their immunogenic properties make them a major target for developing HBV vaccines, and in recent years they have been recognised as valuable targets for new therapeutic approaches. Initial research has already shown promising results in utilising HBsAg isoforms instead of quantitative HBsAg for correctly evaluating chronic infection phases and predicting functional cures. The ratio between surface components was shown to indicate specific outcomes of HBV and HDV infections. Thus, besides traditional HBsAg detection and quantitation, HBsAg isoform quantitation can become a useful non-invasive biomarker for assessing chronically infected patients. This review summarises the current knowledge of HBsAg isoforms, their potential usefulness and aspects deserving further research.
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Affiliation(s)
- Ivana Lazarevic
- Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia; (A.B.); (D.M.); (M.C.)
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10
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Ghaemi Z, Nafiu O, Tajkhorshid E, Gruebele M, Hu J. A computational spatial whole-Cell model for hepatitis B viral infection and drug interactions. Sci Rep 2023; 13:21392. [PMID: 38049515 PMCID: PMC10695947 DOI: 10.1038/s41598-023-45998-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Accepted: 10/26/2023] [Indexed: 12/06/2023] Open
Abstract
Despite a vaccine, hepatitis B virus (HBV) remains a world-wide source of infections and deaths. We develop a whole-cell computational platform combining spatial and kinetic models describing the infection cycle of HBV in a hepatocyte host. We simulate key parts of the infection cycle with this whole-cell platform for 10 min of biological time, to predict infection progression, map out virus-host and virus-drug interactions. We find that starting from an established infection, decreasing the copy number of the viral envelope proteins shifts the dominant infection pathway from capsid secretion to re-importing the capsids into the nucleus, resulting in more nuclear-localized viral covalently closed circular DNA (cccDNA) and boosting transcription. This scenario can mimic the consequence of drugs designed to manipulate viral gene expression. Mutating capsid proteins facilitates capsid destabilization and disassembly at nuclear pore complexes, resulting in an increase in cccDNA copy number. However, excessive destabilization leads to premature cytoplasmic disassembly and does not increase the cccDNA counts. Finally, our simulations can predict the best drug dosage and its administration timing to reduce the cccDNA counts. Our adaptable computational platform can be parameterized to study other viruses and identify the most central viral pathways that can be targeted by drugs.
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Affiliation(s)
- Zhaleh Ghaemi
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
- Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
- National Science Foundation Science and Technology Center for Quantitative Cell Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
| | - Oluwadara Nafiu
- Carle-Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
| | - Emad Tajkhorshid
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- National Science Foundation Science and Technology Center for Quantitative Cell Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
| | - Martin Gruebele
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- National Science Foundation Science and Technology Center for Quantitative Cell Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Carle-Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
- Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
| | - Jianming Hu
- Department of Microbiology and Immunology, Pennsylvania State University, Hershey, PA, 17033, USA
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11
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Roade L, Riveiro-Barciela M, Pfefferkorn M, Sopena S, Palom A, Bes M, Rando-Segura A, Casillas R, Tabernero D, Rodríguez-Frías F, Berg T, Esteban R, van Bömmel F, Buti M. HBsAg protein composition and clinical outcomes in chronic hepatitis D and variations across HBeAg-negative chronic HBsAg carriers. JHEP Rep 2023; 5:100842. [PMID: 37745192 PMCID: PMC10514556 DOI: 10.1016/j.jhepr.2023.100842] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 05/25/2023] [Accepted: 06/24/2023] [Indexed: 09/26/2023] Open
Abstract
BACKGROUND & AIMS HBsAg proteins are useful to identify HBV inactive carriers (ICs), but data on chronic hepatitis D (CHD) are scarce. This study aimed to describe HBsAg composition in CHD, its changes during the evolution, and the potential association with clinical outcomes. In addition, we assess the composition of HBsAg across different HBV genotypes and validate previous results on HBsAg proteins in an independent HBV cohort. METHODS Quantitative HBsAg, medium HBsAg proteins (MHBs), and large HBsAg proteins (LHBs) were measured in two cohorts. The first cohort consisted of patients with CHD. A cross-sectional study of samples from two European institutions (N = 46) was conducted. Outcomes were assessed in a retrospective-prospective study of those patients with a follow-up of >1 year (n = 36), and the longitudinal evolution of HBsAg proteins in those with samples >5 years apart (n = 12) was analysed. The second cohort consisted of patients with HBeAg-negative HBV, and a cross-sectional study was performed (N = 141). RESULTS Forty-one (89%) patients with CHD had detectable HDV-RNA, and the presence of HDV-RNA was associated with higher LHBs proportion (p = 0.010). Baseline MHBs (p = 0.051) and MHBs proportion (p = 0.086) tended to be higher in those developing clinical outcomes (9/36, 25%) after a median follow-up of 5.9 years. Patients in which HDV-RNA became spontaneously undetectable during follow-up (5/31, 16.1%) tended to present lower MHBs proportion (p = 0.085). In the longitudinal study, changes in LHBs proportion were observed (p = 0.041), whereas MHBs proportion remained stable (p = 0.209). Regarding HBV, ICs showed lower LHBs proportion (p = 0.027). LHBs and MHBs differed significantly according to HBV genotype, regardless of the HBV phase. CONCLUSIONS Patients with CHD with detectable HDV-RNA presented higher LHBs proportion than those with undetectable HDV-RNA. A trend toward having higher baseline MHBs proportion was observed in patients who developed clinical outcomes or remained with detectable HDV-RNA. This study validates the different HBsAg composition in HBV ICs and reveals the HBV-genotype influence in HBsAg composition. IMPACT AND IMPLICATIONS The composition of HBsAg in chronic hepatitis D differs in patients with detectable and undetectable HDV viral load and may help predict the likelihood of achieving undetectable HDV viraemia and the development of clinical events such as decompensation. The composition of the surface antigen is also useful to distinguish inactive carriers of HBV, and it varies according to HBV genotype.
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Affiliation(s)
- Luisa Roade
- Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain
- Liver Unit, Internal Medicine Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - Mar Riveiro-Barciela
- Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain
- Liver Unit, Internal Medicine Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - Maria Pfefferkorn
- Division of Hepatology, Department of Medicine, Leipzig University Medical Center, Leipzig, Germany
| | - Sara Sopena
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
| | - Adriana Palom
- Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain
- Liver Unit, Internal Medicine Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - Marta Bes
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Transfusion Safety Laboratory, Banc de Sang i Teixits, Servei Català de la Salut, Barcelona, Spain
| | - Ariadna Rando-Segura
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
| | - Rosario Casillas
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
| | - David Tabernero
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Universitat Autònoma de Barcelona (UAB), Department of Biochemistry and Molecular Biology, Barcelona, Spain
- Liver Diseases-Viral Hepatitis, Liver Unit, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
| | - Francisco Rodríguez-Frías
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Universitat Autònoma de Barcelona (UAB), Department of Biochemistry and Molecular Biology, Barcelona, Spain
- Liver Diseases-Viral Hepatitis, Liver Unit, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
| | - Thomas Berg
- Division of Hepatology, Department of Medicine, Leipzig University Medical Center, Leipzig, Germany
| | - Rafael Esteban
- Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain
- Liver Unit, Internal Medicine Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - Florian van Bömmel
- Division of Hepatology, Department of Medicine, Leipzig University Medical Center, Leipzig, Germany
| | - María Buti
- Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain
- Liver Unit, Internal Medicine Department, Hospital Universitari Vall d'Hebron, Vall d'Hebron Barcelona Hospital Campus, Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
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12
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Liu Y, Potts JL, Bloch D, Nian K, McCormick CA, Fanari O, Rouhanifard SH. Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers. ACS Sens 2023; 8:2563-2571. [PMID: 37368999 PMCID: PMC10621038 DOI: 10.1021/acssensors.3c00239] [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: 02/08/2023] [Accepted: 06/16/2023] [Indexed: 06/29/2023]
Abstract
Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).
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Affiliation(s)
- Yifang Liu
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Jacob L. Potts
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Dylan Bloch
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Keqing Nian
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Caroline A. McCormick
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Oleksandra Fanari
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
| | - Sara H. Rouhanifard
- Department of Bioengineering, Northeastern University, Boston, Massachusetts 02115, United States
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13
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Jana AK, Sharawy M, May ER. Non-equilibrium virus particle dynamics: Microsecond MD simulations of the complete Flock House virus capsid under different conditions. J Struct Biol 2023; 215:107964. [PMID: 37105277 PMCID: PMC10205670 DOI: 10.1016/j.jsb.2023.107964] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Revised: 03/22/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023]
Abstract
Flock House virus (FHV) is an animal virus and considered a model system for non-enveloped viruses. It has a small, icosahedral capsid (T=3) and a bipartite positive-sense RNA genome. We present an extensive study of the FHV capsid dynamics from all-atom molecular dynamics simulations of the complete capsid. The simulations explore different biologically relevant conditions (neutral/low pH, with/without RNA in the capsid) using the CHARMM force field. The results show that low pH destabilizes the capsid, causing radial expansion, and RNA stabilizes the capsid. The finding of low pH destabilization is biologically relevant because the capsid is exposed to low pH in the endosome, where conformational changes occur leading to genome release. We also observe structural changes at the fivefold and twofold symmetry axes that likely relate to the externalization of membrane active γ peptides through the fivefold vertex and extrusion of RNA at the twofold axis. Simulations using the Amber force field at neutral pH are also performed and display similar characteristics to the CHARMM simulations.
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Affiliation(s)
- Asis K Jana
- DepartmentofMolecularandCellBiology, UniversityofConnecticut, Storrs, CT06269-3125, USA; Department of Microbiology and Biotechnology, Sister Nivedita University, New Town, West Bengal 700156, India
| | - Mahmoud Sharawy
- DepartmentofMolecularandCellBiology, UniversityofConnecticut, Storrs, CT06269-3125, USA
| | - Eric R May
- DepartmentofMolecularandCellBiology, UniversityofConnecticut, Storrs, CT06269-3125, USA.
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14
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Zheng Y, Yang L, Yu L, Zhu Y, Wu Y, Zhang Z, Xia T, Deng Q. Canocapavir Is a Novel Capsid Assembly Modulator Inducing a Conformational Change of the Linker Region of HBV Core Protein. Viruses 2023; 15:v15051195. [PMID: 37243280 DOI: 10.3390/v15051195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 05/11/2023] [Accepted: 05/15/2023] [Indexed: 05/28/2023] Open
Abstract
Canocapavir is a novel antiviral agent with characteristics of core protein allosteric modulators (CpAMs) that is currently in a phase II clinical trial for treatment of hepatitis B virus (HBV) infection. Herein, we show that Canocapavir prevented the encapsidation of HBV pregenomic RNA and increased the accumulation of cytoplasmic empty capsids, presumably by targeting the hydrophobic pocket at the dimer-dimer interface of HBV core protein (HBc). Canocapavir treatment markedly reduced the egress of naked capsids, which could be reversed by Alix overexpression through a mechanism other than direct association of Alix with HBc. Moreover, Canocapavir interfered with the interaction between HBc and HBV large surface protein, resulting in diminished production of empty virions. Of particular note, Canocapavir induced a conformational change of capsids, with the C-terminus of HBc linker region fully exposed on the exterior of capsids. We posit that the allosteric effect may have great importance in the anti-HBV activity of Canocapavir, given the emerging virological significance of HBc linker region. In support of this notion, the mutation at HBc V124W typically recapitulated the conformational change of the empty capsid with aberrant cytoplasmic accumulation. Collectively, our results indicate Canocapavir as a mechanistically distinct type of CpAMs against HBV infection.
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Affiliation(s)
- Yuan Zheng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai 200032, China
| | - Le Yang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai 200032, China
| | - Lin Yu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai 200032, China
| | - Yuanfei Zhu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai 200032, China
| | - Yang Wu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
| | - Zhijun Zhang
- Shanghai Zhimeng Biopharma, Inc., 1976 Gaoke Middle Road, Suite A-302, Pudong District, Shanghai 201210, China
| | - Tian Xia
- Shanghai Zhimeng Biopharma, Inc., 1976 Gaoke Middle Road, Suite A-302, Pudong District, Shanghai 201210, China
| | - Qiang Deng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai 200032, China
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15
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Shi Y, Jin X, Wu S, Liu J, Zhang H, Cai X, Yang Y, Zhang X, Wei J, Luo M, Zhou H, Zhou H, Huang A, Wang D. Release of hepatitis B virions is positively regulated by glucose-regulated protein 78 through direct interaction with preS1. J Med Virol 2023; 95:e28271. [PMID: 36321566 PMCID: PMC10107996 DOI: 10.1002/jmv.28271] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 07/19/2022] [Accepted: 08/27/2022] [Indexed: 12/04/2022]
Abstract
In this study, we investigated the mechanism of hepatitis B virus (HBV)-enveloped particle release. Specifically, we used preS1 as a bait protein to screen host proteins using mass spectroscopy, with the results of immunofluorescence, western blot, co-immunoprecipitation, isothermal titration calorimetry, and pull-down assays identifying glucose-regulated protein (GRP)78 as a specific target for preS1 binding. We employed transcriptome sequencing, enzyme-linked immunosorbent assays, and particle gel assays to investigate the mechanism of GRP78-mediated positive regulation of HBV-enveloped particle release. Additionally, we performed phage-display, surface plasmon resonance, and molecular-docking assays to assess peptides inhibiting enveloped-particle release. We found that HBV upregulated GRP78 expression in liver cell lines and the serum of patients with chronic hepatitis B. Furthermore, GRP78 promoted the release of HBV-enveloped particles in vitro and in vivo within an HBV transgenic mouse model. Moreover, we identified interactions of preS1 peptides with GRP78 via hydrogen bonding and hydrophobic interactions, which effectively inhibited its interaction with HBV-enveloped particles and their subsequent release. These findings provide novel insights regarding HBV virion release, and demonstrated that GRP78 interacted with preS1 to positively regulate the release of HBV-enveloped particles, suggesting GRP78 as a potential therapeutic target for inhibiting HBV infection.
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Affiliation(s)
- Yueyuan Shi
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China.,College of Laboratory Medicine, Chongqing Medical University, Yuzhong, Chongqing, China.,Department of Clinical Laboratory, The People's Hospital of Yubei District of Chongqing City, Yubei, Chongqing, China
| | - Xin Jin
- College of Laboratory Medicine, Chongqing Medical University, Yuzhong, Chongqing, China.,Department of Clinical Laboratory, The Second Hospital of Harbin, Harbin City, Heilongjiang Province, China
| | - Shuang Wu
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China.,Department of Clinical Laboratory, The Affiliated Children Hospital of Xi'an Jiaotong University, Xi'an City, Shanxi Province, China
| | - Junye Liu
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China.,Department of Clinical Laboratory, Honghui Hospital, Xi'an Jiaotong University, Xi'an City, Shanxi Province, China
| | - Hongpeng Zhang
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China.,Department of Blood Transfusion, Women and Children's Hospital of Chongqing Medical University, Yubei, Chongqing, China
| | - Xuefei Cai
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China
| | - Yuan Yang
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China
| | - Xiang Zhang
- College of Laboratory Medicine, Chongqing Medical University, Yuzhong, Chongqing, China
| | - Jie Wei
- College of Laboratory Medicine, Chongqing Medical University, Yuzhong, Chongqing, China
| | - Miao Luo
- Department of Clinical Laboratory, The People's Hospital of Yubei District of Chongqing City, Yubei, Chongqing, China
| | - Hua Zhou
- Department of Clinical Laboratory, The Second Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing, China
| | - Huihao Zhou
- Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
| | - Ailong Huang
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China
| | - Deqiang Wang
- Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Yuzhong, Chongqing, China.,College of Laboratory Medicine, Chongqing Medical University, Yuzhong, Chongqing, China
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16
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The Origin of Capsid-Derived Immune Complexes and Their Impact on HBV-Induced Liver Diseases. Viruses 2022; 14:v14122766. [PMID: 36560770 PMCID: PMC9785824 DOI: 10.3390/v14122766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 11/28/2022] [Accepted: 12/09/2022] [Indexed: 12/14/2022] Open
Abstract
Over 240 million people worldwide are chronically infected with Hepatitis B Virus (HBV), a hepatotropic DNA virus with an evolutionary root of over 400 million years. Persistent HBV infection exhibits distinct and diverse phases of disease, from minimal liver pathology to fulminant Hepatitis, that vary in duration and severity among individuals. Although huge progress has been made in HBV research which has yielded an effective prophylactic vaccine and potent antiviral therapy, our understanding of its virology and immunobiology is still far from complete. For example, the recent re-discovery of serum HBV RNA in chronic Hepatitis B (CHB) patients has led to the proposal of noncanonical viral particles such as RNA virion and capsid-derived immune complex (Capsid-Antibody-Complexes, CACs) that contradict long-established basic theory. Furthermore, the existence of capsid-derived immune complex may hint at novel mechanism of HBV-induced liver disease. Here, we summarize the past and recent literature on HBV-induced immune complex. We propose that the release of capsid-derived particles by HBV has its deep evolutionary origin, and the associated complement activation serves as an indispensable trigger for intrahepatic damage and a catalyst for further cell-mediated immunopathology.
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17
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Chen Z, Yuan Y, Yang D, Luo M, Liang Q, Li Z, Lu S, Sun J, Deng M, Liu M, Liang Z, Liu K. Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus. J Med Virol 2022; 94:5987-5999. [PMID: 36000452 DOI: 10.1002/jmv.28087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 08/02/2022] [Accepted: 08/19/2022] [Indexed: 01/06/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.
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Affiliation(s)
- Zhuohang Chen
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Yan Yuan
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Di Yang
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Minhui Luo
- School of Public Health, Southern Medical University, Guangzhou, China
| | - Qian Liang
- Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, China
| | - Zan Li
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Siya Lu
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Jianan Sun
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Maohua Deng
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Miaoya Liu
- College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China
| | - Zongsuo Liang
- College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China
| | - Kuancheng Liu
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
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18
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Pronier C, Bomo J, Besombes J, Genet V, Laperche S, Gripon P, Thibault V. Characterization of hepatitis B viral forms from patient plasma using velocity gradient: Evidence for an excess of capsids in fractions enriched in Dane particles. PLoS One 2022; 17:e0272474. [PMCID: PMC9668129 DOI: 10.1371/journal.pone.0272474] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Accepted: 10/30/2022] [Indexed: 11/17/2022] Open
Abstract
Hepatitis B virus (HBV) morphogenesis is characterized by a large over-production of subviral particles and recently described new forms in parallel of complete viral particles (VP). This study was designed to depict circulating viral forms in HBV infected patient plasmas, using velocity gradients and most sensitive viral markers. Plasmas from chronic hepatitis B (CHB) patients, HBeAg positive or negative, genotype D or E, were fractionated on velocity and equilibrium gradients with or without detergent treatment. Antigenic and molecular markers were measured in plasma and in each collected fraction. Fast Nycodenz velocity gradients revealed good reproducibility and provided additional information to standard equilibrium sucrose gradients. HBV-RNAs circulated as enveloped particles in all plasmas, except one, and at lesser concentrations than VP. Calculations based on standardized measurements and relative virion and subviral particle molecular stoichiometry allowed to refine the experimental approach. For the HBeAg-positive plasma, VP were accompanied by an overproduction of enveloped capsids, either containing HBs, likely corresponding to empty virions, or for the main part, devoid of this viral envelope protein. Similarly, in the HBeAg-negative sample, HBs enveloped capsids, likely corresponding to empty virions, were detected and the presence of enveloped capsids devoid of HBs protein was suspected but not clearly evidenced due to the presence of contaminating high-density subviral particles. While HBeAg largely influences HBcrAg measurement and accounts for two-thirds of HBcrAg reactivity in HBeAg-positive patients, it remains a 10 times more sensitive marker than HBsAg to characterize VP containing fractions. Using Nycodenz velocity gradients and standardized biomarkers, our study proposes a detailed characterization of circulating viral forms in chronically HBV infected patients. We provide evidence for an excess of capsids in fractions enriched in Dane particles, likely due to the presence of empty virions but also by capsids enveloped by an HBs free lipid layer. Identification of this new circulating viral particle sets the basis for studies around the potential role of these entities in hepatitis B pathogeny and their physiological regulation.
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Affiliation(s)
- Charlotte Pronier
- Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
| | - Jérémy Bomo
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
| | - Juliette Besombes
- Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
| | - Valentine Genet
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
| | - Syria Laperche
- Department of Blood-Borne Agents, National Reference Center of Infectious Risks in Blood Transfusion, Institut National de la Transfusion Sanguine, Paris, France
- Etablissement Français du Sang, La Plaine-Saint-Denis, France
| | - Philippe Gripon
- Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
| | - Vincent Thibault
- Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) UMR_S 1085, Rennes, France
- * E-mail:
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19
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Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation. J Virol 2022; 96:e0071822. [PMID: 35867543 PMCID: PMC9364803 DOI: 10.1128/jvi.00718-22] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.
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20
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Hong X, Menne S, Hu J. Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes. PLoS Pathog 2022; 18:e1010739. [PMID: 35901192 PMCID: PMC9362955 DOI: 10.1371/journal.ppat.1010739] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Revised: 08/09/2022] [Accepted: 07/13/2022] [Indexed: 11/19/2022] Open
Abstract
Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes. Hepadnaviruses infect a wide range of hosts. The human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) are two closely related hepadnaviruses. In contrast to the WHV precore protein, which is N-glycosylated, the HBV precore protein lacks N-glycosylation. As precore and core proteins expressed from the overlapping precore/core genes share most of their sequences but have distinct functions, we investigated the roles of the N-linked glycosylation sequons in HBV and WHV precore/core genes. Our results revealed an important role of the N-linked glycosylation sequons in enhancing precore secretion levels and regulating core protein functions in virion secretion and infection. Furthermore, cross-species complementation assays using HBV and WHV core proteins and HBV or WHV genomes defective in core protein expression indicated that both HBV and WHV core proteins could support intracellular viral replication but not virion secretion of the heterologous virus. These results provide novel insights into the evolution of overlapping genes during host adaptation of hepadnaviruses.
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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
| | - 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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21
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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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22
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Xu M, Li C, Ding J, Wu M, Tang Y, Yuan Z, Zhang X. The role of hepatitis B virus surface proteins in regulating the maturation and secretion of complete and incomplete virions. J Gen Virol 2022; 103. [PMID: 35438623 DOI: 10.1099/jgv.0.001733] [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
The expression of various forms of hepatitis B virus (HBV) surface proteins regulates the release of mature virion, but whether they affect the release of other incomplete viral particles, such as naked capsid, is not clear. Here, by stable overexpression of large or middle/small hepatitis B surface proteins (LHBs, M/SHBs) in HepAD38 cells, we evaluated their effects on the release of complete and incomplete viral particles. Overproduction of LHBs inhibited the release of all surface proteins, which increased the ratio of naked capsids/virions. This effect was accompanied by the elevated extracellular HBV RNA. On the other hand, overexpression of M/SHBs greatly improved the secretion of enveloped viral and subviral particles. In situ visualization of viral DNA and LHBs revealed intracellular retention of mature virions when LHBs were overexpressed. These results indicate that the molecular decision on secretion of enveloped or unenveloped viral particles is modulated by the intracellular ratio of large, middle and small surface antigens. This mechanism may be relevant in the progression and resolution of HBV-induced chronic liver disease.
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Affiliation(s)
- Mingzhu Xu
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China
| | - Chang Li
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China
| | - Jiahui Ding
- Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, PR China
| | - Min Wu
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China
| | - Yijie Tang
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, PR China
| | - Xiaonan Zhang
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China.,Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, ACT, Australia
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23
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Luo M, Chen Z, Liu M, Liang Q, Han R, Liang Z, Ye Z, Liu K. Inhibitory Activities of Ranunculus japonicus Thunb. Ethanol Extract against Hepatitis B Virus. J Med Virol 2022; 94:2727-2735. [PMID: 35075662 DOI: 10.1002/jmv.27621] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Revised: 01/16/2022] [Accepted: 01/21/2022] [Indexed: 11/08/2022]
Abstract
The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that Ranunculus japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that Ranunculus japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Minhui Luo
- School of Public Health (Shenzhen), Sun Yat-Sen University, Guangzhou, 510006, China
| | - Zhuohang Chen
- School of Public Health, Southern Medical University, Guangzho, 510000, China
| | - Miaoya Liu
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Qian Liang
- Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, 650224, China
| | - Ruilian Han
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Zongsuo Liang
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Zhuoming Ye
- School of Public Health, Southern Medical University, Guangzho, 510000, China
| | - Kuancheng Liu
- School of Public Health (Shenzhen), Sun Yat-Sen University, Guangzhou, 510006, China.,Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China
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24
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Chang CH, Chou SF, Shih C. A nuanced role of the small loop of hepatitis B virus small envelope protein in virion morphogenesis and secretion. J Biomed Sci 2021; 28:82. [PMID: 34852809 PMCID: PMC8638367 DOI: 10.1186/s12929-021-00780-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2021] [Accepted: 11/23/2021] [Indexed: 01/16/2023] Open
Abstract
BACKGROUND The virion secretion mechanism of human hepatitis B virus (HBV) remains to be investigated. In our current study, we characterized a reverse transcriptase mutant, which changed from the YMDD motif to YMHA. We noted that this mutant YMHA secreted no virions in the medium. Because of the overlapping open reading frame (ORF) between the polymerase and the envelope genes, the lack of virion secretion is likely due to corresponding concurrent mutations in a small loop of the envelope protein (HBsAg, HBV surface antigen). In literature, small loop mutations are thought to affect virion secretion of hepatitis delta virus (HDV), but not HBV. METHODS Here, we revisited the relationship between the small loop and virion secretion by site-directed mutagenesis and native agarose gel electrophoresis. RESULTS A proline substitution at residue 196 or 198 in the small loop blocked both HBV genome-containing and genome-free virion secretion, but not the secretion of 22-nm HBsAg subviral particles. Surprisingly, a leucine substitution at residue 196 enhanced genome-containing virion secretion. It is also intriguing that a proline-197, sandwiched by residue 196 and 198, exhibited no apparent defect in secreted virions, with or without containing an HBV genome. By complementation assay, we demonstrated that the wild type small envelope protein alone is sufficient to rescue the virion secretion defect of a small loop mutant M198P. CONCLUSIONS The effect of the small loop mutation of HBV small envelope protein on virion secretion is position-dependent. It warrants further investigation how the small loop of HBsAg plays a subtle role in HBV morphogenesis and secretion of virions with or without containing an HBV genome.
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Affiliation(s)
- Chih-Hsu Chang
- Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Shu-Fan Chou
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Chiaho Shih
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. .,Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
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25
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Liu H, Cheng J, Viswanathan U, Chang J, Lu F, Guo JT. Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis. PLoS Pathog 2021; 17:e1010057. [PMID: 34752483 PMCID: PMC8604296 DOI: 10.1371/journal.ppat.1010057] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Revised: 11/19/2021] [Accepted: 10/22/2021] [Indexed: 12/19/2022] Open
Abstract
The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.
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Affiliation(s)
- Hui Liu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Junjun Cheng
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Usha Viswanathan
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Jinhong Chang
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Fengmin Lu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- * E-mail: (FL); (J-TG)
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
- * E-mail: (FL); (J-TG)
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26
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Niklasch M, Zimmermann P, Nassal M. The Hepatitis B Virus Nucleocapsid-Dynamic Compartment for Infectious Virus Production and New Antiviral Target. Biomedicines 2021; 9:1577. [PMID: 34829806 PMCID: PMC8615760 DOI: 10.3390/biomedicines9111577] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2021] [Revised: 10/19/2021] [Accepted: 10/21/2021] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) is a small enveloped DNA virus which replicates its tiny 3.2 kb genome by reverse transcription inside an icosahedral nucleocapsid, formed by a single ~180 amino acid capsid, or core, protein (Cp). HBV causes chronic hepatitis B (CHB), a severe liver disease responsible for nearly a million deaths each year. Most of HBV's only seven primary gene products are multifunctional. Though less obvious than for the multi-domain polymerase, P protein, this is equally crucial for Cp with its multiple roles in the viral life-cycle. Cp provides a stable genome container during extracellular phases, allows for directed intracellular genome transport and timely release from the capsid, and subsequent assembly of new nucleocapsids around P protein and the pregenomic (pg) RNA, forming a distinct compartment for reverse transcription. These opposing features are enabled by dynamic post-transcriptional modifications of Cp which result in dynamic structural alterations. Their perturbation by capsid assembly modulators (CAMs) is a promising new antiviral concept. CAMs inappropriately accelerate assembly and/or distort the capsid shell. We summarize the functional, biochemical, and structural dynamics of Cp, and discuss the therapeutic potential of CAMs based on clinical data. Presently, CAMs appear as a valuable addition but not a substitute for existing therapies. However, as part of rational combination therapies CAMs may bring the ambitious goal of a cure for CHB closer to reality.
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Affiliation(s)
| | | | - Michael Nassal
- Internal Medicine II/Molecular Biology, University Hospital Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany; (M.N.); (P.Z.)
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27
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Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97. Viruses 2021; 13:v13112115. [PMID: 34834922 PMCID: PMC8618838 DOI: 10.3390/v13112115] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Revised: 10/13/2021] [Accepted: 10/15/2021] [Indexed: 12/26/2022] Open
Abstract
(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.
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28
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Regulation of Hepatitis B Virus Virion Release and Envelopment Timing by Nucleocapsid and Envelope Interactions. J Virol 2021; 96:e0130521. [PMID: 34643434 DOI: 10.1128/jvi.01305-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Interactions between the N-terminal (assembly) domain (NTD) and the linker region of the hepatitis B virus (HBV) capsid protein and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulates covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA was further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment, and highlights the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. Importance Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases including cancer. The interactions between the HBV capsid and the large (L) envelope protein is required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.
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Inoue T, Yagi S, Tanaka Y. Reply to: "Understanding HBcrAg components improves the interpretation of clinical HBcrAg assay results". J Hepatol 2021; 75:998-999. [PMID: 34246705 DOI: 10.1016/j.jhep.2021.06.047] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 06/24/2021] [Indexed: 02/01/2023]
Affiliation(s)
- Takako Inoue
- Department of Clinical Laboratory Medicine, Nagoya City University Hospital, Nagoya, Japan
| | - Shintaro Yagi
- Research and Development Department, Advanced Life Science Institute, Inc., Hachioji, Tokyo, Japan
| | - Yasuhito Tanaka
- Department of Clinical Laboratory Medicine, Nagoya City University Hospital, Nagoya, Japan; Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
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Philips CA, Ahamed R, Abduljaleel JK, Rajesh S, Augustine P. Critical Updates on Chronic Hepatitis B Virus Infection in 2021. Cureus 2021; 13:e19152. [PMID: 34733599 PMCID: PMC8557099 DOI: 10.7759/cureus.19152] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/30/2021] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection is a global healthcare burden in the form of chronic liver disease, cirrhosis, liver failure and liver cancer. There is no definite cure for the virus and even though extensive vaccination programs have reduced the burden of liver disease in the future population, treatment options to eradicate the virus from the host are still lacking. In this review, we discuss in detail current updates on the structure and applied biology of the virus in the host, examine updates to current treatment and explore novel and state-of-the-art therapeutics in the pipeline for management of chronic HBV. Furthermore, we also specifically review clinical updates on HBV-related acute on chronic liver failure (ACLF). Current treatments for chronic HBV infection have seen important updates in the form of considerations for treating patients in the immune tolerant phase and some clarity on end points for treatment and decisions on finite therapy with nucleos(t)ide inhibitors. Ongoing cutting-edge research on HBV biology has helped us identify novel target areas in the life cycle of the virus for application of new therapeutics. Due to improvements in the area of genomics, the hope for therapeutic vaccines, vector-based treatments and focused management aimed at targeting host integration of the virus and thereby a total cure could become a reality in the near future. Newer clinical prognostic tools have improved our understanding of timing of specific treatment options for the catastrophic syndrome of ACLF secondary to reactivation of HBV. In this review, we discuss in detail pertinent updates regarding virus biology and novel therapeutic targets with special focus on the appraisal of prognostic scores and treatment options in HBV-related ACLF.
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Affiliation(s)
- Cyriac A Philips
- Clinical and Translational Hepatology, The Liver Institute, Rajagiri Hospital, Aluva, IND
| | - Rizwan Ahamed
- Gastroenterology and Advanced Gastrointestinal Endoscopy, Center of Excellence in Gastrointestinal Sciences, Rajagiri Hospital, Aluva, IND
| | - Jinsha K Abduljaleel
- Gastroenterology and Advanced Gastrointestinal Endoscopy, Center of Excellence in Gastrointestinal Sciences, Rajagiri Hospital, Aluva, IND
| | - Sasidharan Rajesh
- Diagnostic and Interventional Radiology, Center of Excellence in Gastrointestinal Sciences, Rajagiri Hospital, Aluva, IND
| | - Philip Augustine
- Gastroenterology and Advanced Gastrointestinal Endoscopy, Center of Excellence in Gastrointestinal Sciences, Rajagiri Hospital, Aluva, IND
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Hong X, Hu J. Understanding HBcrAg components improves the interpretation of clinical HBcrAg assay results. J Hepatol 2021; 75:997-998. [PMID: 33964371 DOI: 10.1016/j.jhep.2021.04.041] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Accepted: 04/27/2021] [Indexed: 01/18/2023]
Affiliation(s)
- Xupeng Hong
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
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Inoue T, Kusumoto S, Iio E, Ogawa S, Suzuki T, Yagi S, Kaneko A, Matsuura K, Aoyagi K, Tanaka Y. Clinical efficacy of a novel, high-sensitivity HBcrAg assay in the management of chronic hepatitis B and HBV reactivation. J Hepatol 2021; 75:302-310. [PMID: 33762167 DOI: 10.1016/j.jhep.2021.02.017] [Citation(s) in RCA: 80] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 01/22/2021] [Accepted: 02/15/2021] [Indexed: 01/14/2023]
Abstract
BACKGROUND & AIMS A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. We demonstrate the clinical utility of iTACT-HBcrAg for monitoring chronic hepatitis B (CHB) and for the early detection of HBV reactivation. METHODS After fundamental assessments, the clinical performance of iTACT-HBcrAg was compared with other HBV markers. i) Serial sera, available from 161 HBeAg-negative patients with CHB and persistently undetectable HBV DNA, were measured by iTACT-HBcrAg and a conventional HBcrAg assay (G-HBcrAg). ii) Serial sera from 13 HBV-reactivated patients were measured by iTACT-HBcrAg and an ultra-high-sensitivity HBsAg immune complex transfer-chemiluminescent enzyme immunoassay (lower limit of detection; 0.0005 IU/ml, ICT-CLEIA) to compare HBV DNA detection. iii) To elucidate the various HBcrAg components detected by iTACT-HBcrAg, OptiPrep density gradient centrifugation analysis was performed on sera obtained before and after HBV reactivation. RESULTS The analytical performance of iTACT-HBcrAg was satisfactory. The sensitivity of iTACT-HBcrAg (2.1 Log U/ml) was approximately 10-fold greater than that of G-HBcrAg (2.8 Log U/ml). i) HBcrAg was detectable in the sera of 97.5% (157/161) of patients with CHB by iTACT-HBcrAg, of whom 75.2% (121/161) had ≥2.8 Log U/ml HBcrAg and 22.4% (36/161) had 2.1-2.8 Log U/ml HBcrAg, which was undetectable by G-HBcrAg. ii) 9 and 2 of 13 HBV-reactivated patients were HBcrAg-positive by iTACT-HBcrAg before and at HBV DNA positivity, respectively; 7 and 4 were HBcrAg-positive by iTACT-HBcrAg before and at HBsAg-positivity by ICT-CLEIA, respectively. iii) The HBcrAg detected by iTACT-HBcrAg before HBV reactivation was contained in empty particles (22 KDa precore protein). CONCLUSIONS iTACT-HBcrAg could be used to better monitor responses to anti-HBV treatments in HBeAg-negative patients and for the early detection of HBV reactivation. LAY SUMMARY A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. iTACT-HBcrAg can be used to monitor HBeAg-negative patients with chronic hepatitis B, as well as for the early detection of HBV reactivation. iTACT-HBcrAg could be used as a general marker of disease progression and treatment response.
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Affiliation(s)
- Takako Inoue
- Department of Clinical Laboratory Medicine, Nagoya City University Hospital, Nagoya, Japan
| | - Shigeru Kusumoto
- Department of Hematology and Oncology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Etsuko Iio
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Shintaro Ogawa
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Takanori Suzuki
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Shintaro Yagi
- Research and Development Department, Advanced Life Science Institute, Inc., Hachioji, Tokyo, Japan
| | - Atsushi Kaneko
- Research and Development Division, Fujirebio Inc., Hachioji, Tokyo, Japan
| | - Kentaro Matsuura
- Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
| | - Katsumi Aoyagi
- Research and Development Department, Advanced Life Science Institute, Inc., Hachioji, Tokyo, Japan; Research and Development Division, Fujirebio Inc., Hachioji, Tokyo, Japan
| | - Yasuhito Tanaka
- Department of Clinical Laboratory Medicine, Nagoya City University Hospital, Nagoya, Japan; Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Department of Gastroenterology and Hepatology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
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Chen J, Liu B, Tang X, Zheng X, Lu J, Zhang L, Wang W, Candotti D, Fu Y, Allain JP, Li C, Li L, Li T. Role of core protein mutations in the development of occult HBV infection. J Hepatol 2021; 74:1303-1314. [PMID: 33453326 DOI: 10.1016/j.jhep.2020.12.023] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 11/04/2020] [Accepted: 12/14/2020] [Indexed: 02/05/2023]
Abstract
BACKGROUND & AIMS Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.
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Affiliation(s)
- Jingna Chen
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China; Department of Laboratory Medicine, General Hospital of Southern Theatre Command of PLA, Guangzhou, China; Department of Clinical Laboratory, The First Affiliated Hospital of Shantou University Medical College, Shantou, China
| | - Bochao Liu
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China
| | - Xi Tang
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China; Department of Infectious Diseases, The First Foshan People's Hospital, Foshan, China
| | - Xin Zheng
- Shenzhen Blood Center, Shenzhen, China
| | - Jinhui Lu
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China
| | - Ling Zhang
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China
| | - Wenjing Wang
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China
| | - Daniel Candotti
- Department of Blood Transmitted Agents, National Institute of Blood Transfusion, Paris, France
| | - Yongshui Fu
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China; Guangzhou Blood Center, Guangzhou, China
| | - Jean-Pierre Allain
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China; Department of Haematology, University of Cambridge, Cambridge, UK
| | - Chengyao Li
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
| | - Linhai Li
- Department of Laboratory Medicine, General Hospital of Southern Theatre Command of PLA, Guangzhou, China.
| | - Tingting Li
- Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
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Regulation of Hepatitis B Virus Replication by Cyclin Docking Motifs in Core Protein. J Virol 2021; 95:JVI.00230-21. [PMID: 33789995 DOI: 10.1128/jvi.00230-21] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Accepted: 03/27/2021] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and, furthermore, is incorporated into the viral capsid, accounting for most of the "endogenous kinase" activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs in the HBc NTD known as cyclin docking motifs (CDMs), which mediate the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection.IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.
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Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype. Microorganisms 2021; 9:microorganisms9050956. [PMID: 33946808 PMCID: PMC8145704 DOI: 10.3390/microorganisms9050956] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2021] [Revised: 04/22/2021] [Accepted: 04/26/2021] [Indexed: 12/31/2022] Open
Abstract
Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.
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Abstract
Viral hepatitis causes more deaths than tuberculosis and HIV-1 infection. Most cases are due to chronic infection with hepatitis B virus (HBV), which afflicts >250 million people. Current therapies are rarely curative, and new approaches are needed. Here, we report the discovery (by nuclear magnetic resonance) of a small molecule binder in the hydrophobic pocket in the HBV capsid. This structural element is, in an unknown manner, central in capsid envelopment. Binding of the pocket factor induces a distinct, stable conformation in the capsid, as expected for a signaling switch. This brings not only a new molecular view on the mechanism underlying capsid envelopment, but it also opens a rationale for its inhibition. Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid–envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the “synergistic double interaction” hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.
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In vitro expression of precore proteins of hepatitis B virus subgenotype A1 is affected by HBcAg, and can affect HBsAg secretion. Sci Rep 2021; 11:8167. [PMID: 33854155 PMCID: PMC8046783 DOI: 10.1038/s41598-021-87529-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Accepted: 03/30/2021] [Indexed: 12/16/2022] Open
Abstract
HBeAg, a non-particulate protein of hepatitis B virus (HBV), is translated from the precore/core region as a precursor, which is post-translationally modified. Subgenotype A1 of HBV, which is a risk factor for hepatocellular carcinoma (HCC), has unique molecular characteristics in the basic core promoter/precore regions. Carriers of A1 exhibit early HBeAg loss. We sought to further characterize the precore proteins of A1 in vitro. HuH-7 cells were transfected with subgenomic constructs expressing individual precore proteins. Western blot analysis using DAKO anti-core antibody showed the expected sizes and a 1 kDa larger band for P22, P20 and P17. Using confocal microscopy, a cytoplasmic accumulation of HBeAg and precursors was observed with P25-expressing plasmid, whereas P22 localized both in the cytoplasm and nucleus. P20 and P17, which lack the carboxy end of P22 showed strong nuclear accumulation, implicating a nuclear localization signal in the N-terminal 10 amino acids. G1862T, unique to subgenotype A1, is frequently found in HBV from HCC patients. P25 with G1862T showed delayed and reduced HBeAg expression/secretion. Knock-out of core in the replication competent clones led to precore protein accumulation in the cytoplasm/perinuclear region, and decreased HBeAg secretion. Knock-out of precore proteins increased HBsAg secretion but intracellular HBsAg expression was unaffected. Over-expression of precore proteins in trans led to decreased HBsAg expression and secretion. Intracellular trafficking of HBV A1 precore proteins was followed. This was unaffected by the CMV promoter and different cell types. In the viral context, precore protein expression was affected by absence of core, and affected HBsAg expression, suggesting an interrelationship between precore proteins, HBcAg and HBsAg. This modulatory role of HBeAg and its precursors may be important in viral persistence and ultimate development of HCC.
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Hong X, Luckenbaugh L, Mendenhall M, Walsh R, Cabuang L, Soppe S, Revill PA, Burdette D, Feierbach B, Delaney W, Hu J. Characterization of Hepatitis B Precore/Core-Related Antigens. J Virol 2021; 95:JVI.01695-20. [PMID: 33148795 PMCID: PMC7925093 DOI: 10.1128/jvi.01695-20] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Accepted: 10/27/2020] [Indexed: 02/07/2023] Open
Abstract
Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities.IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.
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Affiliation(s)
- Xupeng Hong
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Laurie Luckenbaugh
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Megan Mendenhall
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Renae Walsh
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Liza Cabuang
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Sally Soppe
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Peter A Revill
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | | | | | | | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
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Sun H, Chang L, Yan Y, Wang L. Hepatitis B virus pre-S region: Clinical implications and applications. Rev Med Virol 2020; 31. [PMID: 33314434 DOI: 10.1002/rmv.2201] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 11/22/2020] [Accepted: 11/29/2020] [Indexed: 12/12/2022]
Abstract
Hepatitis B virus (HBV) infection is a major threat to global public health, which can result in many acute and chronic liver diseases. HBV, a member of the family Hepadnaviridae, is a small enveloped DNA virus containing a circular genome of 3.2 kb. Located upstream of the S-open-reading frame of the HBV genome is the pre-S region, which is vital to the viral life cycle. The pre-S region has high variability and many mutations in the pre-S region are associated with several liver diseases, such as fulminant hepatitis (FH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). In addition, the pre-S region has been applied in the development of several pre-S-based materials and systems to prevent or treat HBV infection. In conclusion, the pre-S region plays an essential role in the occurrence, diagnosis, and treatment of HBV-related liver diseases, which may provide a novel perspective for the study of HBV infection and relevant diseases.
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Affiliation(s)
- Huizhen Sun
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
- Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, PR China
| | - Le Chang
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
| | - Ying Yan
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
| | - Lunan Wang
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, PR China
- Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China
- Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, PR China
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40
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Glebe D, Goldmann N, Lauber C, Seitz S. HBV evolution and genetic variability: Impact on prevention, treatment and development of antivirals. Antiviral Res 2020; 186:104973. [PMID: 33166575 DOI: 10.1016/j.antiviral.2020.104973] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 10/31/2020] [Accepted: 11/02/2020] [Indexed: 12/15/2022]
Abstract
Hepatitis B virus (HBV) poses a major global health burden with 260 million people being chronically infected and 890,000 dying annually from complications in the course of the infection. HBV is a small enveloped virus with a reverse-transcribed DNA genome that infects hepatocytes and can cause acute and chronic infections of the liver. HBV is endemic in humans and apes representing the prototype member of the viral family Hepadnaviridae and can be divided into 10 genotypes. Hepadnaviruses have been found in all vertebrate classes and constitute an ancient viral family that descended from non-enveloped progenitors more than 360 million years ago. The de novo emergence of the envelope protein gene was accompanied with the liver-tropism and resulted in a tight virus-host association. The oldest HBV genomes so far have been isolated from human remains of the Bronze Age and the Neolithic (~7000 years before present). Despite the remarkable stability of the hepadnaviral genome over geological eras, HBV is able to rapidly evolve within an infected individual under pressure of the immune response or during antiviral treatment. Treatment with currently available antivirals blocking intracellular replication of HBV allows controlling of high viremia and improving liver health during long-term therapy of patients with chronic hepatitis B (CHB), but they are not sufficient to cure the disease. New therapy options that cover all HBV genotypes and emerging viral variants will have to be developed soon. In addition to the antiviral treatment of chronically infected patients, continued efforts to expand the global coverage of the currently available HBV vaccine will be one of the key factors for controlling the rising global spread of HBV. Certain improvements of the vaccine (e.g. inclusion of PreS domains) could counteract known problems such as low or no responsiveness of certain risk groups and waning anti-HBs titers leading to occult infections, especially with HBV genotypes E or F. But even with an optimal vaccine and a cure for hepatitis B, global eradication of HBV would be difficult to achieve because of an existing viral reservoir in primates and bats carrying closely related hepadnaviruses with zoonotic potential.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus Liebig University of Giessen, National Reference Centre for Hepatitis B Viruses and Hepatitis D Viruses, Schubertstr. 81, 35392, Giessen, Germany; German Center for Infection Research (DZIF), Partner Sites Giessen, Heidelberg, Hannover, Germany.
| | - Nora Goldmann
- Institute of Medical Virology, Justus Liebig University of Giessen, National Reference Centre for Hepatitis B Viruses and Hepatitis D Viruses, Schubertstr. 81, 35392, Giessen, Germany
| | - Chris Lauber
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany; Research Group Computational Virology, Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture Between the Helmholtz Centre for Infection Research and the Hannover Medical School, Cluster of Excellence RESIST, Hannover Medical School, 30625, Hannover, Germany; German Center for Infection Research (DZIF), Partner Sites Giessen, Heidelberg, Hannover, Germany
| | - Stefan Seitz
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany; Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120, Heidelberg, Germany; German Center for Infection Research (DZIF), Partner Sites Giessen, Heidelberg, Hannover, Germany.
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41
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Viswanathan U, Mani N, Hu Z, Ban H, Du Y, Hu J, Chang J, Guo JT. Targeting the multifunctional HBV core protein as a potential cure for chronic hepatitis B. Antiviral Res 2020; 182:104917. [PMID: 32818519 DOI: 10.1016/j.antiviral.2020.104917] [Citation(s) in RCA: 74] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2020] [Revised: 08/08/2020] [Accepted: 08/10/2020] [Indexed: 12/14/2022]
Abstract
The core (capsid) protein of hepatitis B virus (HBV) is the building block of nucleocapsids where viral DNA reverse transcriptional replication takes place and mediates virus-host cell interaction important for the persistence of HBV infection. The pleiotropic role of core protein (Cp) in HBV replication makes it an attractive target for antiviral therapies of chronic hepatitis B, a disease that affects more than 257 million people worldwide without a cure. Recent clinical studies indicate that core protein allosteric modulators (CpAMs) have a great promise as a key component of hepatitis B curative therapies. Particularly, it has been demonstrated that modulation of Cp dimer-dimer interactions by several chemical series of CpAMs not only inhibit nucleocapsid assembly and viral DNA replication, but also induce the disassembly of double-stranded DNA-containing nucleocapsids to prevent the synthesis of cccDNA. Moreover, the different chemotypes of CpAMs modulate Cp assembly by interaction with distinct amino acid residues at the HAP pocket between Cp dimer-dimer interfaces, which results in the assembly of Cp dimers into either non-capsid Cp polymers (type I CpAMs) or empty capsids with distinct physical property (type II CpAMs). The different CpAMs also differentially modulate Cp metabolism and subcellular distribution, which may impact cccDNA metabolism and host antiviral immune responses, the critical factors for the cure of chronic HBV infection. This review article highlights the recent research progress on the structure and function of core protein in HBV replication cycle, the mode of action of CpAMs, as well as the current status and perspectives on the discovery and development of core protein-targeting antivirals. This article forms part of a symposium in Antiviral Research on "Wide-ranging immune and direct-acting antiviral approaches to curing HBV and HDV infections."
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Affiliation(s)
- Usha Viswanathan
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Nagraj Mani
- Arbutus Biopharma Inc., 701 Veterans Circle, Warminster, PA, 18974, USA
| | - Zhanying Hu
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Haiqun Ban
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Yanming Du
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Jin Hu
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Jinhong Chang
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, PA, 18902, USA.
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Jiang B, Wen X, Wu Q, Bender D, Carra G, Basic M, Kubesch A, Peiffer KH, Boller K, Hildt E. The N-Terminus Makes the Difference: Impact of Genotype-Specific Disparities in the N-Terminal Part of The Hepatitis B Virus Large Surface Protein on Morphogenesis of Viral and Subviral Particles. Cells 2020; 9:cells9081898. [PMID: 32823751 PMCID: PMC7463600 DOI: 10.3390/cells9081898] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 07/27/2020] [Accepted: 08/11/2020] [Indexed: 12/12/2022] Open
Abstract
The N-terminus of the hepatitis B virus (HBV) large surface protein (LHB) differs with respect to genotypes. Compared to the amino terminus of genotype (Gt)D, in GtA, GtB and GtC, an additional identical 11 amino acids (aa) are found, while GtE and GtG share another similar 10 aa. Variants of GtB and GtC affecting this N-terminal part are associated with hepatoma formation. Deletion of these amino-terminal 11 aa in GtA reduces the amount of LHBs and changes subcellular accumulation (GtA-like pattern) to a dispersed distribution (GtD-like pattern). Vice versa, the fusion of the GtA-derived N-terminal 11 aa to GtD causes a GtA-like phenotype. However, insertion of the corresponding GtE-derived 10 aa to GtD has no effect. Deletion of these 11aa decreases filament size while neither the number of released viral genomes nor virion size and infectivity are affected. A negative regulatory element (aa 2–8) and a dominant positive regulatory element (aa 9–11) affecting the amount of LHBs were identified. The fusion of this motif to eGFP revealed that the effect on protein amount and subcellular distribution is not restricted to LHBs. These data identify a novel region in the N-terminus of LHBs affecting the amount and subcellular distribution of LHBs and identify release-promoting and -inhibiting aa residues within this motive.
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Affiliation(s)
- Bingfu Jiang
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
| | - Xingjian Wen
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
| | - Qingyan Wu
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
| | - Daniela Bender
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
| | - Gert Carra
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
| | - Michael Basic
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
- Department of Gastroenterology and Hepatology, J. W. Goethe University, D-60590 Frankfurt, Germany
| | - Alica Kubesch
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
- Department of Gastroenterology and Hepatology, J. W. Goethe University, D-60590 Frankfurt, Germany
| | - Kai-Henrik Peiffer
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
- Department of Gastroenterology and Hepatology, J. W. Goethe University, D-60590 Frankfurt, Germany
| | - Klaus Boller
- Department of Immunology, Paul-Ehrlich-Institut, D-63225 Langen, Germany;
| | - Eberhard Hildt
- Division of Virology, Paul-Ehrlich-Institut, D-63225 Langen, Germany; (B.J.); (X.W.); (Q.W.); (D.B.); (G.C.); (M.B.); (A.K.); (K.-H.P.)
- TTU Hepatitis, German Center for Infection Research (DZIF), 38124 Braunschweig, Germany
- Correspondence: ; Tel.: +496103772140
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Hepatitis B Virus Exploits ERGIC-53 in Conjunction with COPII to Exit Cells. Cells 2020; 9:cells9081889. [PMID: 32806600 PMCID: PMC7464876 DOI: 10.3390/cells9081889] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 08/05/2020] [Accepted: 08/10/2020] [Indexed: 12/20/2022] Open
Abstract
Several decades after its discovery, the hepatitis B virus (HBV) still displays one of the most successful pathogens in human populations worldwide. The identification and characterization of interactions between cellular and pathogenic components are essential for the development of antiviral treatments. Due to its small-sized genome, HBV highly depends on cellular functions to produce and export progeny particles. Deploying biochemical-silencing methods and molecular interaction studies in HBV-expressing liver cells, we herein identified the cellular ERGIC-53, a high-mannose-specific lectin, and distinct components of the endoplasmic reticulum (ER) export machinery COPII as crucial factors of viral trafficking and egress. Whereas the COPII subunits Sec24A, Sec23B and Sar1 are needed for both viral and subviral HBV particle exit, ERGIC-53 appears as an exclusive element of viral particle propagation, therefore interacting with the N146-glycan of the HBV envelope in a productive manner. Cell-imaging studies pointed to ER-derived, subcellular compartments where HBV assembly initiates. Moreover, our findings provide evidence that HBV exploits the functions of ERGIC-53 and Sec24A after the envelopment of nucleocapsids at these compartments in conjunction with endosomal sorting complexes required for transport (ESCRT) components. These data reveal novel insights into HBV assembly and trafficking, illustrating therapeutic prospects for intervening with the viral life cycle.
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Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid. Viruses 2020; 12:v12070738. [PMID: 32650547 PMCID: PMC7412469 DOI: 10.3390/v12070738] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 07/02/2020] [Accepted: 07/03/2020] [Indexed: 02/08/2023] Open
Abstract
The morphogenesis of Hepatitis B Virus (HBV) viral particles is nucleated by the oligomerization of HBc protein molecules, resulting in the formation of an icosahedral capsid shell containing the replication-competent nucleoprotein complex made of the viral polymerase and the pre-genomic RNA (pgRNA). HBc is a phospho-protein containing two distinct domains acting together throughout the viral replication cycle. The N-terminal domain, (residues 1–140), shown to self-assemble, is linked by a short flexible domain to the basic C-terminal domain (residues 150–183) that interacts with nucleic acids (NAs). In addition, the C-terminal domain contains a series of phospho-acceptor residues that undergo partial phosphorylation and de-phosphorylation during virus replication. This highly dynamic process governs the homeostatic charge that is essential for capsid stability, pgRNA packaging and to expose the C-terminal domain at the surface of the particles for cell trafficking. In this review, we discuss the roles of the N-terminal and C-terminal domains of HBc protein during HBV morphogenesis, focusing on how the C-terminal domain phosphorylation dynamics regulate its interaction with nucleic acids throughout the assembly and maturation of HBV particles.
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Seitz S, Habjanič J, Schütz AK, Bartenschlager R. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. Annu Rev Virol 2020; 7:263-288. [PMID: 32600157 DOI: 10.1146/annurev-virology-092818-015508] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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Affiliation(s)
- Stefan Seitz
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany;
| | - Jelena Habjanič
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Anne K Schütz
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany; .,Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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Yll M, Cortese MF, Guerrero-Murillo M, Orriols G, Gregori J, Casillas R, González C, Sopena S, Godoy C, Vila M, Tabernero D, Quer J, Rando A, Lopez-Martinez R, Esteban R, Riveiro-Barciela M, Buti M, Rodríguez-Frías F. Conservation and variability of hepatitis B core at different chronic hepatitis stages. World J Gastroenterol 2020; 26:2584-2598. [PMID: 32523313 PMCID: PMC7265140 DOI: 10.3748/wjg.v26.i20.2584] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2020] [Revised: 05/08/2020] [Accepted: 05/19/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Since it is currently not possible to eradicate hepatitis B virus (HBV) infection with existing treatments, research continues to uncover new therapeutic strategies. HBV core protein, encoded by the HBV core gene (HBC), intervenes in both structural and functional processes, and is a key protein in the HBV life cycle. For this reason, both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies. Moreover, alterations in the protein sequence could serve as potential markers of disease progression. AIM To detect, by next-generation sequencing, HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies. METHODS Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage [chronic hepatitis B infection without liver damage (CHB, n = 16), liver cirrhosis (LC, n = 5), and hepatocellular carcinoma (HCC, n = 17)]. HBV DNA was extracted from patients' plasma. A region between nucleotide (nt) 1863 and 2483, which includes HBC, was amplified and analyzed by next-generation sequencing (Illumina MiSeq platform). Sequences were genotyped by distance-based discriminant analysis. General and intergroup nt and amino acid (aa) conservation was determined by sliding window analysis. The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences. RESULTS Three nt (nt 1900-1929, 2249-2284, 2364-2398) and 2 aa (aa 117-120, 159-167) hyper-conserved regions were shared by all the clinical groups. All groups showed a similar pattern of conservation, except for five nt regions (nt 1946-1992, 2060-2095, 2145-2175, 2230-2250, 2270-2293) and one aa region (aa 140-160), where CHB and LC, respectively, were less conserved (P < 0.05). Some group-specific conserved regions were also observed at both nt (2306-2334 in CHB and 1935-1976 and 2402-2435 in LC) and aa (between aa 98-103 in CHB and 28-30 and 51-54 in LC) levels. No differences in insertion and deletions frequencies were observed. An aa substitution (P79Q) was observed in the HCC group with a median (interquartile range) frequency of 15.82 (0-78.88) vs 0 (0-0) in the other groups (P < 0.05 vs CHB group). CONCLUSION The differentially conserved HBC and HBV core protein regions and the P79Q substitution could be involved in disease progression. The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies.
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MESH Headings
- Adult
- Aged
- Base Sequence/genetics
- Biomarkers
- Carcinoma, Hepatocellular/blood
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/virology
- Conserved Sequence/genetics
- DNA, Viral/blood
- DNA, Viral/genetics
- DNA, Viral/isolation & purification
- Disease Progression
- Female
- Genes, Viral/genetics
- Hepatitis B virus/genetics
- Hepatitis B virus/isolation & purification
- Hepatitis B, Chronic/blood
- Hepatitis B, Chronic/diagnosis
- Hepatitis B, Chronic/therapy
- Hepatitis B, Chronic/virology
- Humans
- Liver Cirrhosis/blood
- Liver Cirrhosis/pathology
- Liver Cirrhosis/virology
- Liver Neoplasms/blood
- Liver Neoplasms/pathology
- Liver Neoplasms/virology
- Male
- Middle Aged
- Prognosis
- Sequence Analysis, DNA
- Viral Core Proteins/genetics
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Affiliation(s)
- Marçal Yll
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Maria Francesca Cortese
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Mercedes Guerrero-Murillo
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Department of Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Gerard Orriols
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Josep Gregori
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Rosario Casillas
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Carolina González
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Sara Sopena
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Cristina Godoy
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Marta Vila
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - David Tabernero
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Josep Quer
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Ariadna Rando
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Rosa Lopez-Martinez
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Rafael Esteban
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autónoma de Barcelona, Barcelona 08035, Spain
| | - Mar Riveiro-Barciela
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autónoma de Barcelona, Barcelona 08035, Spain
| | - Maria Buti
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autónoma de Barcelona, Barcelona 08035, Spain
| | - Francisco Rodríguez-Frías
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Madrid 28029, Spain
- Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
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Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context. J Mol Biol 2020; 432:3802-3819. [PMID: 32371046 DOI: 10.1016/j.jmb.2020.04.026] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 04/25/2020] [Accepted: 04/28/2020] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.
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Liu Y, Wang H, Zhang J, Yang J, Bai L, Zheng B, Zheng T, Wang Y, Li J, Zhang W. SERINC5 Inhibits the Secretion of Complete and Genome-Free Hepatitis B Virions Through Interfering With the Glycosylation of the HBV Envelope. Front Microbiol 2020; 11:697. [PMID: 32431673 PMCID: PMC7216740 DOI: 10.3389/fmicb.2020.00697] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Accepted: 03/25/2020] [Indexed: 12/22/2022] Open
Abstract
Serine incorporator 3 (SERINC3) and SERINC5 were recently identified as host intrinsic factors against human immunodeficiency virus (HIV)-1 and counteracted by HIV-1 Nef. However, whether they inhibit hepatitis B virus (HBV), which is a severe health problem worldwide, is unknown. Here, we demonstrate that SERINC5 potently inhibited HBV virion secretion in the supernatant without affecting intracellular core particle-associated DNA and the total RNA, but SERINC3 and SERINC1 did not. Further investigation discovered that SERINC5 increased the non-glycosylation of LHB, MHB, and SHB proteins of HBV and slightly decreased HBs proteins levels, which led to the decreased HBV secretion. Importantly, SERINC5 co-localized with LHB proteins in the Golgi apparatus, which is important for glycan processing and transport. In addition, we determined the functional domain in SERINC5 required for HBV inhibition, which was completely different from that required for HIV-1 restriction, whereas phosphorylation and glycosylation sites in SERINC5 were dispensable for HBV restriction. Taken together, our results demonstrate that SERINC5 suppresses HBV virion secretion through interfering with the glycosylation of HBV proteins, suggesting that SERINC5 might possess broad-spectrum antiviral activity.
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Affiliation(s)
- Yue Liu
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
- Department of Echocardiography, The First Hospital of Jilin University, Changchun, China
| | - Hong Wang
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
| | - Jun Zhang
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
| | - Jing Yang
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
| | - Lu Bai
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China
| | - Baisong Zheng
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
| | - Tianhang Zheng
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
| | - Yingchao Wang
- Department of Hepatobiliary Pancreatic Surgery, The First Hospital of Jilin University, Changchun, China
| | - Jianhua Li
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, China
| | - Wenyan Zhang
- Institute of Virology and AIDS Research, The First Hospital of Jilin University, Changchun, China
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Ou Q, Guo J, Zeng Y, Chen H. Insights for clinical diagnostic indicators of virus and host in chronic hepatitis B infection. J Viral Hepat 2020; 27:224-232. [PMID: 31954089 DOI: 10.1111/jvh.13260] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/22/2019] [Revised: 10/19/2019] [Accepted: 12/17/2019] [Indexed: 02/06/2023]
Abstract
Covalently closed circular DNA (cccDNA), which is stably present in the nucleus of hepatocytes, is an important indicator for evaluating antiviral efficacy. Since cccDNA quantification requires an invasive procedure, serum biological markers that can effectively reflect the transcriptional activity of intrahepatic virus and the efficacy of treatment are required. Here, from the aspects of virus and host, we outline the focus of clinical research of HBV in recent years, including HBV RNA, empty virus, hepatitis B core-related antigen and changes in the immune response. We briefly discuss their significance in predicting disease activity and monitoring treatment response in chronic hepatitis B. On this basis, some issues worthy of attention in laboratory diagnosis are proposed.
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Affiliation(s)
- Qishui Ou
- Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,The Genetic Diagnostic Laboratory, Fujian Medical University, Fuzhou, China.,First Clinical College, Fujian Medical University, Fuzhou, China
| | - Jianhui Guo
- Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,The Genetic Diagnostic Laboratory, Fujian Medical University, Fuzhou, China.,First Clinical College, Fujian Medical University, Fuzhou, China
| | - Yongbin Zeng
- Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,The Genetic Diagnostic Laboratory, Fujian Medical University, Fuzhou, China.,First Clinical College, Fujian Medical University, Fuzhou, China
| | - Huijuan Chen
- Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,The Genetic Diagnostic Laboratory, Fujian Medical University, Fuzhou, China.,First Clinical College, Fujian Medical University, Fuzhou, China
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Dusheiko G. Will we need novel combinations to cure HBV infection? Liver Int 2020; 40 Suppl 1:35-42. [PMID: 32077595 DOI: 10.1111/liv.14371] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2019] [Revised: 01/22/2020] [Accepted: 12/31/2019] [Indexed: 12/15/2022]
Abstract
Chronic hepatitis B is a numerically important cause of cirrhosis and hepatocellular carcinoma. Nucleoside analogue therapy may modify the risk. However, maintenance suppressive therapy is required, as a functional cure (generally defined as loss of HBsAg off treatment) is an uncommon outcome of antiviral treatment. Chronic hepatitis B is a numerically important cause of cirrhosis and hepatocellular carcinoma. Nucleoside analogue therapy may modify the risk. However, maintenance suppressive therapy is required, as a functional cure (generally defined as loss of HBsAg off treatment) is an uncommon outcome of antiviral treatment. Currently numerous investigational agents being developed to either interfere with specific steps in HBV replication or as host cellular targeting agents, that inhibit viral replication, and deplete or inactivate cccDNA, or as immune modulators. Synergistic mechanisms will be needed to incorporate a decrease in HBV transcription, impairment of transcription from HBV genomes, loss of cccDNA or altered epigenetic regulation of cccDNA transcription, and immune modulation or immunologically stimulated hepatocyte cell turnover. Nucleoside analogue suppressed patients are being included in many current trials. Trials are progressing to combination therapy as additive or synergistic effects are sought. These trials will provide important insights into the biology of HBV and perturbations of the immune response, required to effect HBsAg loss at different stages of the disease. The prospect of cures of hepatitis B would ensure that a wide range of patients could be deemed candidates for treatment with new compounds if these were highly effective, finite and safe. Withdrawal of therapy in short-term trials is challenging because short-term therapies may risk severe hepatitis flares, and hepatic decompensation. The limited clinical trial data to date suggest that combination therapy is inevitable.
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Affiliation(s)
- Geoffrey Dusheiko
- Kings College Hospital, London, UK.,University College London Medical School, London, UK
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