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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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Hsieh CL, Chang LY, Chen PJ, Yeh SH. HBV polymerase recruits the phosphatase PP1 to dephosphorylate HBc-Ser170 to complete encapsidation. PLoS Pathog 2025; 21:e1012905. [PMID: 39932960 PMCID: PMC11813143 DOI: 10.1371/journal.ppat.1012905] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2024] [Accepted: 01/14/2025] [Indexed: 02/13/2025] Open
Abstract
The HBV core (HBc) protein contains an N-terminal domain (NTD) for capsid assembly and an arginine-rich C-terminal domain (CTD) for pregenomic RNA (pgRNA) encapsidation. Phosphorylation of the HBc CTD, especially at Ser162 and Ser170, is essential for nucleation with the polymerase (Pol) to initiate pgRNA encapsidation. As capsids mature, the HBc CTD undergoes dephosphorylation, suggesting the involvement of a phosphatase in the late stage of encapsidation, which remains to be determined. Using a C-S170 antibody specific for non-phosphorylated HBc-Ser170, we observed a transition from a phosphorylated to a dephosphorylated state during pgRNA packaging. The Pol-dependent dephosphorylation of HBc-Ser170 was confirmed by the substitution of one single amino acid at Val782 in the RNase H domain, which abolished the dephosphorylation of HBc-Ser170. Immunoprecipitation, mass spectrometry analyses, and the protein structural analyses showed that the recruitment of the host phosphatase PP1 is dependent on the Pol-Val782 domain. This recruitment does not require HBc but does require Pol via epsilon RNA signal, suggesting that the Pol-pgRNA complex plays a key role in PP1 recruitment. Pol-pgRNA-PP1-mediated dephosphorylation of HBc-Ser170 is essential for the completion of pgRNA encapsidation and appears to be associated with late endosomes/multivesicular bodies (MVBs). Therefore, HBV Pol may play a dual role by initially bringing pgRNA to phosphorylated HBc and recruiting PP1 for later completion of RNA packaging into the capsids. These findings not only decipher the mechanism by which Pol-mediated dephosphorylation of HBc regulates pgRNA encapsulation, but also reveal the possibility of PP1 as a potential target for antiviral development.
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Affiliation(s)
- Chi-Ling Hsieh
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Li-Yang Chang
- Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Pei-Jer Chen
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
- Center of Precision Medicine, National Taiwan University, Taipei, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Shiou-Hwei Yeh
- Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan
- Center of Precision Medicine, National Taiwan University, Taipei, Taiwan
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan
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Yan H, Liu C, Li Y, Tang S, Guo H, Zhou B, Fan Q, Wang H, Ge X, Wang X, Liao X, Li J, Zhang Z, Ju B. The characterization and structural basis of a human broadly binding antibody to HBV core protein. J Virol 2025; 99:e0169424. [PMID: 39601613 PMCID: PMC11784010 DOI: 10.1128/jvi.01694-24] [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] [Received: 09/25/2024] [Accepted: 10/31/2024] [Indexed: 11/29/2024] Open
Abstract
Hepatitis B virus (HBV) core protein (HBc) plays a crucial role in the virus life cycle, making it an important detection marker for HBV infection and a potential target for treatment. However, several commercially available monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) targeting HBc have certain limitations in detecting HBV across different genotypes in various biochemical assays, such as enzyme-linked immunosorbent assay, western blot, immunofluorescence assay, flow cytometry, and immune spot assay. In this study, we identified 12 human anti-HBc mAbs and evaluated their potential application in multiple biochemical assays. These mAbs mainly recognized the epitopes near residues 20-22 amino acids (a.a.) and 77-78 a.a. of HBc, demonstrating a broadly cross-genotypic activity. Notably, three Group II mAbs, named cAbA1, cAbD4, and cAbF9, displayed excellent capacities for the detection of HBV infection in all tested biochemical assays. Furthermore, we determined a 3.22 Å of cryo-electron microscopy (cryo-EM) structure of the fragment of antigen binding (Fab) of cAbD4 complexed with HBc dimer, which was the highest resolution of the structural model for Fab-HBc to date. Collectively, our findings provided excellent and reliable antibody candidates for live HBV detection and revealed the recognition mechanism and the detailed interaction information of a potent human anti-HBc mAb binding to the spike tips of HBc dimer.IMPORTANCEThe lack of excellent detection Abs for live hepatitis B virus (HBV) infection and high-resolution structures of the Ab-HBV core protein (HBc) complex largely limited the development of HBV-related research. This study reports a panel of anti-HBc monoclonal antibodies (mAbs) with excellent capacities for detecting HBV infection in multiple biochemical assays and determines a 3.22 Å of cryo-EM structure of HBc with a potent binding mAb. These findings provide excellent and reliable detecting tools for HBV-related research and promote the understanding of the recognition mechanism of anti-HBc mAbs to HBc particles.
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Affiliation(s)
- Hu Yan
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Congcong Liu
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Yuxiao Li
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Shilong Tang
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Huimin Guo
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Bing Zhou
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Qing Fan
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Haiyan Wang
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Xiangyang Ge
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Xin Wang
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Xuejiao Liao
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Jin Li
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
| | - Zheng Zhang
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
- Guangdong Key Laboratory for Anti-infection Drug Quality Evaluation, Shenzhen, Guangdong Province, China
- Shenzhen Research Center for Communicable Disease Diagnosis and Treatment, Chinese Academy of Medical Sciences, Shenzhen, Guangdong Province, China
| | - Bin Ju
- Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen, Guangdong Province, China
- The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China
- Guangdong Key Laboratory for Anti-infection Drug Quality Evaluation, Shenzhen, Guangdong Province, China
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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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Liu Y, Yang J, Wang Y, Zeng Q, Fan Y, Huang A, Fan H. The proteasome activator subunit PSME1 promotes HBV replication by inhibiting the degradation of HBV core protein. Genes Dis 2024; 11:101142. [PMID: 39281837 PMCID: PMC11400625 DOI: 10.1016/j.gendis.2023.101142] [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: 04/15/2023] [Revised: 08/27/2023] [Accepted: 09/10/2023] [Indexed: 09/18/2024] Open
Abstract
Chronic hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma, representing a global health problem for which a functional cure is difficult to achieve. The HBV core protein (HBc) is essential for multiple steps in the viral life cycle. It is the building block of the nucleocapsid in which viral DNA reverse transcription occurs, and its mediation role in viral-host cell interactions is critical to HBV infection persistence. However, systematic studies targeting HBc-interacting proteins remain lacking. Here, we combined HBc with the APEX2 to systematically identify HBc-related host proteins in living cells. Using functional screening, we confirmed that proteasome activator subunit 1 (PSME1) is a potent HBV-associated host factor. PSME1 expression was up-regulated upon HBV infection, and the protein level of HBc decreased after PSME1 knockdown. Mechanistically, the interaction between PSME1 and HBc inhibited the degradation of HBc by the 26S proteasome, thereby improving the stability of the HBc protein. Furthermore, PSME1 silencing inhibits HBV transcription in the HBV infection system. Our findings reveal an important mechanism by which PSME1 regulates HBc proteins and may facilitate the development of new antiviral therapies targeting PSME1 function.
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Affiliation(s)
- Yu Liu
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
| | - Jiaxin Yang
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
| | - Yanyan Wang
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
| | - Qiqi Zeng
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
| | - Yao Fan
- The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China
| | - Ailong Huang
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
| | - Hui Fan
- The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing 400016, China
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Hossain MG, Ueda K. Regulation of Hepatitis B Virus Replication by Modulating Endoplasmic Reticulum Stress (ER-Stress). Int J Microbiol 2024; 2024:9117453. [PMID: 39246409 PMCID: PMC11379510 DOI: 10.1155/2024/9117453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Accepted: 08/21/2024] [Indexed: 09/10/2024] Open
Abstract
Hepatitis B virus (HBV), resistant to several antiviral drugs due to viral genomic mutations, has been reported, which aggravates chronic infection and leads to hepatocellular carcinoma. Therefore, host cellular factors/signaling modulation might be an alternative way of treatment for drug-resistant HBV. Here, we investigated the viral protein expression, replication, and virion production using endoplasmic reticulum (ER) stress-modulating chemicals, tunicamycin (an ER-stress inducer), and salubrinal (an ER-stress inhibitor). We found that ER-stress could be induced by HBV replication in transfected HepG2 cells as well as by tunicamycin as demonstrated by dual luciferase assay. HBV intracellular core-associated DNA quantified by qPCR has been significantly increased by tunicamycin in transfected HepG2 cells. Inversely, intracellular core associated and extracellular particle DNA has been significantly decreased in a dose-dependent manner in salubrinal-treated HepG2 cells transfected with HBV-replicating plasmid pHBI. Similar results were found in stably HBV-expressing hepatoblastoma (HB611) cells treated with salubrinal. However, increased or decreased ER-stress by tunicamycin or salubrinal treatment, respectively, has been confirmed by expression analysis of grp78 using Western blot. In addition, Western blot results demonstrated that the expression of HBV core protein and large HBsAg is increased and decreased by tunicamycin and salubrinal, respectively. In conclusion, the sal-mediated inhibition of the HBV replication and virion production might be due to the simultaneous reduction of core and large HBsAg expression and maintaining the ER homeostasis. These results of HBV replication regulation by modulation of ER-stress dynamics would be useful for designing/identifying anti-HBV drugs targeting cellular signaling pathways.
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Affiliation(s)
- Md Golzar Hossain
- Department of Microbiology and Hygiene Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
| | - Keiji Ueda
- Division of Virology Department of Microbiology and Immunology Graduate School of Medicine Osaka University, Osaka, Japan
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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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Nayak S, Gowda J, Abbas SA, Kim H, Han SB. Recent Advances in the Development of Sulfamoyl-Based Hepatitis B Virus Nucleocapsid Assembly Modulators. Viruses 2023; 15:2367. [PMID: 38140607 PMCID: PMC10747759 DOI: 10.3390/v15122367] [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: 10/15/2023] [Revised: 11/24/2023] [Accepted: 11/27/2023] [Indexed: 12/24/2023] Open
Abstract
Hepatitis B virus (HBV) is the primary contributor to severe liver ailments, encompassing conditions such as cirrhosis and hepatocellular carcinoma. Globally, 257 million people are affected by HBV annually and 887,000 deaths are attributed to it, representing a substantial health burden. Regrettably, none of the existing therapies for chronic hepatitis B (CHB) have achieved satisfactory clinical cure rates. This issue stems from the existence of covalently closed circular DNA (cccDNA), which is difficult to eliminate from the nucleus of infected hepatocytes. HBV genetic material is composed of partially double-stranded DNA that forms complexes with viral polymerase inside an icosahedral capsid composed of a dimeric core protein. The HBV core protein, consisting of 183 to 185 amino acids, plays integral roles in multiple essential functions within the HBV replication process. In this review, we describe the effects of sulfamoyl-based carboxamide capsid assembly modulators (CAMs) on capsid assembly, which can suppress HBV replication and disrupt the production of new cccDNA. We present research on classical, first-generation sulfamoyl benzocarboxamide CAMs, elucidating their structural composition and antiviral efficacy. Additionally, we explore newly identified sulfamoyl-based CAMs, including sulfamoyl bicyclic carboxamides, sulfamoyl aromatic heterocyclic carboxamides, sulfamoyl aliphatic heterocyclic carboxamides, cyclic sulfonamides, and non-carboxamide sulfomoyl-based CAMs. We believe that certain molecules derived from sulfamoyl groups have the potential to be developed into essential components of a well-suited combination therapy, ultimately yielding superior clinical efficacy outcomes in the future.
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Affiliation(s)
- Sandesha Nayak
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Jayaraj Gowda
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Syed Azeem Abbas
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Hyejin Kim
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
| | - Soo Bong Han
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
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10
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Saeed U, Piracha ZZ, Alrokayan S, Hussain T, Almajhdi FN, Waheed Y. Immunoinformatics and Evaluation of Peptide Vaccines Derived from Global Hepatitis B Viral HBx and HBc Proteins Critical for Covalently Closed Circular DNA Integrity. Microorganisms 2023; 11:2826. [PMID: 38137971 PMCID: PMC10745757 DOI: 10.3390/microorganisms11122826] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Revised: 11/07/2023] [Accepted: 11/17/2023] [Indexed: 12/24/2023] Open
Abstract
The Hepatitis B virus (HBV) HBx and HBc proteins play a crucial role in associating with covalently closed circular DNA (cccDNA), the primary factor contributing to intrahepatic viral persistence and a major obstacle in achieving a cure for HBV. The cccDNA serves as a reservoir for viral persistence. Targeting the viral HBc and HBx proteins' interaction with cccDNA could potentially limit HBV replication. In this study, we present epitopes identified from global consensus sequences of HBx and HBc proteins that have the potential to serve as targets for the development of effective vaccine candidates. Furthermore, conserved residues identified through this analysis can be utilized in designing novel, site-specific anti-HBV agents capable of targeting all major genotypes of HBV. Our approach involved designing global consensus sequences for HBx and HBc proteins, enabling the analysis of variable regions and highly conserved motifs. These identified motifs and regions offer potent sites for the development of peptide vaccines, the design of site-specific RNA interference, and the creation of anti-HBV inhibitors. The epitopes derived from global consensus sequences of HBx and HBc proteins emerge as promising targets for the development of effective vaccine candidates. Additionally, the conserved residues identified provide valuable insights for the development of innovative, site-specific anti-HBV agents capable of targeting all major genotypes of HBV from A to J.
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Affiliation(s)
- Umar Saeed
- Clinical and Biomedical Research Center (CBRC) and Multidisciplinary Laboratory (MDL), Foundation University Islamabad, Islamabad 44000, Pakistan;
- Department of Microbiology, Ajou University School of Medicine, Suwon 443-749, Republic of Korea;
| | - Zahra Zahid Piracha
- Department of Microbiology, Ajou University School of Medicine, Suwon 443-749, Republic of Korea;
- International Center of Medical Sciences Research (ICMSR), Islamabad 44000, Pakistan
| | - Salman Alrokayan
- Research Chair for Biomedical Application of Nanomaterials, Biochemistry Department, College of Sciences, King Saud University, Riyadh 11362, Saudi Arabia;
| | - Tajamul Hussain
- Research Chair for Biomedical Application of Nanomaterials, Biochemistry Department, College of Sciences, King Saud University, Riyadh 11362, Saudi Arabia;
- Center of Excellence in Biotechnology Research, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 11451, Saudi Arabia
| | - Fahad N. Almajhdi
- Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia;
| | - Yasir Waheed
- Office of Research, Innovation, and Commercialization, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad 44000, Pakistan;
- Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, Byblos 1401, Lebanon
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11
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Wang M, Zhang J, Dou Y, Liang M, Xie Y, Xue P, Liu L, Li C, Wang Y, Tao F, Zhang X, Hu H, Feng K, Zhang L, Wu Z, Chen Y, Zhan P, Jia H. Design, Synthesis, and Biological Evaluation of Novel Thioureidobenzamide (TBA) Derivatives as HBV Capsid Assembly Modulators. J Med Chem 2023; 66:13968-13990. [PMID: 37839070 DOI: 10.1021/acs.jmedchem.3c01022] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2023]
Abstract
Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a promising therapeutic approach for the treatment of HBV infection. In this study, we designed and synthesized five series of benzamide derivatives based on a multisite-binding strategy at the tolerant region and diversity modification in the solvent-exposed region. Among them, thioureidobenzamide compound 17i exhibited significantly increased anti-HBV activity in HepAD38 (EC50 = 0.012 μM) and HBV-infected HLCZ01 cells (EC50 = 0.033 μM). Moreover, 17i displayed a better inhibitory effect on the assembly of HBV capsid protein compared with NVR 3-778 and a inhibitory effect similar to the clinical drug GLS4. In addition, 17i showed moderate metabolic stability in human microsomes, had excellent oral bioavailability in Sprague-Dawley (SD) rats, and inhibited HBV replication in the HBV carrier mice model, which could be considered as a promising candidate drug for further development.
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Affiliation(s)
- Mei Wang
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Jian Zhang
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Yutong Dou
- Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunity of Shandong Province and Dept. Immunology, School of Basic Medical Sciences, Qilu Hospital, Cheeloo Medical College, Shandong University, Jinan 250012, Shandong, P. R. China
| | - Minghui Liang
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Yong Xie
- State Key Laboratory of Anti-Infective Drug Development (NO. 2015DQ780357), Sunshine Lake Pharma Co., Ltd, Dongguan 523871, P. R. China
| | - Peng Xue
- School of Public Health, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Linyue Liu
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Chuanju Li
- Department of Pharmacy, Affiliated Hospital of Jining Medical University, Jining 272000, Shandong, P. R. China
| | - Yuanze Wang
- Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou 510000, Guangdong, P. R. China
| | - Feiyan Tao
- School of Public Health, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Xiaohui Zhang
- Key Laboratory of Experimental Teratology, Ministry of Education and Department of Genetics, School of Basic Medical Sciences, Qilu Hospital, Cheeloo Medical College, Shandong University, Jinan 250012, P. R. China
- The Research Center of Stem Cell and Regenerative Medicine, Department of Systems Biomedicine, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, P. R. China
| | - Huili Hu
- Key Laboratory of Experimental Teratology, Ministry of Education and Department of Genetics, School of Basic Medical Sciences, Qilu Hospital, Cheeloo Medical College, Shandong University, Jinan 250012, P. R. China
- The Research Center of Stem Cell and Regenerative Medicine, Department of Systems Biomedicine, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, P. R. China
| | - Kairui Feng
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Lei Zhang
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
| | - Zhuanchang Wu
- Key Laboratory for Experimental Teratology of Ministry of Education, Key Laboratory of Infection and Immunity of Shandong Province and Dept. Immunology, School of Basic Medical Sciences, Qilu Hospital, Cheeloo Medical College, Shandong University, Jinan 250012, Shandong, P. R. China
| | - Yunfu Chen
- State Key Laboratory of Anti-Infective Drug Development (NO. 2015DQ780357), Sunshine Lake Pharma Co., Ltd, Dongguan 523871, P. R. China
| | - Peng Zhan
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, P. R. China
| | - Haiyong Jia
- School of Pharmacy, Weifang Medical University, Weifang 261053, Shandong, P. R. China
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12
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Kim C, Schlicksup CJ, Pérez-Segura C, Hadden-Perilla JA, Wang JCY, Zlotnick A. Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit. J Biol Chem 2023; 299:105104. [PMID: 37517693 PMCID: PMC10463254 DOI: 10.1016/j.jbc.2023.105104] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2023] [Revised: 07/08/2023] [Accepted: 07/11/2023] [Indexed: 08/01/2023] Open
Abstract
Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/β complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impβ as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impβ-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.
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Affiliation(s)
- Christine Kim
- Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA
| | | | - Carolina Pérez-Segura
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Jodi A Hadden-Perilla
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Joseph Che-Yen Wang
- Department of Microbiology & Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Adam Zlotnick
- Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA.
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13
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Kwon H, Kim J, Song C, Sajjad MA, Ha J, Jung J, Park S, Shin HJ, Kim K. Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication. Front Cell Infect Microbiol 2023; 13:1195063. [PMID: 37404723 PMCID: PMC10315659 DOI: 10.3389/fcimb.2023.1195063] [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: 03/28/2023] [Accepted: 06/01/2023] [Indexed: 07/06/2023] Open
Abstract
Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.
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Affiliation(s)
- Hyeonjoong Kwon
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jumi Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Chanho Song
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Muhammad Azhar Sajjad
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jiseon Ha
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jaesung Jung
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Sun Park
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Ho-Joon Shin
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Kyongmin Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
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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: 5] [Impact Index Per Article: 2.5] [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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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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16
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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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17
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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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18
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Molecular characteristics of the full-length genome of occult hepatitis B virus from blood donors in China. Sci Rep 2022; 12:8194. [PMID: 35581341 PMCID: PMC9114411 DOI: 10.1038/s41598-022-12288-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Accepted: 05/06/2022] [Indexed: 11/22/2022] Open
Abstract
The characteristics of a large sample size of the full-length genome of occult hepatitis B virus (HBV) infection (OBI) have not been extensively explored in China. Voluntary blood donors who were HBsAg-negative/HBV NAT-positive (HBsAg−/HBV NAT+) were identified by blood screening and recruited. Blood samples were tested for HBV serologic markers, viral loads, and PCR to identify OBI. HBV full-length genomes were obtained by amplifying two fragments using nested PCR. The characterization of OBI strains was based on sequence analyses compared with HBsAg+ strains obtained from the same donor population. Of the 50 full-length genomes of 172 identified OBI strains, 33 were classified as genotype B (OBIB) and 17 strains as genotype C (OBIC). Significantly higher nucleotide variabilities were observed in the Pre-S2/S promoter region (SP2) and core upstream regulatory sequence (CURS) in OBIB than in their HBsAg+ controls (P < 0.05). Both OBIB and OBIC showed higher amino acid (aa) variabilities in Pol and Pre-S/S regions than their controls (P < 0.05). In addition, 19 novel OBI-related mutations were found spanning the four open reading frames (ORFs) of the HBV genome. Four novel deletions and one novel insertion were also found in OBIC strains. Several novel OBI-related mutations spanning the four ORFs of the virus were identified by characterizing a large sample size of the full-length OBI genome, which may affect the production of HBsAg and contribute to the occult infection of HBV.
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19
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Xi J, Cui X, Liu K, Liu H, Wang J, Hu J. Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating. J Virol 2022; 96:e0039922. [PMID: 35389266 PMCID: PMC9044944 DOI: 10.1128/jvi.00399-22] [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: 03/07/2022] [Accepted: 03/09/2022] [Indexed: 11/20/2022] Open
Abstract
Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.
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Affiliation(s)
- Ji Xi
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Xiuji Cui
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Kuancheng Liu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Haitao Liu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Joseph Wang
- 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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20
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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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21
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Saeed U, Piracha ZZ, Kwon H, Kim J, Kalsoom F, Chwae YJ, Park S, Shin HJ, Lee HW, Lim JH, Kim K. The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication. Front Microbiol 2022; 12:795047. [PMID: 34970249 PMCID: PMC8713550 DOI: 10.3389/fmicb.2021.795047] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Accepted: 11/17/2021] [Indexed: 12/14/2022] Open
Abstract
We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are important for HBV replication.
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Affiliation(s)
- Umar Saeed
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Zahra Zahid Piracha
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Hyeonjoong Kwon
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Jumi Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Fadia Kalsoom
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Yong-Joon Chwae
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Sun Park
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Ho-Joon Shin
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
| | - Hyun Woong Lee
- Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea
| | - Jin Hong Lim
- Department of General Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea
| | - Kyongmin Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.,Department of Biomedical Science, Graduate School of Ajou University, Suwon, South Korea
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22
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Kim H, Ko C, Lee JY, Kim M. Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure, Mode-of-Action and Efficacy. Molecules 2021; 26:molecules26247420. [PMID: 34946502 PMCID: PMC8705634 DOI: 10.3390/molecules26247420] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Revised: 11/24/2021] [Accepted: 12/02/2021] [Indexed: 12/15/2022] Open
Abstract
Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.
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Affiliation(s)
- Hyejin Kim
- Correspondence: (H.K.); (M.K.); Tel.: +82-42-860-7130 (H.K.); +82-42-860-7540 (M.K.)
| | | | | | - Meehyein Kim
- Correspondence: (H.K.); (M.K.); Tel.: +82-42-860-7130 (H.K.); +82-42-860-7540 (M.K.)
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23
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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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24
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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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25
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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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26
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Barski MS, Minnell JJ, Maertens GN. PP2A Phosphatase as an Emerging Viral Host Factor. Front Cell Infect Microbiol 2021; 11:725615. [PMID: 34422684 PMCID: PMC8371333 DOI: 10.3389/fcimb.2021.725615] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Accepted: 07/20/2021] [Indexed: 12/12/2022] Open
Abstract
Protein phosphatase 2A (PP2A) is one of the most ubiquitous cellular proteins and is responsible for the vast majority of Ser/Thr phosphatase activity in eukaryotes. PP2A is a heterotrimer, and its assembly, intracellular localization, enzymatic activity, and substrate specificity are subject to dynamic regulation. Each of its subunits can be targeted by viral proteins to hijack and modulate its activity and downstream signaling to the advantage of the virus. Binding to PP2A is known to be essential to the life cycle of many viruses and seems to play a particularly crucial role for oncogenic viruses, which utilize PP2A to transform infected cells through controlling the cell cycle and apoptosis. Here we summarise the latest developments in the field of PP2A viral targeting; in particular recent discoveries of PP2A hijacking through molecular mimicry of a B56-specific motif by several different viruses. We also discuss the potential as well as shortcomings for therapeutic intervention in the face of our current understanding of viral PP2A targeting.
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Affiliation(s)
| | | | - Goedele Noella Maertens
- Department of Infectious Disease, Section of Molecular Virology, St Mary’s Hospital, Imperial College London, London, United Kingdom
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27
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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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28
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Abstract
Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.
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29
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Xi J, Luckenbaugh L, Hu J. Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication. PLoS Pathog 2021; 17:e1009230. [PMID: 33493210 PMCID: PMC7861550 DOI: 10.1371/journal.ppat.1009230] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2020] [Revised: 02/04/2021] [Accepted: 12/08/2020] [Indexed: 12/18/2022] Open
Abstract
Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication. Hepatitis B virus (HBV) causes acute and chronic viral hepatitis, liver fibrosis, cirrhosis and cancer. The dynamic phosphorylation and dephosphorylation of the viral capsid protein (HBc), which are controlled by host cell protein kinases and phosphatases, play a critical role in regulating multiple stages of HBV replication. While a number of cellular kinases have been identified that mediate HBc phosphorylation, there is little information on cellular phosphatases that mediate its dephosphorylation. Herein we have identified a consensus binding motif in HBc for one of the major cellular phosphatases, the protein phosphatase 2A (PP2A). Genetic analysis of this motif revealed that it played multiple roles in regulating CTD phosphorylation state, as well as viral RNA packaging, reverse transcription, virion secretion, and formation of the nuclear HBV episome responsible for viral persistence. Furthermore, pharmacological inhibition of PP2A decreased HBc dephosphorylation and increased the nuclear episome, further supporting a role of PP2A in HBc dephosphorylation and HBV persistence. These results thus suggest that HBc recruits PP2A, among other host factors, to regulate HBc phosphorylation and dephosphorylation dynamics and HBV replication and persistence.
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Affiliation(s)
- Ji Xi
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Laura Luckenbaugh
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, 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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30
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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: 48] [Impact Index Per Article: 12.0] [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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Intracellular Trafficking of HBV Particles. Cells 2020; 9:cells9092023. [PMID: 32887393 PMCID: PMC7563130 DOI: 10.3390/cells9092023] [Citation(s) in RCA: 53] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 08/31/2020] [Accepted: 09/02/2020] [Indexed: 12/15/2022] Open
Abstract
The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.
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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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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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Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target. Viruses 2020; 12:v12050570. [PMID: 32455999 PMCID: PMC7291194 DOI: 10.3390/v12050570] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 05/17/2020] [Accepted: 05/19/2020] [Indexed: 12/15/2022] Open
Abstract
Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure.
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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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Luo J, Xi J, Gao L, Hu J. Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation. PLoS Pathog 2020; 16:e1008459. [PMID: 32226051 PMCID: PMC7145273 DOI: 10.1371/journal.ppat.1008459] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Revised: 04/09/2020] [Accepted: 03/05/2020] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.
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Affiliation(s)
- Jun Luo
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Ji Xi
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Lu Gao
- Roche Pharma Research and Early Development, Roche Innovation Center Shanghai, Shanghai, China
| | - 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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Pastor F, Herrscher C, Patient R, Eymieux S, Moreau A, Burlaud-Gaillard J, Seigneuret F, de Rocquigny H, Roingeard P, Hourioux C. Direct interaction between the hepatitis B virus core and envelope proteins analyzed in a cellular context. Sci Rep 2019; 9:16178. [PMID: 31700077 PMCID: PMC6838148 DOI: 10.1038/s41598-019-52824-z] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Accepted: 10/23/2019] [Indexed: 01/01/2023] Open
Abstract
Hepatitis B virus (HBV) production requires intricate interactions between the envelope and core proteins. Analyses of mutants of these proteins have made it possible to map regions involved in the formation and secretion of virions. Tests of binding between core and envelope peptides have also been performed in cell-free conditions, to study the interactions potentially underlying these mechanisms. We investigated the residues essential for core-envelope interaction in a cellular context in more detail, by transiently producing mutant or wild-type L, S, or core proteins separately or in combination, in Huh7 cells. The colocalization and interaction of these proteins were studied by confocal microscopy and co-immunoprecipitation, respectively. The L protein was shown to constitute a molecular platform for the recruitment of S and core proteins in a perinuclear environment. Several core amino acids were found to be essential for direct interaction with L, including residue Y132, known to be crucial for capsid formation, and residues L60, L95, K96 and I126. Our results confirm the key role of L in the tripartite core-S-L interaction and identify the residues involved in direct core-L interaction. This model may be valuable for studies of the potential of drugs to inhibit HBV core-envelope interaction.
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Affiliation(s)
- Florentin Pastor
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | - Charline Herrscher
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | - Romuald Patient
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | - Sebastien Eymieux
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | - Alain Moreau
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | - Julien Burlaud-Gaillard
- Plate-Forme IBiSA des Microscopies, PPF ASB - University of Tours and CHRU of Tours, Tours, France
| | - Florian Seigneuret
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France
| | | | - Philippe Roingeard
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France. .,Plate-Forme IBiSA des Microscopies, PPF ASB - University of Tours and CHRU of Tours, Tours, France.
| | - Christophe Hourioux
- INSERM U1259 MAVIVH - University of Tours and CHRU of Tours, Tours, France. .,Plate-Forme IBiSA des Microscopies, PPF ASB - University of Tours and CHRU of Tours, Tours, France.
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Abstract
With a yearly death toll of 880,000, hepatitis B virus (HBV) remains a major health problem worldwide, despite an effective prophylactic vaccine and well-tolerated, effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long-term antiviral therapy, and its integration, though no longer able to support viral replication, destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome, which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products, and thus, it is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.
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Liu K, Hu J. Secretion of empty or complete hepatitis B virions: envelopment of empty capsids versus mature nucleocapsids. Future Virol 2019. [DOI: 10.2217/fvl-2018-0128] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
HBV replicates its DNA genome, a partially double-stranded, relaxed circular DNA, via reverse transcription of an RNA intermediate called pre-genomic RNA by its reverse transcriptase. A major characteristic of HBV replication is the selective envelopment and secretion of relaxed circular DNA-containing mature capsids and empty capsids with no DNA or RNA, but not those containing pre-genomic RNA or the single-stranded DNA replication intermediate. In this review, the potential mechanisms of HBV virion morphogenesis will be discussed, with a focus on key determinants of both the capsid and envelope proteins for the selective secretion of complete and empty virions.
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Affiliation(s)
- Kuancheng Liu
- Department of Biochemistry & Molecular Biology, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018 China
| | - Jianming Hu
- Department of Microbiology & Immunology, Penn State University College of Medicine, Hershey, PA 17033, USA
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Yang F. Post-translational Modification Control of HBV Biological Processes. Front Microbiol 2018; 9:2661. [PMID: 30443247 PMCID: PMC6222169 DOI: 10.3389/fmicb.2018.02661] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Accepted: 10/18/2018] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus infection remains a global healthy issue that needs to be urgently solved. Novel strategies for anti-viral therapy are based on exploring the effective diagnostic markers and therapeutic targets of diseases caused by hepatitis B virus (HBV) infection. It is well-established that not only viral proteins themselves but also key factors from the host control the biological processes associated with HBV, including replication, transcription, packaging, and secretion. Protein post-translational modifications (PTMs), such as phosphorylation, acetylation, methylation, and ubiquitination, have been shown to control protein activity, regulate protein stability, promote protein interactions and alter protein subcellular localization, leading to the modulation of crucial signaling pathways and affected cellular processes. This review focuses on the functions and effects of diverse PTMs in regulating important processes in the HBV life cycle. The potential roles of PTMs in the pathogenesis of HBV-associated liver diseases are also discussed.
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Affiliation(s)
- Fan Yang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions. J Virol 2018; 92:JVI.00272-18. [PMID: 29743374 DOI: 10.1128/jvi.00272-18] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Accepted: 05/04/2018] [Indexed: 02/06/2023] Open
Abstract
During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.
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