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Zhang H, Long Q, Liu Y, Marchetti AL, Liu CD, Sun N, Guo H. Host 3' flap endonuclease Mus81 plays a critical role in trimming the terminal redundancy of hepatitis B virus relaxed circular DNA during covalently closed circular DNA formation. PLoS Pathog 2025; 21:e1012918. [PMID: 39913382 PMCID: PMC11801639 DOI: 10.1371/journal.ppat.1012918] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2024] [Accepted: 01/17/2025] [Indexed: 02/11/2025] Open
Abstract
Hepatitis B virus (HBV) relaxed circular DNA (rcDNA) possesses an 8-9 nucleotide-long terminal redundancy (TR, or r) on the negative (-) strand DNA derived from the reverse transcription of viral pregenomic RNA (pgRNA). It remains unclear whether the TR forms a 5' or 3' flap structure on HBV rcDNA and which TR copy is removed during covalently closed circular DNA (cccDNA) formation. To address these questions, a mutant HBV cell line HepDES-C1822G was established with a C1822G mutation in the pgRNA coding sequence, altering the sequence of 3' TR of (-) strand DNA while the 5' TR remained wild type (wt). The production of HBV rcDNA and cccDNA in HepDES-C1822G cells was comparable to wt levels. Next-generation sequencing (NGS) analysis revealed that the positive (+) strand DNA of rcDNA and both strands of cccDNA predominantly carried the wt nt1822 residue, indicating that the 5' TR of (-) strand DNA serves as the template during rcDNA replication, forming a duplex with the (+) strand DNA, while the 3' TR forms a flap-like structure, which is subsequently removed during cccDNA formation. In a survey of known cellular flap endonucleases using a loss-of-function study, we found that the 3' flap endonuclease Mus81 plays a critical role in cccDNA formation in wild-type HBV replicating cells, alongside the 5' flap endonuclease FEN1. Additionally, we have mapped the potential Mus81 and FEN1 cleavage sites within the TR of nuclear DP-rcDNA by RACE-NGS analyses. The overlapping function between Mus81 and FEN1 in cccDNA formation indicates that the putative 5' and 3' flap formed by TR are dynamically interchangeable on rcDNA precursor. These findings shed light on HBV rcDNA structure and cccDNA formation mechanisms, contributing to our understanding of HBV replication cycle.
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Affiliation(s)
- 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
| | - Quanxin Long
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - 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
| | - Alexander L. Marchetti
- 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
| | - Cheng-Der 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
| | - Ning Sun
- 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
| | - 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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2
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Zhang X, Lim K, Qiu Y, Hazawa M, Wong RW. Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways. Viruses 2025; 17:151. [PMID: 40006906 PMCID: PMC11860923 DOI: 10.3390/v17020151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Revised: 01/20/2025] [Accepted: 01/21/2025] [Indexed: 02/27/2025] Open
Abstract
Viruses frequently exploit the host's nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins and other components, they strategically target host nuclear transport receptors (NTRs) and nucleoporins within the spiderweb-like inner channel of the nuclear pore complex (NPC), enabling efficient access to the host nucleus. This review explores the intricate mechanisms governing the nuclear import and export of viral components, with a focus on the interplay between viral factors and host determinants that are essential for these processes. Given the pivotal role of nucleocytoplasmic shuttling in the viral life cycle, we also examine therapeutic strategies aimed at disrupting the host's nuclear transport pathways. This includes evaluating the efficacy of pharmacological inhibitors in impairing viral replication and assessing their potential as antiviral treatments. Furthermore, we emphasize the need for continued research to develop targeted therapies that leverage vulnerabilities in nucleocytoplasmic trafficking. Emerging high-resolution techniques, such as advanced imaging and computational modeling, are transforming our understanding of the dynamic interactions between viruses and the NPC. These cutting-edge tools are driving progress in identifying novel therapeutic opportunities and uncovering deeper insights into viral pathogenesis. This review highlights the importance of these advancements in paving the way for innovative antiviral strategies.
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Affiliation(s)
- Xin Zhang
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
| | - Keesiang Lim
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
| | - Yujia Qiu
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
| | - Masaharu Hazawa
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
| | - Richard W. Wong
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
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3
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Welch SR, Bilello JP, Carter K, Delang L, Dirr L, Durantel D, Feng JY, Gowen BB, Herrero LJ, Janeba Z, Kleymann G, Lee AA, Meier C, Moffat J, Schang LM, Schiffer JT, Seley-Radtke KL, Sheahan TP, Spengler JR. Meeting report of the 37th International Conference on Antiviral Research in Gold Coast, Australia, May 20-24, 2024, organized by the International Society for Antiviral Research. Antiviral Res 2024; 232:106037. [PMID: 39542140 PMCID: PMC11871649 DOI: 10.1016/j.antiviral.2024.106037] [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: 10/12/2024] [Revised: 11/07/2024] [Accepted: 11/07/2024] [Indexed: 11/17/2024]
Abstract
The 37th International Conference on Antiviral Research (ICAR) was held in Gold Coast, Australia, May 20-24, 2024. ICAR 2024 featured over 75 presentations along with two poster sessions and special events, including those specifically tailored for trainees and early-career scientists. The meeting served as a platform for the exchange of cutting-edge research, with presentations and discussions covering novel antiviral compounds, vaccine development, clinical trials, and therapeutic advancements. A comprehensive array of topics in antiviral science was covered, from the latest breakthroughs in antiviral drug development to innovative strategies for combating emerging viral threats. The keynote presentations provided fascinating insight into two diverse areas fundamental to medical countermeasure development and use, including virus emergence at the human-animal interface and practical considerations for bringing antivirals to the clinic. Additional sessions addressed a variety of timely post-pandemic topics, such as the hunt for broad spectrum antivirals, combination therapy, pandemic preparedness, application of in silico tools and AI in drug discovery, the virosphere, and more. Here, we summarize all the presentations and special sessions of ICAR 2024 and introduce the 38th ICAR, which will be held in Las Vegas, USA, March 17-21, 2025.
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Affiliation(s)
- Stephen R Welch
- Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA.
| | | | | | - Leen Delang
- Virus-Host Interactions & Therapeutic Approaches Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
| | - Larissa Dirr
- Institute for Biomedicine and Glycomics, Griffith University, Southport, QLD, Australia
| | - David Durantel
- Centre International de Recherche en Infectiologie (CIRI), Inserm_U1111, CNRS_UMR5308, Université Claude Bernard Lyon 1, F-69007, Lyon, France
| | - Joy Y Feng
- Division of the Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, GA, USA
| | - Brian B Gowen
- Institute for Antiviral Research and Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT, USA
| | - Lara J Herrero
- Institute for Biomedicine and Glycomics, Griffith University, Southport, QLD, Australia
| | - Zlatko Janeba
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nám. 2, 160 00, Prague, Czech Republic
| | - Gerald Kleymann
- Innovative Molecules GmbH, Lipowsky Str. 10, 81373, Munich, Bavaria, Germany
| | | | - Chris Meier
- Organic Chemistry, Department of Chemistry, Faculty of Sciences, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg, Germany
| | - Jennifer Moffat
- Department of Microbiology & Immunology, SUNY Upstate Medical University, Syracuse, NY, USA
| | - Luis M Schang
- Baker Institute and Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA
| | - Joshua T Schiffer
- Fred Hutchinson Cancer Research Center, Vaccine and Infectious Diseases Division, Seattle, WA, USA; Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA
| | - Katherine L Seley-Radtke
- Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, Baltimore, MD, USA
| | - Timothy P Sheahan
- Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA; Rapidly Emerging Antiviral Drug Development Initiative, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Jessica R Spengler
- Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA.
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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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5
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Khaykelson D, Asor R, Zhao Z, Schlicksup CJ, Zlotnick A, Raviv U. Guanidine Hydrochloride-Induced Hepatitis B Virus Capsid Disassembly Hysteresis. Biochemistry 2024; 63:1543-1552. [PMID: 38787909 PMCID: PMC11191408 DOI: 10.1021/acs.biochem.4c00077] [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: 02/14/2024] [Revised: 04/20/2024] [Accepted: 04/25/2024] [Indexed: 05/26/2024]
Abstract
Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.
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Affiliation(s)
- Daniel Khaykelson
- Institute
of Chemistry and the Center for Nanoscience and Nanotechnology, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 9190401, Israel
| | - Roi Asor
- Institute
of Chemistry and the Center for Nanoscience and Nanotechnology, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 9190401, Israel
| | - Zhongchao Zhao
- Department
of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Christopher John Schlicksup
- Department
of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Adam Zlotnick
- Department
of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Uri Raviv
- Institute
of Chemistry and the Center for Nanoscience and Nanotechnology, the Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 9190401, Israel
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Chung CY, Sun CP, Tao MH, Wu HL, Wang SH, Yeh SH, Zheng QB, Yuan Q, Xia NS, Ogawa K, Nakashima K, Suzuki T, Chen PJ. Major HBV splice variant encoding a novel protein important for infection. J Hepatol 2024; 80:858-867. [PMID: 38336347 DOI: 10.1016/j.jhep.2024.01.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 01/16/2024] [Accepted: 01/23/2024] [Indexed: 02/12/2024]
Abstract
BACKGROUND & AIMS HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.
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Affiliation(s)
- Chen-Yen Chung
- National Taiwan University College of Medicine, Taipei, Taiwan
| | - Cheng-Pu Sun
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Mi-Hua Tao
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Hui-Lin Wu
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
| | - Sheng-Han Wang
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
| | - Shiou-Hwei Yeh
- National Taiwan University College of Medicine, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
| | - Qing-Bing Zheng
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen, P.R. China
| | - Quan Yuan
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen, P.R. China
| | - Ning-Shao Xia
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen, P.R. China
| | - Kenji Ogawa
- RIKEN Center for Sustainable Resource Science (CSRS), RIKEN, Wako, Saitama, Japan
| | | | | | - Pei-Jer Chen
- National Taiwan University College of Medicine, Taipei, Taiwan.
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7
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Shen S, Yan R, Xie Z, Yu X, Liang H, You Q, Zhang H, Hou J, Zhang X, Liu Y, Sun J, Guo H. Tripartite Motif-Containing Protein 65 (TRIM65) Inhibits Hepatitis B Virus Transcription. Viruses 2024; 16:890. [PMID: 38932182 PMCID: PMC11209081 DOI: 10.3390/v16060890] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Revised: 05/28/2024] [Accepted: 05/29/2024] [Indexed: 06/28/2024] Open
Abstract
Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65's antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins' capacity to inhibit HBV transcription and highlight TRIM65's pivotal role in this process.
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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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; (X.Y.); (H.Z.); (Y.L.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - Ran Yan
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - Zhanglian Xie
- 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - Xiaoyang Yu
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; (X.Y.); (H.Z.); (Y.L.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
| | - Qiuhong You
- 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
| | - Hu Zhang
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; (X.Y.); (H.Z.); (Y.L.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - Jinlin Hou
- 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
| | - Xiaoyong Zhang
- 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
| | - Yuanjie Liu
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; (X.Y.); (H.Z.); (Y.L.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
| | - 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 510515, China; (S.S.); (Z.X.); (H.L.); (Q.Y.); (J.H.); (X.Z.)
| | - Haitao Guo
- Department of Microbiology and Molecular Genetics; Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; (X.Y.); (H.Z.); (Y.L.)
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA;
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8
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Hong X, Mendenhall MA, Hu J. Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation. Methods Mol Biol 2024; 2837:99-111. [PMID: 39044078 DOI: 10.1007/978-1-0716-4027-2_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/25/2024]
Abstract
Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and of its reliable detection due to its low abundance and the presence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA and all DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.
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Affiliation(s)
- Xupeng Hong
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Megan A Mendenhall
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, USA.
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9
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Shen S, Liu W, Zeng G, Liang H, Yu X, Zhang H, Sun J, Guo H. Conditional replication and secretion of hepatitis B virus genome uncover the truncated 3' terminus of encapsidated viral pregenomic RNA. J Virol 2023; 97:e0076023. [PMID: 37754759 PMCID: PMC10617516 DOI: 10.1128/jvi.00760-23] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2023] [Accepted: 08/07/2023] [Indexed: 09/28/2023] Open
Abstract
IMPORTANCE The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics.
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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, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - 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
| | - 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
| | - 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
| | - Xiaoyang Yu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Hu Zhang
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - 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, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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10
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Jin Y, Wang S, Xu S, Zhao S, Xu X, Poongavanam V, Menéndez-Arias L, Zhan P, Liu X. Targeting hepatitis B virus cccDNA levels: Recent progress in seeking small molecule drug candidates. Drug Discov Today 2023; 28:103617. [PMID: 37196762 DOI: 10.1016/j.drudis.2023.103617] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 03/29/2023] [Accepted: 05/10/2023] [Indexed: 05/19/2023]
Abstract
Hepatitis B virus (HBV) infection is a major global health problem that puts people at high risk of death from cirrhosis and liver cancer. The presence of covalently closed circular DNA (cccDNA) in infected cells is considered to be the main obstacle to curing chronic hepatitis B. At present, the cccDNA cannot be completely eliminated by standard treatments. There is an urgent need to develop drugs or therapies that can reduce HBV cccDNA levels in infected cells. We summarize the discovery and optimization of small molecules that target cccDNA synthesis and degradation. These compounds are cccDNA synthesis inhibitors, cccDNA reducers, core protein allosteric modulators, ribonuclease H inhibitors, cccDNA transcriptional modulators, HBx inhibitors and other small molecules that reduce cccDNA levels.
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Affiliation(s)
- Yu Jin
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
| | - Shuo Wang
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
| | - Shujing Xu
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
| | - Shujie Zhao
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
| | - Xiangrui Xu
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
| | - Vasanthanathan Poongavanam
- Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M DK-5230, Denmark
| | - Luis Menéndez-Arias
- Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), 28049 Madrid, Spain.
| | - Peng Zhan
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China.
| | - Xinyong Liu
- Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China.
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11
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Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA. J Virol 2023; 97:e0126122. [PMID: 36519896 PMCID: PMC9888207 DOI: 10.1128/jvi.01261-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.
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12
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Pregenomic RNA Launch Hepatitis B Virus Replication System Facilitates the Mechanistic Study of Antiviral Agents and Drug-Resistant Variants on Covalently Closed Circular DNA Synthesis. J Virol 2022; 96:e0115022. [PMID: 36448800 PMCID: PMC9769369 DOI: 10.1128/jvi.01150-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2022] Open
Abstract
Hepatitis B virus (HBV) replicates its genomic DNA by reverse transcription of an RNA intermediate, termed pregenomic RNA (pgRNA), within nucleocapsid. It had been shown that transfection of in vitro-transcribed pgRNA initiated viral replication in human hepatoma cells. We demonstrated here that viral capsids, single-stranded DNA, relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA) became detectable sequentially at 3, 6, 12, and 24 h post-pgRNA transfection into Huh7.5 cells. The levels of viral DNA replication intermediates and cccDNA peaked at 24 and 48 h post-pgRNA transfection, respectively. HBV surface antigen (HBsAg) became detectable in culture medium at day 4 posttransfection. Interestingly, the early robust viral DNA replication and cccDNA synthesis did not depend on the expression of HBV X protein (HBx), whereas HBsAg production was strictly dependent on viral DNA replication and expression of HBx, consistent with the essential role of HBx in the transcriptional activation of cccDNA minichromosomes. While the robust and synchronized HBV replication within 48 h post-pgRNA transfection is particularly suitable for the precise mapping of the HBV replication steps, from capsid assembly to cccDNA formation, targeted by distinct antiviral agents, the treatment of cells starting at 48 h post-pgRNA transfection allows the assessment of antiviral agents on mature nucleocapsid uncoating, cccDNA synthesis, and transcription, as well as viral RNA stability. Moreover, the pgRNA launch system could be used to readily assess the impacts of drug-resistant variants on cccDNA formation and other replication steps in the viral life cycle. IMPORTANCE Hepadnaviral pgRNA not only serves as a template for reverse transcriptional replication of viral DNA but also expresses core protein and DNA polymerase to support viral genome replication and cccDNA synthesis. Not surprisingly, cytoplasmic expression of duck hepatitis B virus pgRNA initiated viral replication leading to infectious virion secretion. However, HBV replication and antiviral mechanism were studied primarily in human hepatoma cells transiently or stably transfected with plasmid-based HBV replicons. The presence of large amounts of transfected HBV DNA or transgenes in cellular chromosomes hampered the robust analyses of HBV replication and cccDNA function. As demonstrated here, the pgRNA launch HBV replication system permits the accurate mapping of antiviral target and investigation of cccDNA biosynthesis and transcription using secreted HBsAg as a convenient quantitative marker. The effect of drug-resistant variants on viral capsid assembly, genome replication, and cccDNA biosynthesis and function can also be assessed using this system.
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13
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Chen Z, Yuan Y, Yang D, Luo M, Liang Q, Li Z, Lu S, Sun J, Deng M, Liu M, Liang Z, Liu K. Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus. J Med Virol 2022; 94:5987-5999. [PMID: 36000452 DOI: 10.1002/jmv.28087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 08/02/2022] [Accepted: 08/19/2022] [Indexed: 01/06/2023]
Abstract
Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.
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Affiliation(s)
- Zhuohang Chen
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Yan Yuan
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Di Yang
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Minhui Luo
- School of Public Health, Southern Medical University, Guangzhou, China
| | - Qian Liang
- Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, China
| | - Zan Li
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Siya Lu
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Jianan Sun
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Maohua Deng
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Miaoya Liu
- College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China
| | - Zongsuo Liang
- College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China
| | - Kuancheng Liu
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
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14
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Nakanishi A, Okumura H, Hashita T, Yamashita A, Nishimura Y, Watanabe C, Kamimura S, Hayashi S, Murakami S, Ito K, Iwao T, Ikeda A, Hirose T, Sunazuka T, Tanaka Y, Matsunaga T. Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2. Viruses 2022; 14:2468. [PMID: 36366568 PMCID: PMC9695645 DOI: 10.3390/v14112468] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 10/28/2022] [Accepted: 11/04/2022] [Indexed: 11/09/2022] Open
Abstract
Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1-6 suppressed the production of cccDNA. These results suggest that KPNA1-6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.
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Affiliation(s)
- Anna Nakanishi
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Hiroki Okumura
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Tadahiro Hashita
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Aya Yamashita
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Yuka Nishimura
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Chihiro Watanabe
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Sakina Kamimura
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Sanae Hayashi
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
- Department of Gastroenterology and Hepatology, Kumamoto University, Kumamoto 860-8556, Japan
| | - Shuko Murakami
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
| | - Kyoko Ito
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
| | - Takahiro Iwao
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
| | - Akari Ikeda
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Tomoyasu Hirose
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Toshiaki Sunazuka
- Ōmura Satoshi Memorial Institute, Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan
- Department of Gastroenterology and Hepatology, Kumamoto University, Kumamoto 860-8556, Japan
| | - Tamihide Matsunaga
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
- Educational Research Center for Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
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15
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HIRA Supports Hepatitis B Virus Minichromosome Establishment and Transcriptional Activity in Infected Hepatocytes. Cell Mol Gastroenterol Hepatol 2022; 14:527-551. [PMID: 35643233 PMCID: PMC9304598 DOI: 10.1016/j.jcmgh.2022.05.007] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 05/11/2022] [Accepted: 05/18/2022] [Indexed: 12/10/2022]
Abstract
BACKGROUND & AIMS Upon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes. METHODS Detailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na+-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2hNTCP) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription. RESULTS Our results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production. CONCLUSIONS Altogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes.
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16
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Song Y, Shou S, Guo H, Gao Z, Liu N, Yang Y, Wang F, Deng Q, Liu J, Xie Y. Establishment and Characterization of a New Cell Culture System for Hepatitis B Virus Replication and Infection. Virol Sin 2022; 37:558-568. [PMID: 35568375 PMCID: PMC9437612 DOI: 10.1016/j.virs.2022.05.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 04/18/2022] [Indexed: 11/24/2022] Open
Abstract
Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents.
BEL7404 cells are characterized by a high transfection efficiency, but do not support canonical HBV replication. BEL7404 cells lack endogenous HNF4α expression, and exogenous HNF4α rescues canonical HBV replication. BEL7404 cells with stable hNTCP and inducible HNF4α expression support HBV infection and inducible replication. BEL7404-derived cell lines supporting HBV infection retain high transfection efficiencies and allow testing of antivirals.
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Affiliation(s)
- Yingying Song
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Shuyu Shou
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Huimin Guo
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China; Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, Shenzhen 518112, China; The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen 518112, China
| | - Zixiang Gao
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Nannan Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Yang Yang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Feifei Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Qiang Deng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Jing Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China; Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China.
| | - Youhua Xie
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China; Children's Hospital, Fudan University, Shanghai 201102, China.
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17
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Luo M, Chen Z, Liu M, Liang Q, Han R, Liang Z, Ye Z, Liu K. Inhibitory Activities of Ranunculus japonicus Thunb. Ethanol Extract against Hepatitis B Virus. J Med Virol 2022; 94:2727-2735. [PMID: 35075662 DOI: 10.1002/jmv.27621] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Revised: 01/16/2022] [Accepted: 01/21/2022] [Indexed: 11/08/2022]
Abstract
The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that Ranunculus japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that Ranunculus japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Minhui Luo
- School of Public Health (Shenzhen), Sun Yat-Sen University, Guangzhou, 510006, China
| | - Zhuohang Chen
- School of Public Health, Southern Medical University, Guangzho, 510000, China
| | - Miaoya Liu
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Qian Liang
- Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, 650224, China
| | - Ruilian Han
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Zongsuo Liang
- College of Life Sciences & Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China
| | - Zhuoming Ye
- School of Public Health, Southern Medical University, Guangzho, 510000, China
| | - Kuancheng Liu
- School of Public Health (Shenzhen), Sun Yat-Sen University, Guangzhou, 510006, China.,Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China
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18
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Zhang X, Wang Y, Yang G. Research progress in hepatitis B virus covalently closed circular DNA. Cancer Biol Med 2021; 19:j.issn.2095-3941.2021.0454. [PMID: 34931766 PMCID: PMC9088183 DOI: 10.20892/j.issn.2095-3941.2021.0454] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 11/16/2021] [Indexed: 11/28/2022] Open
Abstract
Hepatitis B virus (HBV) infections are a global public health issue. HBV covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is a key factor in the HBV replication cycle. Notably, many host factors involved in HBV cccDNA epigenetic modulation promote the development of hepatocellular carcinoma (HCC). The HBV cccDNA minichromosome is a clinical obstacle that cannot be efficiently eliminated. In this review, we provide an update on the advances in research on HBV cccDNA and further discuss factors affecting the modulation of HBV cccDNA. Hepatitis B virus X protein (HBx) contributes to HBV cccDNA transcription and the development of hepatocarcinogenesis through modulating host epigenetic regulatory factors, thus linking the cccDNA to hepatocarcinogenesis. The measurable serological biomarkers of continued transcription of cccDNA, the effects of anti-HBV drugs on cccDNA, and potential therapeutic strategies targeting cccDNA are discussed in detail. Thus, this review describes new insights into HBV cccDNA mechanisms and therapeutic strategies for cleaning cccDNA, which will benefit patients with liver diseases.
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Affiliation(s)
- Xiaodong Zhang
- Department of Gastrointestinal Cancer Biology, Liver Cancer Center, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
| | - Yufei Wang
- Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Guang Yang
- Department of Gastrointestinal Cancer Biology, Liver Cancer Center, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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19
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Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation. J Virol 2021; 96:e0136021. [PMID: 34705558 DOI: 10.1128/jvi.01360-21] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify said host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the co-precipitated proteins by mass spectrometry. The DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported shRNA screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Trans-complementation of wild type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA-binding, DDB1-binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified a handful of host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNAi screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay we discovered a novel relationship between the UV-DDB complex and cccDNA formation, hence, providing a proof-of-concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.
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20
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Zhao F, Xie X, Tan X, Yu H, Tian M, Lv H, Qin C, Qi J, Zhu Q. The Functions of Hepatitis B Virus Encoding Proteins: Viral Persistence and Liver Pathogenesis. Front Immunol 2021; 12:691766. [PMID: 34456908 PMCID: PMC8387624 DOI: 10.3389/fimmu.2021.691766] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Accepted: 07/26/2021] [Indexed: 12/14/2022] Open
Abstract
About 250 million people worldwide are chronically infected with Hepatitis B virus (HBV), contributing to a large burden on public health. Despite the existence of vaccines and antiviral drugs to prevent infection and suppress viral replication respectively, chronic hepatitis B (CHB) cure remains a remote treatment goal. The viral persistence caused by HBV is account for the chronic infection which increases the risk for developing liver cirrhosis and hepatocellular carcinoma (HCC). HBV virion utilizes various strategies to escape surveillance of host immune system therefore enhancing its replication, while the precise mechanisms involved remain elusive. Accumulating evidence suggests that the proteins encoded by HBV (hepatitis B surface antigen, hepatitis B core antigen, hepatitis B envelope antigen, HBx and polymerase) play an important role in viral persistence and liver pathogenesis. This review summarizes the major findings in functions of HBV encoding proteins, illustrating how these proteins affect hepatocytes and the immune system, which may open new venues for CHB therapies.
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Affiliation(s)
- Fenglin Zhao
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China
| | - Xiaoyu Xie
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Xu Tan
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Hongli Yu
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China
| | - Miaomiao Tian
- Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Huanran Lv
- Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Chengyong Qin
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Jianni Qi
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
| | - Qiang Zhu
- Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.,Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, China.,Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.,The First Affiliated Hospital of Xinjiang Medical University, Urumqi, China
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21
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Wei L, Ploss A. Mechanism of Hepatitis B Virus cccDNA Formation. Viruses 2021; 13:v13081463. [PMID: 34452329 PMCID: PMC8402782 DOI: 10.3390/v13081463] [Citation(s) in RCA: 68] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 07/14/2021] [Accepted: 07/21/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.
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22
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RNA Helicase DDX17 inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation. J Virol 2021; 95:e0044421. [PMID: 34287051 DOI: 10.1128/jvi.00444-21] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
DDX17 is a member of the DEAD-Box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a co-factor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley Fever Virus though binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits Hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of DEAD-box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17, but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.
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23
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Sajidah ES, Lim K, Wong RW. How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System. Cells 2021; 10:1424. [PMID: 34200500 PMCID: PMC8230057 DOI: 10.3390/cells10061424] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2021] [Revised: 05/31/2021] [Accepted: 06/03/2021] [Indexed: 12/14/2022] Open
Abstract
The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.
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Affiliation(s)
- Elma Sakinatus Sajidah
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
| | - Keesiang Lim
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
| | - Richard W. Wong
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
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24
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Hu J, Tang L, Cheng J, Zhou T, Li Y, Chang J, Zhao Q, Guo JT. Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases. Emerg Microbes Infect 2021; 10:852-864. [PMID: 33870849 PMCID: PMC8812769 DOI: 10.1080/22221751.2021.1919034] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5′ end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.
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Affiliation(s)
- Jin Hu
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA.,Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Liudi Tang
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA.,Microbiology and Immunology Graduate Program, Drexel University College of Medicine, Philadelphia, PA, USA
| | - Junjun Cheng
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
| | - Tianlun Zhou
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
| | - Yuhuan Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Jinhong Chang
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
| | - Qiong Zhao
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
| | - Ju-Tao Guo
- Department of Experimental Medicine, Baruch S. Blumberg Institute, Doylestown, PA, USA
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25
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Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation. Viruses 2021; 13:v13050757. [PMID: 33925977 PMCID: PMC8145197 DOI: 10.3390/v13050757] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) remains a major public health concern, with more than 250 million chronically infected people who are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma. Although antiviral treatments efficiently control virus replication and improve liver function, they cannot cure HBV infection. Viral persistence is due to the maintenance of the viral circular episomal DNA, called covalently closed circular DNA (cccDNA), in the nuclei of infected cells. cccDNA not only resists antiviral therapies, but also escapes innate antiviral surveillance. This viral DNA intermediate plays a central role in HBV replication, as cccDNA is the template for the transcription of all viral RNAs, including pregenomic RNA (pgRNA), which in turn feeds the formation of cccDNA through a step of reverse transcription. The establishment and/or expression of cccDNA is thus a prime target for the eradication of HBV. In this review, we provide an update on the current knowledge on the initial steps of HBV infection, from the nuclear import of the nucleocapsid to the formation of the cccDNA.
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26
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Maepa MB, Bloom K, Ely A, Arbuthnot P. Hepatitis B virus: promising drug targets and therapeutic implications. Expert Opin Ther Targets 2021; 25:451-466. [PMID: 33843412 DOI: 10.1080/14728222.2021.1915990] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Introduction: Current therapy for infection with hepatitis B virus (HBV) rarely clears the virus, and viremia commonly resurges following treatment withdrawal. To prevent serious complications of the infection, research has been aimed at identifying new viral and host targets that can be exploited to inactivate HBV replication.Areas covered: This paper reviews the use of these new molecular targets to advance anti-HBV therapy. Emphasis is on appraising data from pre-clinical and early clinical studies described in journal articles published during the past 10 years and available from PubMed.Expert opinion: The wide range of viral and host factors that can be targeted to disable HBV is impressive and improved insight into HBV molecular biology continues to provide the basis for new drug design. In addition to candidate therapies that have direct or indirect actions on HBV covalently closed circular DNA (cccDNA), compounds that inhibit HBsAg secretion, viral entry, destabilize viral RNA and effect enhanced immune responses to HBV show promise. Preclinical and clinical evaluation of drug candidates, as well as investigating use of treatment combinations, are encouraging. The field is poised at an interesting stage and indications are that reliably achieving functional cure from HBV infection is a tangible goal.
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Affiliation(s)
- Mohube Betty Maepa
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Kristie Bloom
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Abdullah Ely
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- School of Pathology, Faculty of Health Sciences, Wits/SAMRC Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
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27
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Li M, Xu Z, Zou X, Wang Y, Li Y, Ou X, Deng Y, Guo Y, Gan W, Chen D, Peng T, Xiao J, Cai M. Intracellular distribution of pseudorabies virus UL2 and detection of its nuclear import mechanism. Biol Chem 2021; 401:309-317. [PMID: 31665103 DOI: 10.1515/hsz-2019-0311] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Accepted: 10/10/2019] [Indexed: 11/15/2022]
Abstract
Pseudorabies virus (PRV) UL2 (pUL2) is a multifunctional protein, which is homologous with herpes simplex virus 1 early protein UL2 (hUL2) and crucial for the viral propagation. Yet, how pUL2 executes its roles in the viral life cycle remain inadequately understood. In order to uncover its effect on the procedure of PRV infection, investigation was performed to examine the subcellular distribution of pUL2 and establish its trafficking mechanism. In the present study, enhanced yellow fluorescent protein or Myc tag fused pUL2 was transiently overexpressed in transfected cells and exhibited an absolutely nuclear accumulation without the existence of other PRV proteins. Additionally, the nuclear trafficking of pUL2 was proved to rely on Ran-, transportin-1, importin β1, importin α1, α3 and α5. Accordingly, these data will benefit the knowledge of pUL2-mediated biological effects in PRV infection cycle.
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Affiliation(s)
- Meili Li
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Zuo Xu
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Xingmei Zou
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yuanfang Wang
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yiwen Li
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Xiaowen Ou
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yangxi Deng
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yingjie Guo
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Weidong Gan
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Daixiong Chen
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Tao Peng
- State Key Laboratory of Respiratory Diseases, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Xinzao Town, Panyu, Guangzhou 511436, Guangdong, China.,South China Vaccine Corporation Limited, Guangzhou Science Park, Guangzhou 510663, Guangdong, China
| | - Jing Xiao
- State Key Laboratory of Respiratory Diseases, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Xinzao Town, Panyu, Guangzhou 511436, Guangdong, China
| | - Mingsheng Cai
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
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Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA. J Virol 2020; 95:JVI.00922-20. [PMID: 33055252 DOI: 10.1128/jvi.00922-20] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Accepted: 10/05/2020] [Indexed: 12/13/2022] Open
Abstract
The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand [(-)strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand [(+)strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.IMPORTANCE The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
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29
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Guo F, Li K, Wu J, He L, Zhang L. Effects of Topological Constraints on Penetration Structures of Semi-Flexible Ring Polymers. Polymers (Basel) 2020; 12:E2659. [PMID: 33187232 PMCID: PMC7696204 DOI: 10.3390/polym12112659] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Revised: 11/03/2020] [Accepted: 11/08/2020] [Indexed: 11/24/2022] Open
Abstract
The effects of topological constraints on penetration structures of semi-flexible ring polymers in a melt are investigated using molecular dynamics simulations, considering simultaneously the effects of the chain stiffness. Three topology types of rings are considered: 01-knot (the unknotted), 31-knot and 61-knot ring polymers, respectively. With the improved algorithm to detect and quantify the inter-ring penetration (or inter-ring threading), the degree of ring threading does not increase monotonously with the chain stiffness, existing a peak value at the intermediate stiffness. It indicates that rings interpenetrate most at intermediate stiffness where there is a balance between coil expansion (favoring penetrations) and stiffness (inhibiting penetrations). Meanwhile, the inter-ring penetration would be suppressed with the knot complexity of the rings. The analysis of effective potential between the rings provides a better understanding for this non-monotonous behavior in inter-ring penetration.
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Affiliation(s)
- Fuchen Guo
- Department of Physics, Zhejiang University, Hangzhou 310027, China; (F.G.); (K.L.); (J.W.)
| | - Ke Li
- Department of Physics, Zhejiang University, Hangzhou 310027, China; (F.G.); (K.L.); (J.W.)
| | - Jiaxin Wu
- Department of Physics, Zhejiang University, Hangzhou 310027, China; (F.G.); (K.L.); (J.W.)
| | - Linli He
- Department of Physics, Wenzhou University, Wenzhou 325035, China
| | - Linxi Zhang
- Department of Physics, Zhejiang University, Hangzhou 310027, China; (F.G.); (K.L.); (J.W.)
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30
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Marchetti AL, Guo H. New Insights on Molecular Mechanism of Hepatitis B Virus Covalently Closed Circular DNA Formation. Cells 2020; 9:cells9112430. [PMID: 33172220 PMCID: PMC7694973 DOI: 10.3390/cells9112430] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 11/03/2020] [Accepted: 11/04/2020] [Indexed: 12/15/2022] Open
Abstract
The chronic factor of the Hepatitis B Virus (HBV), specifically the covalently closed circular DNA (cccDNA), is a highly stable and active viral episomal genome established in the livers of chronic hepatitis B patients as a constant source of disease. Being able to target and eliminate cccDNA is the end goal for a genuine cure for HBV. Yet how HBV cccDNA is formed from the viral genomic relaxed circular DNA (rcDNA) and by what host factors had been long-standing research questions. It is generally acknowledged that HBV hijacks cellular functions to turn the open circular DNA conformation of rcDNA into cccDNA through DNA repair mechanisms. With great efforts from the HBV research community, there have been several recent leaps in our understanding of cccDNA formation. It is our goal in this review to analyze the recent reports showing evidence of cellular factor's involvement in the molecular pathway of cccDNA biosynthesis.
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Affiliation(s)
- Alexander L. Marchetti
- Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA;
- Cancer Virology Program, Hillman Cancer Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Haitao Guo
- Cancer Virology Program, Hillman Cancer Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Correspondence:
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31
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Shen S, Xie Z, Cai D, Yu X, Zhang H, Kim ES, Zhou B, Hou J, Zhang X, Huang Q, Sun J, Guo H. Biogenesis and molecular characteristics of serum hepatitis B virus RNA. PLoS Pathog 2020; 16:e1008945. [PMID: 33079954 PMCID: PMC7575114 DOI: 10.1371/journal.ppat.1008945] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 08/28/2020] [Indexed: 02/06/2023] Open
Abstract
HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope-knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3' RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3'-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
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Affiliation(s)
- Sheng Shen
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Zhanglian Xie
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
| | - Dawei Cai
- Assembly Biosciences, Inc., South San Francisco, CA, United States of America
| | - Xiaoyang Yu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Hu Zhang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Elena S. Kim
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Bin Zhou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
| | - Jinlin Hou
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Xiaoyong Zhang
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Qi Huang
- Assembly Biosciences, Inc., South San Francisco, CA, United States of America
| | - Jian Sun
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
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32
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Intracellular Trafficking of HBV Particles. Cells 2020; 9:cells9092023. [PMID: 32887393 PMCID: PMC7563130 DOI: 10.3390/cells9092023] [Citation(s) in RCA: 53] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 08/31/2020] [Accepted: 09/02/2020] [Indexed: 12/15/2022] Open
Abstract
The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.
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33
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Zhao Q, Guo JT. Have the Starting Lineup of Five for Hepatitis B Virus Covalently Closed Circular DNA Synthesis Been Identified? Hepatology 2020; 72:1142-1144. [PMID: 32502295 DOI: 10.1002/hep.31408] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Qiong Zhao
- Baruch S. Blumberg Institute, Doylestown, PA
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, PA
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34
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Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription. J Virol 2020; 94:JVI.00442-20. [PMID: 32581092 DOI: 10.1128/jvi.00442-20] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Accepted: 06/15/2020] [Indexed: 12/12/2022] Open
Abstract
Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.
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35
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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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36
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Xia Y, Guo H. Hepatitis B virus cccDNA: Formation, regulation and therapeutic potential. Antiviral Res 2020; 180:104824. [PMID: 32450266 PMCID: PMC7387223 DOI: 10.1016/j.antiviral.2020.104824] [Citation(s) in RCA: 125] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 05/03/2020] [Accepted: 05/18/2020] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) infection remains a major public health concern worldwide with about 257 million individuals chronically infected. Current therapies can effectively control HBV replication and slow down disease progress, but cannot cure HBV infection. Upon infection, HBV establishes a pool of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. The cccDNA exists as a minichromosome and resists to antivirals, thus a therapeutic eradication of cccDNA from the infected cells remains unattainable. In this review, we summarize the state of knowledge on the mechanisms underlying cccDNA formation and regulation, and discuss the possible strategies that may contribute to the eradication of HBV through targeting cccDNA.
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Affiliation(s)
- Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, China.
| | - Haitao Guo
- UPMC Hillman Cancer Center, Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA.
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37
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Wang Z, Wang W, Wang L. Epigenetic regulation of covalently closed circular DNA minichromosome in hepatitis B virus infection. BIOPHYSICS REPORTS 2020. [DOI: 10.1007/s41048-020-00112-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
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38
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Abstract
Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth. The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5′ truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production.
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39
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Cai M, Wang P, Wang Y, Chen T, Xu Z, Zou X, Ou X, Li Y, Chen D, Peng T, Li M. Identification of the molecular determinants for nuclear import of PRV EP0. Biol Chem 2020; 400:1385-1394. [PMID: 31120855 DOI: 10.1515/hsz-2019-0201] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Accepted: 05/12/2019] [Indexed: 12/12/2022]
Abstract
Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, β1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.
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Affiliation(s)
- Mingsheng Cai
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Ping Wang
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yuanfang Wang
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Tao Chen
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Zuo Xu
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Xingmei Zou
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Xiaowen Ou
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Yiwen Li
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Daixiong Chen
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
| | - Tao Peng
- State Key Laboratory of Respiratory Diseases, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Xinzao Town, Panyu, Guangzhou 511436, Guangdong, China.,South China Vaccine Corporation Limited, Guangzhou Science Park, Guangzhou 510663, Guangdong, China
| | - Meili Li
- Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China.,Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, No. 250 Changgang Dong Road, Haizhu District, Guangzhou 510260, Guangdong, China
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Brezgin S, Kostyusheva A, Bayurova E, Gordeychuk I, Isaguliants M, Goptar I, Nikiforova A, Smirnov V, Volchkova E, Glebe D, Kostyushev D, Chulanov V. Replenishment of Hepatitis B Virus cccDNA Pool Is Restricted by Baseline Expression of Host Restriction Factors In Vitro. Microorganisms 2019; 7:E533. [PMID: 31698767 PMCID: PMC6920784 DOI: 10.3390/microorganisms7110533] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2019] [Revised: 11/03/2019] [Accepted: 11/05/2019] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. METHODS HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. RESULTS Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. CONCLUSION Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.
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Affiliation(s)
- Sergey Brezgin
- National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; (A.K.); (V.C.)
- Institute of Immunology, Federal Medical Biological Agency, 115522 Moscow, Russia;
| | - Anastasiia Kostyusheva
- National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; (A.K.); (V.C.)
| | - Ekaterina Bayurova
- NF Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; (E.B.); (I.G.); (M.I.)
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
| | - Ilya Gordeychuk
- NF Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; (E.B.); (I.G.); (M.I.)
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
- Sechenov First Moscow State Medical University, 119146 Moscow, Russia;
| | - Maria Isaguliants
- NF Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; (E.B.); (I.G.); (M.I.)
- Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia
- Riga Stradins University, LV-1007 Riga, Latvia
- Karolinska Institutet, SE-171 76 Stockholm, Sweden
| | - Irina Goptar
- Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia; (I.G.); (A.N.)
| | - Anastasiia Nikiforova
- Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia; (I.G.); (A.N.)
| | - Valery Smirnov
- Institute of Immunology, Federal Medical Biological Agency, 115522 Moscow, Russia;
| | - Elena Volchkova
- Sechenov First Moscow State Medical University, 119146 Moscow, Russia;
| | - Dieter Glebe
- Institute of Medical Virology, University of Giessen, 35392 Giessen, Germany;
| | - Dmitry Kostyushev
- National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; (A.K.); (V.C.)
| | - Vladimir Chulanov
- National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; (A.K.); (V.C.)
- Sechenov First Moscow State Medical University, 119146 Moscow, Russia;
- Central Research Institute of Epidemiology, 111123 Moscow, Russia
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Abstract
With a yearly death toll of 880,000, hepatitis B virus (HBV) remains a major health problem worldwide, despite an effective prophylactic vaccine and well-tolerated, effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long-term antiviral therapy, and its integration, though no longer able to support viral replication, destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome, which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products, and thus, it is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.
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Mitra B, Wang J, Kim ES, Mao R, Dong M, Liu Y, Zhang J, Guo H. Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation. J Virol 2019; 93:e00196-19. [PMID: 31019054 PMCID: PMC6580977 DOI: 10.1128/jvi.00196-19] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Accepted: 04/18/2019] [Indexed: 02/07/2023] Open
Abstract
Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg-) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg- patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy.IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.
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Affiliation(s)
- Bidisha Mitra
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Jinyu Wang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Elena S Kim
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Richeng Mao
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Medical Molecular Virology of the Ministry of Health and Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Minhui Dong
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Yuanjie Liu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Jiming Zhang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Medical Molecular Virology of the Ministry of Health and Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA. J Virol 2019; 93:JVI.02230-18. [PMID: 30867306 DOI: 10.1128/jvi.02230-18] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Accepted: 03/01/2019] [Indexed: 12/18/2022] Open
Abstract
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
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Hu J, Cheng J, Tang L, Hu Z, Luo Y, Li Y, Zhou T, Chang J, Guo JT. Virological Basis for the Cure of Chronic Hepatitis B. ACS Infect Dis 2019; 5:659-674. [PMID: 29893548 DOI: 10.1021/acsinfecdis.8b00081] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Hepatitis B virus (HBV) has infected one-third of world population, and 240 million people are chronic carriers, to whom a curative therapy is still not available. Similar to other viruses, persistent HBV infection relies on the virus to exploit host cell functions to support its replication and efficiently evade host innate and adaptive antiviral immunity. Understanding HBV replication and concomitant host cell interactions is thus instrumental for development of therapeutics to disrupt the virus-host interactions critical for its persistence and cure chronic hepatitis B. Although the currently available cell culture systems of HBV infection are refractory to genome-wide high throughput screening of key host cellular factors essential for and/or regulating HBV replication, classic one-gene (or pathway)-at-a-time studies in the last several decades have already revealed many aspects of HBV-host interactions. An overview of the landscape of HBV-hepatocyte interaction indicates that, in addition to more tightly suppressing viral replication by directly targeting viral proteins, disruption of key viral-host cell interactions to eliminate or inactivate the covalently closed circular (ccc) DNA, the most stable HBV replication intermediate that exists as an episomal minichromosome in the nucleus of infected hepatocyte, is essential to achieve a functional cure of chronic hepatitis B. Moreover, therapeutic targeting of integrated HBV DNA and their transcripts may also be required to induce hepatitis B virus surface antigen (HBsAg) seroclearance and prevent liver carcinogenesis.
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Affiliation(s)
- Jin Hu
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, 1 Tian-tan Xi-li, Beijing, 100050, China
| | - Junjun Cheng
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Liudi Tang
- Microbiology and Immunology Graduate Program, Drexel University College of Medicine, 2900 West Queen Lane, Philadelphia, Pennsylvania 19129, United States
| | - Zhanying Hu
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Yue Luo
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
- Institute of Hepatology, Second Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, China
| | - Yuhuan Li
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, 1 Tian-tan Xi-li, Beijing, 100050, China
| | - Tianlun Zhou
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Jinhong Chang
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
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Zhang X, Cheng J, Ma J, Hu Z, Wu S, Hwang N, Kulp J, Du Y, Guo JT, Chang J. Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors. ACS Infect Dis 2019; 5:759-768. [PMID: 30525438 DOI: 10.1021/acsinfecdis.8b00269] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.
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Affiliation(s)
- Xuexiang Zhang
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Junjun Cheng
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Julia Ma
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Zhanying Hu
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Shuo Wu
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Nicky Hwang
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - John Kulp
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Yanming Du
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
| | - Jinhong Chang
- Baruch S. Blumberg Institute, 3805 Old Eastern Road, Doylestown, Pennsylvania 18902, United States
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Tang L, Sheraz M, McGrane M, Chang J, Guo JT. DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA. PLoS Pathog 2019; 15:e1007742. [PMID: 31026293 PMCID: PMC6505960 DOI: 10.1371/journal.ppat.1007742] [Citation(s) in RCA: 67] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2018] [Revised: 05/08/2019] [Accepted: 03/29/2019] [Indexed: 02/07/2023] Open
Abstract
Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ. CCC DNA is the most refractory HBV replication intermediate under long-term antiviral therapies and is responsible for the viral rebound after treatment cessation. Therefore, understanding the biosynthesis and maintenance of cccDNA minichromosome is crucial for the development of novel antiviral therapeutics to cure chronic HBV infection. Although it has been clearly demonstrated that cccDNA biosynthesis relies on host cellular DNA repair machinery, the molecular pathways that convert rcDNA into cccDNA remain to be identified. Here we report that DNA polymerase alpha (Pol α) as well as Pol δ and ɛ are required for converting rcDNA into cccDNA through intracellular cccDNA amplification. This finding adds novel molecular insights on cccDNA biosynthesis. Further understanding the mechanism of cccDNA synthesis should reveal molecular targets for developing therapeutic agents to eradicate cccDNA and cure chronic hepatitis B.
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Affiliation(s)
- Liudi Tang
- Microbiology and Immunology Graduate Program, Drexel University College of Medicine, Philadelphia, PA, United States of America
| | - Muhammad Sheraz
- Microbiology and Immunology Graduate Program, Drexel University College of Medicine, Philadelphia, PA, United States of America
| | - Michael McGrane
- FlowMetric Diagnostics, Doylestown, PA, United States of America
| | - Jinhong Chang
- Baruch S. Blumberg Institute, Doylestown, PA, United States of America
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, PA, United States of America
- * E-mail:
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Dezhbord M, Lee S, Kim W, Seong BL, Ryu WS. Characterization of the molecular events of covalently closed circular DNA synthesis in de novo Hepatitis B virus infection of human hepatoma cells. Antiviral Res 2019; 163:11-18. [PMID: 30639437 DOI: 10.1016/j.antiviral.2019.01.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2018] [Revised: 12/19/2018] [Accepted: 01/09/2019] [Indexed: 12/27/2022]
Abstract
Despite the utmost importance of cccDNA in HBV biology, the mechanism by which cccDNA synthesis is regulated is not completely understood. Here we explored HepG2-NTCP cell line and performed a time-course HBV infection experiment (up to 30 days) to follow the conversion of the input viral DNA into cccDNA. We found that a protein-free RC DNA (PF-RC DNA) become detectable as early as 12 h post infection (hpi) prior to the detection of cccDNA, which become evident only at 2-3 dpi. Intriguingly, the PF-RC DNA detected at 12 hpi was abundantly located in the cytoplasm, implicating that the protein-removal from the input viral DNA takes place in the cytoplasm, perhaps inside the nucleocapsid. Notably, during the early time points of HBV infection, the PF-RC DNA accumulated at significantly higher levels and appeared in a peak followed by a plateau at late time points with dramatically lower levels, implicating the presence of two distinct populations of the PF-RC DNA. Importantly, the PF-RC DNA at earlier peak is entecavir (ETV)-resistant, whereas the PF-RC DNA at posterior days is ETV-sensitive. An interpretation is that the PF-RC DNA at earlier peak represents "input viral DNA" derived from HBV inoculum, whereas the PF-RC DNA at late time points represents the de novo product of the viral reverse transcription. The existence of two populations of the PF-RC DNA having a distinct kinetic profile and ETV-sensitivity implicated that intracellular amplification via the viral reverse transcription greatly contributes to the maintenance of cccDNA pool during HBV infection. As such, we concluded that the cccDNA level is stably maintained by continuing replenishment of cccDNA primarily through intracellular amplification in the HepG2-NTCP cell line.
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Affiliation(s)
- Mehrangiz Dezhbord
- Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea
| | - Sooyoung Lee
- Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea
| | - Woohyun Kim
- Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea
| | - Baik Lin Seong
- Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea.
| | - Wang-Shick Ryu
- Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, South Korea.
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Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication. J Virol 2018; 92:JVI.01255-18. [PMID: 30232184 DOI: 10.1128/jvi.01255-18] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 09/15/2018] [Indexed: 12/22/2022] Open
Abstract
Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocyte cell lines appeared suboptimal. In the present study, we constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1α and HNF4α, which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B virus infection.IMPORTANCE HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we constructed NTCP-expressing HepG2 and AML12 cell lines that are highly permissive to HBV infection. More significantly, we found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to the cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards the ultimate elimination of chronic hepatitis B virus infection.
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Mitra B, Thapa RJ, Guo H, Block TM. Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B. Antiviral Res 2018; 158:185-198. [PMID: 30145242 PMCID: PMC6193490 DOI: 10.1016/j.antiviral.2018.08.014] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Revised: 08/21/2018] [Accepted: 08/22/2018] [Indexed: 02/06/2023]
Abstract
Similar to other mammalian viruses, the life cycle of hepatitis B virus (HBV) is heavily dependent upon and regulated by cellular (host) functions. These cellular functions can be generally placed in to two categories: (a) intrinsic host restriction factors and innate defenses, which must be evaded or repressed by the virus; and (b) gene products that provide functions necessary for the virus to complete its life cycle. Some of these functions may apply to all viruses, but some may be specific to HBV. In certain cases, the virus may depend upon the host function much more than does the host itself. Knowing which host functions regulate the different steps of a virus' life cycle, can lead to new antiviral targets and help in developing novel treatment strategies, in addition to improving a fundamental understanding of viral pathogenesis. Therefore, in this review we will discuss known host factors which influence key steps of HBV life cycle, and further elucidate therapeutic interventions targeting host-HBV interactions.
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Affiliation(s)
- Bidisha Mitra
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | | | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.
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Duan Z, Xu H, Ji X, Zhao J, Xu H, Hu Y, Deng S, Hu S, Liu X. Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts. Virulence 2018. [PMID: 29532715 PMCID: PMC5955436 DOI: 10.1080/21505594.2018.1449507] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.
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Affiliation(s)
- Zhiqiang Duan
- a Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education , Guizhou University , Guiyang , China.,b College of Animal Science , Guizhou University , Guiyang , China
| | - Haixu Xu
- c Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture , Yangzhou University , Yangzhou , China
| | - Xinqin Ji
- a Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education , Guizhou University , Guiyang , China.,b College of Animal Science , Guizhou University , Guiyang , China
| | - Jiafu Zhao
- a Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education , Guizhou University , Guiyang , China.,b College of Animal Science , Guizhou University , Guiyang , China
| | - Houqiang Xu
- a Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education , Guizhou University , Guiyang , China.,b College of Animal Science , Guizhou University , Guiyang , China
| | - Yan Hu
- b College of Animal Science , Guizhou University , Guiyang , China
| | - Shanshan Deng
- b College of Animal Science , Guizhou University , Guiyang , China
| | - Shunlin Hu
- c Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture , Yangzhou University , Yangzhou , China
| | - Xiufan Liu
- c Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture , Yangzhou University , Yangzhou , China
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