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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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Shen S, Cai D, Liang H, Zeng G, Liu W, Yan R, Yu X, Zhang H, Liu S, Li W, Deng R, Lu X, Liu Y, Sun J, Guo H. NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner. PLoS Pathog 2024; 20:e1012485. [PMID: 39259704 PMCID: PMC11389946 DOI: 10.1371/journal.ppat.1012485] [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: 05/28/2024] [Accepted: 08/06/2024] [Indexed: 09/13/2024] Open
Abstract
Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.
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Affiliation(s)
- Sheng Shen
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Dawei Cai
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Hongyan Liang
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Ge Zeng
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Wendong Liu
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Ran Yan
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Xiaoyang Yu
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Hu Zhang
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Shi Liu
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Wanying Li
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Rui Deng
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xingyu Lu
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yuanjie Liu
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Jian Sun
- Department of Infectious Diseases, State Key Laboratory of Organ Failure Research, Key Laboratory of Infectious Diseases Research in South China, Ministry of Education, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haitao Guo
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
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Foko S. Dynamical analysis of a general delayed HBV infection model with capsids and adaptive immune response in presence of exposed infected hepatocytes. J Math Biol 2024; 88:75. [PMID: 38689137 PMCID: PMC11061075 DOI: 10.1007/s00285-024-02096-7] [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: 07/28/2021] [Revised: 04/03/2024] [Accepted: 04/08/2024] [Indexed: 05/02/2024]
Abstract
The aim of this paper is to develop and investigate a novel mathematical model of the dynamical behaviors of chronic hepatitis B virus infection. The model includes exposed infected hepatocytes, intracellular HBV DNA-containing capsids, uses a general incidence function for viral infection covering a variety of special cases available in the literature, and describes the interaction of cytotoxic T lymphocytes that kill the infected hepatocytes and the magnitude of B-cells that send antibody immune defense to neutralize free virions. Further, one time delay is incorporated to account for actual capsids production. The other time delays are used to account for maturation of capsids and free viruses. We start with the analysis of the proposed model by establishing the local and global existence, uniqueness, non-negativity and boundedness of solutions. After defined the threshold parameters, we discuss the stability properties of all possible steady state constants by using the crafty Lyapunov functionals, the LaSalle's invariance principle and linearization methods. The impacts of the three time delays on the HBV infection transmission are discussed through local and global sensitivity analysis of the basic reproduction number and of the classes of infected states. Finally, an application is provided and numerical simulations are performed to illustrate and interpret the theoretical results obtained. It is suggested that, a good strategy to eradicate or to control HBV infection within a host should concentrate on any drugs that may prolong the values of the three delays.
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Affiliation(s)
- Severin Foko
- Committed Mathematics Team, Research Unit in Mathematics and Applications, Department of Mathematics and Computer Science, Faculty of Science, University of Dschang, P.O. Box: 67, Dschang, Cameroon.
- School of Computer Science and Applied Mathematics, University of the Witwatersrand, 1 Jan Smuts Avenue, Braamfontein, Johannesburg, Gauteng, 2000, South Africa.
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Romero S, Unchwaniwala N, Evans EL, Eliceiri KW, Loeb DD, Sherer NM. Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division. mBio 2023; 14:e0330322. [PMID: 36809075 PMCID: PMC10127671 DOI: 10.1128/mbio.03303-22] [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: 11/29/2022] [Accepted: 01/17/2023] [Indexed: 02/23/2023] Open
Abstract
Hepatitis B virus (HBV) capsid assembly is traditionally thought to occur predominantly in the cytoplasm, where the virus gains access to the virion egress pathway. To better define sites of HBV capsid assembly, we carried out single cell imaging of HBV Core protein (Cp) subcellular trafficking over time under conditions supporting genome packaging and reverse transcription in Huh7 hepatocellular carcinoma cells. Time-course analyses including live cell imaging of fluorescently tagged Cp derivatives showed Cp to accumulate in the nucleus at early time points (~24 h), followed by a marked re-distribution to the cytoplasm at 48 to 72 h. Nucleus-associated Cp was confirmed to be capsid and/or high-order assemblages using a novel dual label immunofluorescence strategy. Nuclear-to-cytoplasmic re-localization of Cp occurred predominantly during nuclear envelope breakdown in conjunction with cell division, followed by strong cytoplasmic retention of Cp. Blocking cell division resulted in strong nuclear entrapment of high-order assemblages. A Cp mutant, Cp-V124W, predicted to exhibit enhanced assembly kinetics, also first trafficked to the nucleus to accumulate at nucleoli, consistent with the hypothesis that Cp's transit to the nucleus is a strong and constitutive process. Taken together, these results provide support for the nucleus as an early-stage site of HBV capsid assembly, and provide the first dynamic evidence of cytoplasmic retention after cell division as a mechanism underpinning capsid nucleus-to-cytoplasm relocalization. IMPORTANCE Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized. Here, we developed a combination of fixed and long-term (>24 h) live cell imaging technologies to study the single cell trafficking dynamics of the HBV Core Protein (Cp). We demonstrate that Cp first accumulates in the nucleus, and forms high-order structures consistent with capsids, with the predominant route of nuclear egress being relocalization to the cytoplasm during cell division in conjunction with nuclear membrane breakdown. Single cell video microscopy demonstrated unequivocally that Cp's localization to the nucleus is constitutive. This study represents a pioneering application of live cell imaging to study HBV subcellular transport, and demonstrates links between HBV Cp and the cell cycle.
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Affiliation(s)
- Sofia Romero
- McArdle Laboratory for Cancer Research (Department of Oncology), University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Carbone Cancer Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
| | - Nuruddin Unchwaniwala
- McArdle Laboratory for Cancer Research (Department of Oncology), University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Carbone Cancer Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
| | - Edward L. Evans
- Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Morgridge Institute for Research, Madison, Wisconsin, USA
| | - Kevin W. Eliceiri
- Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Morgridge Institute for Research, Madison, Wisconsin, USA
| | - Daniel D. Loeb
- McArdle Laboratory for Cancer Research (Department of Oncology), University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
- Carbone Cancer Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
| | - Nathan M. Sherer
- McArdle Laboratory for Cancer Research (Department of Oncology), University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA
- Carbone Cancer Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA
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Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation. J Virol 2022; 96:e0071822. [PMID: 35867543 PMCID: PMC9364803 DOI: 10.1128/jvi.00718-22] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.
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Alternative hepatitis B virus DNA confirmatory algorithm identified occult hepatitis B virus infection in Chinese blood donors with non-discriminatory nucleic acid testing. BLOOD TRANSFUSION = TRASFUSIONE DEL SANGUE 2022; 20:8-17. [PMID: 33370226 PMCID: PMC8796838 DOI: 10.2450/2020.0213-20] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Accepted: 09/17/2020] [Indexed: 01/24/2023]
Abstract
BACKGROUND Multiplex viral nucleic acid testing (NAT) and a discriminatory testing algorithm have been used to detect viral infections in blood donors. Non-discriminated reactive (NDR) results may arise from low hepatitis B virus (HBV) DNA levels and are challenging for donor management by blood services. The aim of this study was to evaluate the performance and feasibility of alternative viral particle concentration methods to confirm and to characterise HBV infection status in NDR donors from Dalian, China, in order to improve routine donor management according to the potential residual risk estimate. MATERIALS AND METHODS Individual donations were tested with ULTRIO Plus, and discriminated when reactive. Virions were concentrated from 12 and 6 mL plasma samples by ultracentrifugation (UC) and polyethylene glycol (PEG) precipitation, respectively. HBV DNA was detected with four nested polymerase chain reactions (95% limit of detection: 5-25 IU/mL). Amplified products were sequenced for definitive confirmation. Anti-HBc and anti-HBs were tested. RESULTS Of 77,556 donors, 79 (0.1%) were NAT NDR. After viral particle concentration by UC and PEG precipitation, HBV DNA was detected in 46 (58.2%) and 34 (43.0%) NDR donors, respectively, including 61.7% of samples that were repeatedly non-reactive with multiple NAT testing. Anti-HBc and anti-HBs (median titre: 37 mIU/mL) were detected in 87.3% and 46.8% of NDR donors, respectively. Sequencing confirmed HBV DNA in 65.8% of NDR donors, of whom 96.2% were occult HBV carriers with rare mutations in S and core proteins. DISCUSSION A HBV DNA confirmatory procedure with limited technical constraints was implemented successfully. The majority of NDR donors had occult HBV infections with extremely low viral DNA levels, which may constitute a potential residual threat for blood safety. Only a minority of anti-HBc+ NDR donors had anti-HBs levels high enough to consider their reinstatement as donors. The data support the permanent deferral of NDR donors to ensure maximum blood safety in areas of high HBV endemicity.
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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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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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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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10
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Seitz S, Habjanič J, Schütz AK, Bartenschlager R. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. Annu Rev Virol 2020; 7:263-288. [PMID: 32600157 DOI: 10.1146/annurev-virology-092818-015508] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.
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Affiliation(s)
- Stefan Seitz
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany;
| | - Jelena Habjanič
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Anne K Schütz
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, University of Heidelberg, 69120 Heidelberg, Germany; .,Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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11
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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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12
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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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13
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Zhang Y, Zhang H, Zhang J, Zhang J, Guo H. Naturally occurring core protein mutations compensate for the reduced replication fitness of a lamivudine-resistant HBV isolate. Antiviral Res 2019; 165:47-54. [PMID: 30902704 DOI: 10.1016/j.antiviral.2019.03.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Revised: 02/05/2019] [Accepted: 03/14/2019] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of an RNA intermediate. The lack of proofreading capacity of the viral DNA polymerase results in a high mutation rate of HBV genome. Under the selective pressure created by the nucleos(t)ide analogue (NA) antiviral drugs, viruses with resistance mutations are selected. However, the replication fitness of NA-resistant mutants is markedly reduced compared to wild-type. Compensatory mutations in HBV polymerase, which restore the viral replication capacity, have been reported to arise under continuous treatment with lamivudine (LMV). We have previously identified a highly replicative LMV-resistant HBV isolate from a chronic hepatitis B patient experiencing acute disease exacerbation. Besides the common YMDD drug-resistant mutations, this isolate possesses multiple additional mutations in polymerase and core regions. The transcomplementation assay demonstrated that the enhanced viral replication is due to the mutations of core protein. Further mutagenesis study revealed that the P5T mutation of core protein plays an important role in the enhanced viral replication through increasing the levels of capsid formation and pregenomic RNA encapsidation. However, the LMV-resistant virus harboring compensatory core mutations remains sensitive to capsid assembly modulators (CpAMs). Taken together, our study suggests that the enhanced HBV nucleocapsid formation resulting from core mutations represents an important viral strategy to surmount the antiviral drug pressure and contribute to viral pathogenesis, and CpAMs hold promise for developing the combinational antiviral therapy for hepatitis B.
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Affiliation(s)
- Yongmei Zhang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Hu Zhang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Junjie Zhang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Jiming Zhang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China; Key Laboratory of Medical Molecular Virology (MOH & MOE), Fudan University, Shanghai, China.
| | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.
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14
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Li C, Wang Y, Liu T, Niklasch M, Qiao K, Durand S, Chen L, Liang M, Baumert TF, Tong S, Nassal M, Wen YM, Wang YX. An E. coli-produced single-chain variable fragment (scFv) targeting hepatitis B virus surface protein potently inhibited virion secretion. Antiviral Res 2019; 162:118-129. [DOI: 10.1016/j.antiviral.2018.12.019] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Revised: 12/06/2018] [Accepted: 12/28/2018] [Indexed: 01/14/2023]
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15
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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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16
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Heger-Stevic J, Zimmermann P, Lecoq L, Böttcher B, Nassal M. Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure. PLoS Pathog 2018; 14:e1007488. [PMID: 30566530 PMCID: PMC6317823 DOI: 10.1371/journal.ppat.1007488] [Citation(s) in RCA: 62] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Revised: 01/03/2019] [Accepted: 11/27/2018] [Indexed: 12/19/2022] Open
Abstract
Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.
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Affiliation(s)
- Julia Heger-Stevic
- University Hospital Freiburg, Department of Medicine II / Molecular Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Biological Faculty, University of Freiburg, Freiburg, Germany
| | - Peter Zimmermann
- University Hospital Freiburg, Department of Medicine II / Molecular Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Biological Faculty, University of Freiburg, Freiburg, Germany
| | - Lauriane Lecoq
- Institut de Biologie et Chimie des Protéines, University of Lyon1, Lyon, France
| | - Bettina Böttcher
- Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany
| | - Michael Nassal
- University Hospital Freiburg, Department of Medicine II / Molecular Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany
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17
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Zheng CL, Fu YM, Xu ZX, Zou Y, Deng K. Hepatitis B virus core protein dimer‑dimer interface is critical for viral replication. Mol Med Rep 2018; 19:262-270. [PMID: 30387827 PMCID: PMC6297743 DOI: 10.3892/mmr.2018.9620] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 09/07/2018] [Indexed: 12/18/2022] Open
Abstract
Hepatitis B virus (HBV) core protein (HBc) serves pivotal roles in the viral life cycle, particularly serving as the basic unit for capsid assembly, and is closely associated with HBV genome replication and progeny virion production. Previous studies have demonstrated that HBc has at least two functional interfaces; two HBc monomers form a homodimer via an intradimer interface, and then 90 or 120 homodimers form an icosahedral capsid via a dimer-dimer interface. In the present study, the role of the HBc dimer-dimer interface in HBV replication was investigated. A panel of residues located at the dimer-dimer interface were identified based on the crystal structure of HBc. Native gel electrophoresis and western blotting revealed that, despite mutations in the dimer-dimer interface, HBc formed a capsid-like structure, whereas mutations at amino acid residues 23–39 completely disrupted capsid assembly. Using denaturing gel electrophoresis, Southern and Northern blotting, and quantitative polymerase chain reaction, it was demonstrated that none of the mutations in the dimer-dimer interface supported pregenomic RNA encapsidation or DNA replication. In addition, these mutants interacted with the wild-type (WT) HBc monomer and inhibited WT genome replication and virion production in a dose-dependent manner. However, the quantity of covalently closed circular DNA in the nucleus was not affected. The present study highlighted the importance of the HBc dimer-dimer interface for normal capsid function and demonstrated that the HBc dimer-dimer interface may be a novel antiviral target.
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Affiliation(s)
- Chang-Long Zheng
- Department of Emergency, The Third Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Yong-Mei Fu
- Department of Emergency, The Third Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Zhan-Xue Xu
- Institute of Human Virology and Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat‑Sen University, Guangzhou, Guangdong 510080, P.R. China
| | - Yong Zou
- Department of Blood Transfusion, The Third Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510630, P.R. China
| | - Kai Deng
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510060, P.R. China
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18
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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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Liu K, Luckenbaugh L, Ning X, Xi J, Hu J. Multiple roles of core protein linker in hepatitis B virus replication. PLoS Pathog 2018; 14:e1007085. [PMID: 29782550 PMCID: PMC5983865 DOI: 10.1371/journal.ppat.1007085] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Revised: 06/01/2018] [Accepted: 05/09/2018] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) core protein (HBc) contains an N-terminal domain (NTD, assembly domain) and a C-terminal domain (CTD), which are linked by a flexible linker region. HBc plays multiple essential roles in viral replication, including capsid assembly, packaging of the viral pregenomic RNA (pgRNA) into nucleocapsids, viral reverse transcription that converts pgRNA to the genomic DNA, and secretion of DNA-containing (complete) virions or genome-free (empty) virions. The HBc linker is generally assumed to act merely as a spacer between NTD and CTD but some results suggest that the linker may affect NTD assembly. To determine its role in viral replication, we have made a number of deletion and substitution mutants in the linker region, in either the presence or absence of CTD, and tested their abilities to support capsid assembly and viral replication in human cells. Our results indicate that the linker could indeed impede NTD assembly in the absence of CTD, which could be partially relieved by partial linker deletion. In contrast, when CTD was present, the linker deletions or substitutions did not affect capsid assembly. Deletion of the entire linker or its C-terminal part resulted in a partial defect in pgRNA packaging and severely impaired viral DNA synthesis. In contrast, deletion of the N-terminal part of the linker, or substitutions of the linker sequence, had little to no effect on RNA packaging or first-strand DNA synthesis. However, the N-terminal linker deletion and two linker substitution mutants were defective in the production of mature double-stranded viral DNA. Secretion of empty virions was blocked by all the linker deletions and substitutions tested. In particular, a conservative linker substitution that allowed mature viral DNA synthesis and secretion of complete virions severely impaired the secretion of empty virions, thus increasing the ratio of complete to empty virions that were secreted. Together, these results demonstrate that the HBc linker region plays critical and complex roles at multiple stages of HBV replication. The hepatitis B virus (HBV) is a major human pathogen that infects hundreds of millions of people worldwide and represents a major cause of viral hepatitis, liver cirrhosis, and liver cancer. The HBV capsid protein (HBc) plays multiple roles in the viral life cycle and has emerged recently as a major target for developing antiviral therapies against HBV infection. HBc is divided into three separate regions, an N-terminal domain (NTD) responsible for capsid assembly, a C-terminal domain (CTD) that plays critical roles in the specific packaging of the viral pregenomic RNA (pgRNA) into replication-competent nucleocapsids and the subsequent reverse transcription of the pgRNA into the viral genomic DNA, and a linker region between the NTD and CTD. In contrast to the prevailing assumption that the linker merely serves to connect the NTD and CTD, we have discovered here that it plays a critical role in almost every stage of HBV replication. The linker likely exerted its pleiotropic effects via affecting the NTD and CTD as well as via direct interactions with other viral factors independent of the NTD or CTD. Our results thus not only deepen understanding of HBc structure and functions but also implicate the linker as a potential novel target for antiviral development against HBV infection.
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Affiliation(s)
- Kuancheng Liu
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA, United States of America
- College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China
| | - Laurie Luckenbaugh
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA, United States of America
| | - Xiaojun Ning
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA, United States of America
| | - Ji Xi
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA, United States of America
| | - Jianming Hu
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, PA, United States of America
- * E-mail:
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20
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Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly. J Virol 2017; 91:JVI.00519-17. [PMID: 28566379 DOI: 10.1128/jvi.00519-17] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Accepted: 05/19/2017] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV.IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B.
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Berke JM, Tan Y, Verbinnen T, Dehertogh P, Vergauwen K, Vos A, Lenz O, Pauwels F. Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro. Antiviral Res 2017. [PMID: 28647474 DOI: 10.1016/j.antiviral.2017.06.016] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The HBV core protein represents an attractive target for new antiviral therapies due to its multiple functions within the viral life-cycle. Here, we report the antiviral activity of the capsid assembly modulator (CAM) BAY41-4109 and two nucleos(t)ide analogues (NAs) on a diverse panel of 54 HBV clinical isolates from genotype (GT) A-H and assessed the impact of core amino acid (aa) substitutions using site-directed mutants (SDMs). The median EC50 values of BAY41-4109 across genotypes ranged from 26 nM in GT G to 215 nM in GT F irrespective of the presence of NA resistance mutations compared to 43 nM for the GT D reference construct. Combined analyses of clinical isolates and SDMs identified aa changes at positions 29, 33 and 118 led to reduced antiviral activity of BAY41-4109 with fold changes in EC50 values of 6, 46, and 9 for D29G, T33N, and Y118F, respectively. These aa substitutions are located within the CAM binding pocket, and are expected to have an effect on CAM binding based on structural modeling. Importantly aa variations at these positions were rarely (<0.3%) observed as naturally occurring in public sequence databases. NA's remained fully active against these variants. Our study demonstrated that BAY41-4109 generally remained fully active across GT A-H clinical isolates. In addition, core aa substitutions within the CAM-binding pocket replicated in vitro and variants at positions 29, 33, and 118 were identified to reduce antiviral activity.
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Affiliation(s)
- Jan Martin Berke
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium.
| | - Ying Tan
- Janssen China Research & Development Center, 5F North Building #1 Jinchuang Mansion, 4560 Jinke Road, Shanghai 201210, China
| | - Thierry Verbinnen
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Pascale Dehertogh
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Karen Vergauwen
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Ann Vos
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Oliver Lenz
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
| | - Frederik Pauwels
- Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium
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22
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Chong CK, Cheng CYS, Tsoi SYJ, Huang FY, Liu F, Seto WK, Lai CL, Yuen MF, Wong DKH. Role of hepatitis B core protein in HBV transcription and recruitment of histone acetyltransferases to cccDNA minichromosome. Antiviral Res 2017; 144:1-7. [PMID: 28499864 DOI: 10.1016/j.antiviral.2017.05.003] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2017] [Revised: 04/12/2017] [Accepted: 05/08/2017] [Indexed: 12/16/2022]
Abstract
The hepatitis B core protein (HBc) has been suggested to interact with covalently closed circular DNA (cccDNA) and regulate hepatitis B virus (HBV) transcription. However, direct evidence is lacking. We aimed to identify the specific HBc region(s) responsible for transcription regulation and its interaction with cccDNA. Seventeen mutants with mutations at the four arginine-rich clusters of the HBc carboxyl-terminal domain (CTD) were created. The effect of HBc mutations on the levels of HBV DNA, RNA, and hepatitis B surface antigen (HBsAg) were measured. The association of cccDNA with mutant HBc and histone acetyltransferases (HATs) was assessed by chromatin immunoprecipitation (ChIP). Compared with wild-type HBc, HBc mutants with mutations in clusters III and IV resulted in a significant reduction in HBV RNA levels (all P < 0.05). HBc arginine clusters III and IV mutants also had a significantly lower levels of intracellular HBV DNA (<5% of wild-type; P < 0.001) and HBsAg (<10% of wild-type; P < 0.0001). cccDNA-ChIP assay demonstrated that HBc clusters III and IV mutants had a smaller degree of association with cccDNA (P < 0.001). In the HBc mutants, the association between HATs with cccDNA were reduced. In conclusion, HBc-CTD arginine residues at clusters III and IV play an important role in the regulation of HBV transcription as well as subsequent replication steps, likely through the reduced interaction of HBc with cccDNA and reduced acetylation of cccDNA-bound histones. These findings may provide clues to the identification of novel therapeutic targets against HBV.
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Affiliation(s)
- Chun Kong Chong
- Department of Medicine, The University of Hong Kong, Hong Kong
| | | | | | - Fung-Yu Huang
- Department of Medicine, The University of Hong Kong, Hong Kong
| | - Fen Liu
- Department of Medicine, The University of Hong Kong, Hong Kong
| | - Wai-Kay Seto
- Department of Medicine, The University of Hong Kong, Hong Kong; State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong
| | - Ching-Lung Lai
- Department of Medicine, The University of Hong Kong, Hong Kong; State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong
| | - Man-Fung Yuen
- Department of Medicine, The University of Hong Kong, Hong Kong; State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong.
| | - Danny Ka-Ho Wong
- Department of Medicine, The University of Hong Kong, Hong Kong; State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong.
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Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses 2017; 9:v9030056. [PMID: 28335554 PMCID: PMC5371811 DOI: 10.3390/v9030056] [Citation(s) in RCA: 209] [Impact Index Per Article: 26.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Revised: 03/11/2017] [Accepted: 03/17/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.
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24
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Blondot ML, Bruss V, Kann M. Intracellular transport and egress of hepatitis B virus. J Hepatol 2016; 64:S49-S59. [PMID: 27084037 DOI: 10.1016/j.jhep.2016.02.008] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 01/27/2016] [Accepted: 02/03/2016] [Indexed: 12/23/2022]
Abstract
Hepatitis B virus (HBV) replicates its genomic information in the nucleus via transcription and therefore has to deliver its partially double stranded DNA genome into the nucleus. Like other viruses with a nuclear replication phase, HBV genomes are transported inside the viral capsids first through the cytoplasm towards the nuclear envelope. Following the arrival at the nuclear pore, the capsids are transported through, using classical cellular nuclear import pathways. The arrest of nuclear import at the nucleoplasmic side of the nuclear pore is unique, however, and is where the capsids efficiently disassemble leading to genome release. In the latter phase of the infection, newly formed nucleocapsids in the cytosol have to move to budding sites at intracellular membranes carrying the three viral envelope proteins. Capsids containing single stranded nucleic acid are not enveloped, in contrast to empty and double stranded DNA containing capsids. A small linear domain in the large envelope protein and two areas on the capsid surface have been mapped, where point mutations strongly block nucleocapsid envelopment. It is possible that these domains are involved in the envelope--with capsid interactions driving the budding process. Like other enveloped viruses, HBV also uses the cellular endosomal sorting complexes required for transport (ESCRT) machinery for catalyzing budding through the membrane and away from the cytosol.
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Affiliation(s)
- Marie-Lise Blondot
- Univ. de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France
| | - Volker Bruss
- Institute for Virology, Helmholtz Zentrum München, Technische Universität Muenchen, Neuherberg, Germany
| | - Michael Kann
- Univ. de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; CHU de Bordeaux, Bordeaux, France.
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A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation. Antimicrob Agents Chemother 2015; 59:7061-72. [PMID: 26349829 DOI: 10.1128/aac.01558-15] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Accepted: 09/02/2015] [Indexed: 12/18/2022] Open
Abstract
Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection.
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26
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Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation. J Virol 2015. [PMID: 26202253 DOI: 10.1128/jvi.01481-15] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.
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Thedja MD, Muljono DH, Ie SI, Sidarta E, Turyadi, Verhoef J, Marzuki S. Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific. PLoS One 2015; 10:e0132533. [PMID: 26162099 PMCID: PMC4498642 DOI: 10.1371/journal.pone.0132533] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Accepted: 06/15/2015] [Indexed: 12/18/2022] Open
Abstract
Distribution of hepatitis B virus (HBV) genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C) is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates—East-Southeast Asia and Papua-Pacific—were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG). This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-.
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Affiliation(s)
- Meta Dewi Thedja
- Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Eijkman Winkler Institute, University Medical Centre (UMC) Utrecht, Utrecht, The Netherlands
| | - David Handojo Muljono
- Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | | | - Erick Sidarta
- Eijkman Institute for Molecular Biology, Jakarta, Indonesia
| | - Turyadi
- Eijkman Institute for Molecular Biology, Jakarta, Indonesia
| | - Jan Verhoef
- Eijkman Winkler Institute, University Medical Centre (UMC) Utrecht, Utrecht, The Netherlands
| | - Sangkot Marzuki
- Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Department of Medicine, Monash Medical Centre, Monash University, Clayton, Victoria, Australia
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28
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An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation. J Virol 2015; 89:9281-7. [PMID: 26136564 DOI: 10.1128/jvi.00466-15] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2015] [Accepted: 06/18/2015] [Indexed: 12/29/2022] Open
Abstract
UNLABELLED The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain [MD]) in the large surface protein L and a narrow region (matrix binding domain [MBD]) including isoleucine 126 on the capsid surface have been mapped, in which point mutations such as core I126A specifically blocked nucleocapsid envelopment. It is possible that the two domains interact with each other during virion morphogenesis. By the systematic evolution of ligands by exponential enrichment (SELEX) method, we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications, 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50% of the clones. The main aptamer, AO-01 (13 clones, 18%), showed the lowest dissociation constant (Kd) of 180 ± 82 nM for capsid binding among the four molecules. Its Kd for I126A capsids was 1,306 ± 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47% inhibition of virion production at 3 days posttransfection, but there was no inhibition by cotransfection of an aptamer with a random sequence. The half-life of AO-01 in cells was 2 h, which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction with a low-molecular-weight substance might represent a new approach for an antiviral therapy. IMPORTANCE Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55-base-long single-stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The results substantiate the current model for HBV morphogenesis and show that the capsid envelope interaction is a potential antiviral target.
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29
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Zlotnick A, Venkatakrishnan B, Tan Z, Lewellyn E, Turner W, Francis S. Core protein: A pleiotropic keystone in the HBV lifecycle. Antiviral Res 2015; 121:82-93. [PMID: 26129969 DOI: 10.1016/j.antiviral.2015.06.020] [Citation(s) in RCA: 203] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2015] [Revised: 06/22/2015] [Accepted: 06/26/2015] [Indexed: 12/21/2022]
Abstract
Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates almost every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals - while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story."
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Affiliation(s)
- Adam Zlotnick
- Molecular & Cellular Biology, Indiana University, Bloomington, IN, United States.
| | | | - Zhenning Tan
- Assembly BioSciences, Bloomington, IN, United States; Assembly BioSciences, San Francisco, CA, United States
| | - Eric Lewellyn
- Assembly BioSciences, Bloomington, IN, United States; Assembly BioSciences, San Francisco, CA, United States
| | - William Turner
- Assembly BioSciences, Bloomington, IN, United States; Assembly BioSciences, San Francisco, CA, United States
| | - Samson Francis
- Molecular & Cellular Biology, Indiana University, Bloomington, IN, United States; Assembly BioSciences, Bloomington, IN, United States; Assembly BioSciences, San Francisco, CA, United States
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30
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Shen L, Zhou J, Wang Y, Kang N, Ke X, Bi S, Ren L. Efficient encapsulation of Fe₃O₄ nanoparticles into genetically engineered hepatitis B core virus-like particles through a specific interaction for potential bioapplications. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2015; 11:1190-1196. [PMID: 25155647 DOI: 10.1002/smll.201401952] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Revised: 07/23/2014] [Indexed: 06/03/2023]
Abstract
Self-assembly of viral coating proteins encapsulating functional nanoparticles provides a new class of biomaterials with robust chemical and physical properties for potential applications in functional imaging, and therapeutic or diagnostic agent delivery. Herein, a straightforward method is demonstrated for efficient encapsidation of magnetic nanoparticles into the engineered virus-like particle (VLP) through the affinity of histidine tags for the nickel- nitrilotriacetic acid (NTA) chelate. Monodispersed, uniformly sized, magnetic core-containing VLPs are obtained at high efficiency (>85%) and used as the cellular T2 contrast agents for MR imaging applications thanks to their biocompatibility, higher cellular uptake, as well as higher r2 values.
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Affiliation(s)
- Lihua Shen
- Department of Biomaterials, College of Materials, Xiamen University, Xiamen, 361005, P.R. China
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31
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Glebe D, König A. Molecular virology of hepatitis B virus and targets for antiviral intervention. Intervirology 2014; 57:134-40. [PMID: 25034480 DOI: 10.1159/000360946] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The members of the viral family Hepadnaviridae comprise one of the smallest enveloped DNA viruses and cause acute and chronic infections in mammals and birds, leading to large virus and antigen loads in the blood. They have a restricted host range and depend on differentiated hepatocytes for replication. Hepatitis B virus (HBV) is the prototype of the Hepadnaviridae. HBV can persist in infected hepatocytes and has evolved elaborate strategies to evade the immune system. HBV replicates like HIV (family of Retroviridae) via reverse transcription. Drugs licensed for inhibition of HIV reverse transcriptase lower the viral load of chronic HBV patients, but they do not cure the infection. HBV genomes are archived in the nucleus of hepatocytes as episomal DNA before reverse transcription. In contrast, the RNA genome of HIV first needs reverse transcription before proviral integration within the host genome. Wild-type HBV remains relatively stable in chronic HBV patients during the immunotolerant state, but is able to evolve mutants rapidly upon selective pressure due to therapy or immune reactions. Current therapies for chronic hepatitis B are far from optimal. To extend therapeutic options, further studies on HBV and its interaction with the host are urgently needed.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research (DZIF), Biomedical Research Center Seltersberg, Giessen, Germany
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32
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Glebe D, Geipel A. Selected phenotypic assays used to evaluate antiviral resistance and viral fitness of hepatitis B virus and its variants. Intervirology 2014; 57:225-31. [PMID: 25034492 DOI: 10.1159/000360950] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Currently available antiviral therapies specifically target viral replication by blocking reverse transcription with orally given nucleos(t)ide analogues and are able to specifically suppress viral replication. The unique replication strategy of hepatitis B virus (HBV), however, allows long-term persistence of the viral genome within infected hepatocytes in spite of successful therapy. Thus, antiviral therapy needs to be continued for years. Therapy can result either in the emergence and selection of antiviral-resistant variants or the relapse of viral replication after the termination of antiviral therapy. Resistance is a major problem for 4 of the 5 approved HBV nucleos(t)ide analogues, but it is not the only reason for therapy failure. An accurate phenotypic in vitro assay for resistance allows the identification of a viral variant selected in vivo during antiviral therapy and helps to find therapeutic alternatives. Furthermore, these assays can be used to measure viral fitness and pathogenicity in vitro. With the help of these assays, the prediction of emerging viral variants with drug resistance or increased pathogenic potential can be realized. Phenotypic resistance tests for HBV are not trivial because the virus cannot be readily grown in cell culture. This review focuses on currently available phenotypic assays to evaluate antiviral resistance of HBV and fitness of viral variants in general.
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Affiliation(s)
- Dieter Glebe
- Institute of Medical Virology, Justus Liebig University Giessen, National Reference Center for Hepatitis B and D Viruses, German Center for Infection Research (DZIF), Biomedical Research Center Seltersberg, Giessen, Germany
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33
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Schittl B, Bruss V. Mutational profiling of the variability of individual amino acid positions in the hepatitis B virus matrix domain. Virology 2014; 458-459:183-9. [PMID: 24928050 DOI: 10.1016/j.virol.2014.04.030] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 02/12/2014] [Accepted: 04/22/2014] [Indexed: 01/20/2023]
Abstract
The hepatitis B virus (HBV) is formed by budding. A stretch of 22 amino acids (aa) (matrix domain, MD, R103 - S124) in the large envelope protein L is crucial for virion formation and probably establishes contact to the nucleocapsid. Here, we assess the impact of sequence variations at numerous individual aa positions within the MD on virion formation. We generated panels of L mutants covering all 19 possible aa for 11 positions and tested the capacity of these mutants to rescue virus production by an L-defective HBV genome. At four positions (L112, R113, P117, W122), any replacement of the wild type (WT) aa reduced virus assembly to undetectable levels. Virus production was strongly diminished by substitutions at five other positions (R103, T106, S115, H116, A119). Only two tested positions (D114, Q118) tolerated several substitutions. The restricted positions may represent promising targets for the development of novel antiviral strategies.
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Affiliation(s)
- Beate Schittl
- Institute of Virology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany
| | - Volker Bruss
- Institute of Virology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
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Homs M, Buti M, Tabernero D, Quer J, Sanchez A, Corral N, Esteban R, Rodriguez-Frias F. Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing. World J Gastroenterol 2012; 18:6096-6105. [PMID: 23155338 PMCID: PMC3496886 DOI: 10.3748/wjg.v18.i42.6096] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/16/2012] [Revised: 07/25/2012] [Accepted: 07/28/2012] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS). METHODS Four samples (2 genotype A and 2 genotype D) from 4 treatment-naïve patients were assessed for baseline variability. Two additional samples from one patient (patient 4, genotype D) were selected for analysis: one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment. The HBV region analyzed covered amino acids 40 to 95 of the core gene, and included the two main epitopic regions, Th50-69 and B74-84. UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences, Roche). After computer filtering of UDPS data based on a Poisson statistical model, 122,813 sequences were analyzed. The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture. RESULTS Positions with highest variability rates were mainly located in the main core epitopes, confirming their role as immune-stimulating regions. In addition, the distribution of variability showed a relationship with HBV genotype. Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%. Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%, respectively. The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed, approaching significance (P = 0.07, sample 1 and P = 0.05, sample 2). In contrast, there were no significant differences in variability between the epitopic and other positions in genotype D cases. Interestingly, patient 1 presented a completely mutated motif from amino acid 64 to 67 (E₆₄LMT₆₇), which is commonly recognized by T helper cells. Additionally, the variability observed in all 4 patients was particularly associated with the E₆₄LMT₆₇ motif. Codons 78 and 79 were highly conserved in all samples, in keeping with their involvement in the interaction between the HBV virion capsid and the surface antigens (HBsAg). Of note, codon 76 was even more conserved than codons 78 and 79, suggesting a possible role in HBsAg interactions or even in hepatitis B e antigen conformation. Sequential analysis of samples from patient 4 (genotype D) illustrated the dynamism of the HBV quasispecies, with strong selection of one minor baseline variant coinciding with a decrease in core variability during the treatment-free and lamivudine-treated period. The drop in variability seemed to result from a "steady state" situation of the HBV quasispecies after selection of the variant with greatest fitness. CONCLUSION Host immune pressure seems to be the main cause of HBV core evolution. UDPS analysis is a useful technique for studying viral quasispecies.
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Prange R. Host factors involved in hepatitis B virus maturation, assembly, and egress. Med Microbiol Immunol 2012; 201:449-61. [PMID: 22965171 DOI: 10.1007/s00430-012-0267-9] [Citation(s) in RCA: 96] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2012] [Accepted: 08/24/2012] [Indexed: 01/12/2023]
Abstract
Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Recent work has illuminated a multiplicity of spatially and temporally coordinated virus-host interactions that accompany HBV particle genesis. These interactions include the requirement of cellular chaperones for the maturation of the three viral envelope proteins, the cellular factors involved in dynamic modification, maturation, and intracellular trafficking of the nucleocapsids, and the host components of the multivesicular body (MVB) pathway enabling virion budding at intracellular compartments. Beside infectious virions, HBV produces at least two other types of particles, subviral empty envelope particles and subviral naked capsid particles, likely as a result of the engagement of different host factors by the viral structural proteins. Accordingly, HBV exploits distinct cellular pathways to release its particle types. Here, I review recent progress in these areas of the cell biology of HBV genesis.
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Affiliation(s)
- Reinhild Prange
- Institute of Medical Microbiology and Hygiene, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz, 55131 Mainz, Germany.
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Xie N, Huang K, Zhang T, Lei Y, Liu R, Wang K, Zhou S, Li J, Wu J, Wu H, Deng C, Zhao X, Nice EC, Huang C. Comprehensive proteomic analysis of host cell lipid rafts modified by HBV infection. J Proteomics 2012; 75:725-39. [DOI: 10.1016/j.jprot.2011.09.011] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2011] [Revised: 08/26/2011] [Accepted: 09/17/2011] [Indexed: 12/29/2022]
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A theoretical model for the dynamic structure of hepatitis B nucleocapsid. Biophys J 2011; 101:2476-84. [PMID: 22098746 DOI: 10.1016/j.bpj.2011.10.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2011] [Revised: 10/01/2011] [Accepted: 10/06/2011] [Indexed: 12/13/2022] Open
Abstract
The genomic material of hepatitis B virus (HBV) is confined within a fenestrated nucleocapsid consisting of 240 identical copies of the capsid protein, which has a rigid core and a positively charged and highly flexible C-terminal domain (CTD). Although previous mutagenesis studies have demonstrated the importance of the CTD in viral RNA packaging and reverse transcription, the microscopic structure of the CTD and its interaction with encapsidated nucleic acids at various stages of viral maturation remain poorly understood. Here, we present a theoretical analysis of the radial distributions of the CTD chains and nucleic acids in the hepatitis B virus nucleocapsid at the beginning and final stages of viral reverse transcription based on classical density functional theory and a coarse-gained model for the pertinent biomolecules. We find that a significant portion of the CTD is exposed at the surface of the RNA-containing immature nucleocapsid and that the CTD is mostly confined within the DNA-containing mature nucleocapsid. Large accumulation of cations is predicted inside both immature and mature nucleocapsids. The theoretical results provide new insights into the molecular mechanism of CTD regulation of viral reverse transcription and nucleocapsid trafficking during various stages of the viral replication processes.
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Bardens A, Döring T, Stieler J, Prange R. Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner. Cell Microbiol 2010; 13:602-19. [PMID: 21129143 PMCID: PMC7162389 DOI: 10.1111/j.1462-5822.2010.01557.x] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV‐replicating cells release non‐enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non‐vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release‐mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT‐III binding site is disrupted. Together, the boomerang‐shaped Bro1 domain of Alix appears to escort capsids without ESCRT.
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Affiliation(s)
- Andreas Bardens
- Department of Medical Microbiology and Hygiene,University Medical Center of the Johannes Gutenberg University, Mainz, Germany
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Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol 2010; 84:9332-40. [PMID: 20610715 DOI: 10.1128/jvi.00918-10] [Citation(s) in RCA: 106] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Alpha interferon (IFN-alpha) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-gamma) is a key mediator of host antiviral immunity against hepatitis B virus (HBV) infection in vivo. However, the molecular mechanism by which these antiviral cytokines suppress HBV replication remains elusive. Using an immortalized murine hepatocyte (AML12)-derived cell line supporting tetracycline-inducible HBV replication, we show in this report that both IFN-alpha and IFN-gamma efficiently reduce the amount of intracellular HBV nucleocapsids. Furthermore, we provide evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings thus reveal a novel antiviral mechanism of IFNs and provide a basis for a better understanding of HBV pathobiology.
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