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Hong X, Schneider WM, Rice CM. Hepatitis B Virus Nucleocapsid Assembly. J Mol Biol 2025:169182. [PMID: 40316009 DOI: 10.1016/j.jmb.2025.169182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2025] [Revised: 04/13/2025] [Accepted: 04/28/2025] [Indexed: 05/04/2025]
Abstract
Hepatitis B virus (HBV), the prototypical member of the Hepadnaviridae family, is a DNA virus that replicates its genome through reverse transcription of a pregenomic RNA (pgRNA) precursor. The selective packaging of pgRNA and viral polymerase (Pol) into assembling capsids formed by the viral core protein-a process known as nucleocapsid assembly-is an essential step in the HBV lifecycle. Advances in cellular and cell-free systems have provided significant insights into the mechanisms underlying capsid assembly, Pol binding to pgRNA, Pol-pgRNA packaging, and initiation of genome replication. However, the absence of a cell-free system capable of reconstituting selective HBV Pol-pgRNA packaging into fully assembled capsids leaves fundamental questions about nucleocapsid assembly unanswered. This review summarizes the current knowledge of HBV nucleocapsid assembly, focusing on the interplay between Pol-pgRNA interactions, capsid formation, and regulation by host factors. It also highlights the contribution of cellular and cell-free systems to these discoveries and underscores the need for new approaches that reconstitute the complete HBV nucleocapsid assembly process. With the growing interest in developing nucleocapsid assembly inhibitors, some of which are currently in clinical trials, targeting Pol-pgRNA interactions and nucleocapsid assembly represents a promising therapeutic strategy for curing chronic hepatitis B.
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Affiliation(s)
- Xupeng Hong
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.
| | - William M Schneider
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA
| | - Charles M Rice
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA
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2
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Seigneuret F, Eymieux S, Sarabia-Vega V, Lemoine R, Burlaud-Gaillard J, Raynal P, Hourioux C, Sureau C, Roingeard P, de Rocquigny H. The HBV large envelope protein initiates virion assembly by recruiting capsids at membrane rich domains related to late endosome. Cell Mol Life Sci 2025; 82:128. [PMID: 40128454 PMCID: PMC11933560 DOI: 10.1007/s00018-025-05574-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 12/06/2024] [Accepted: 01/02/2025] [Indexed: 03/26/2025]
Abstract
A crucial step of HBV (Hepatitis B Virus) virion morphogenesis is the envelopment of the nucleocapsid by the viral envelope proteins, which is triggered by an interaction between the HBV core protein and the large HBV envelope protein. To document this protein-protein interaction, we co-expressed core and large HBV envelope (LHBs) in Huh-7 cells and subjected the cells to microscopy examination by Fluorescence Resonance Energy Transfer (FRET) and Transmission Electron Microscopy (TEM). Our results show that the sole expression of the core protein leads to assembly of capsids that remain individually isolated within the whole cell, but particularly within the nucleus. In the presence of LHBs, capsids were observed as large clusters in a membrane rich region peripheral to the nucleus. In this context, core-LHBs complex co-localize with markers of the late endosome/multivesicular bodies, this co-localization being driven by LHBs. These results thus show that LHBs binds to the core proteins when preassembled into capsid, at membranes of the late endosome, where the inner capsid and the outer envelope meet to assemble a virion.
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Affiliation(s)
- Florian Seigneuret
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Sébastien Eymieux
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Vanessa Sarabia-Vega
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Roxane Lemoine
- Département Cytométrie et Single-Cell Immunobiologie, Plateforme Scientifique et Technique - Analyse des Systèmes Biologiques (PST-ASB), Université de Tours, 10 Boulevard Tonnellé, 37032, Tours Cedex 1, France
| | - Julien Burlaud-Gaillard
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Pierre Raynal
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Christophe Hourioux
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Camille Sureau
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Philippe Roingeard
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
- Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France
| | - Hugues de Rocquigny
- INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé, BP 3223, 37032, Tours Cedex 1, France.
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Morita C, Wada M, Ohsaki E, Kimura-Ohba S, Ueda K. Generation of Replication-Competent Hepatitis B Virus Harboring Tagged Polymerase for Visualization and Quantification of the Infection. Microbiol Immunol 2025; 69:43-58. [PMID: 39620377 DOI: 10.1111/1348-0421.13183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2024] [Revised: 11/03/2024] [Accepted: 11/04/2024] [Indexed: 01/07/2025]
Abstract
Hepatitis B virus (HBV) infection is a serious global health problem causing acute and chronic hepatitis and related diseases. Approximately, 296 million patients have been chronically infected with the virus, leading to cirrhosis and hepatocellular carcinoma. Although HBV polymerase (HBVpol, pol) plays a pivotal role in HBV replication and must be a definite therapeutic target. The problems are that the detailed functions and intracellular dynamics of HBVpol remain unclear. Here, we constructed two kinds of tagged HBVpol, PA-tagged and HiBiT-tagged pol, and the HBV-producing vectors. Each PA tag and HiBiT tag were inserted into N-terminus of spacer region on HBVpol open reading frame. Transfection of the plasmids into HepG2 cells led to production of HBV. These tagged HBVpol were detectable in HBV replicating cells and pol-HiBiT enabled quantitative analysis. Furthermore, these recombinant HBV were infectious to primary human hepatocytes. Thus, we successfully designed infectious and replication-competent recombinant HBV harboring detectable tagged HBVpol. Such infectious recombinant HBV will provide a novel tool to study HBVpol dynamics and develop new therapeutics against HBV.
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Grants
- This research was supported by Grants from the Japan Agency for Medical Research and Development (AMED) Grants (16fk0310504h0005, 17fk0310105h0001, 18fk0310105h0002, 19fk0310105h0003, 20fk0310105h0004, 21fk0310105h005, 22fk0310505h0001, 23fk0310505h0002, and 24fk0310505h003) to K.U. and from JST SPRING, Grant Number JPMJSP2138, to C.M. and from the Osaka University Transdisciplinary Program for Biomedical Entrepreneurship and Innovation (WISE program) to C.M.
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Affiliation(s)
- Chiharu Morita
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Masami Wada
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
- Center for Infectious Disease Education and Research (CiDER), Osaka, Japan
| | - Shihoko Kimura-Ohba
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
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Whitworth IT, Romero S, Kissi-Twum A, Knoener R, Scalf M, Sherer NM, Smith LM. Identification of Host Proteins Involved in Hepatitis B Virus Genome Packaging. J Proteome Res 2024; 23:4128-4138. [PMID: 39078123 PMCID: PMC11693245 DOI: 10.1021/acs.jproteome.4c00505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/31/2024]
Abstract
A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.
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Affiliation(s)
- Isabella T Whitworth
- Department of Chemistry, University of Wisconsin-Madison College of Letters and Sciences, Madison, Wisconsin, 53706, United States
| | - Sofia Romero
- McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, 53705, United States
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
- Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
| | - Abena Kissi-Twum
- McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, 53705, United States
- Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
| | - Rachel Knoener
- Department of Chemistry, University of Wisconsin-Madison College of Letters and Sciences, Madison, Wisconsin, 53706, United States
- McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, 53705, United States
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
| | - Mark Scalf
- Department of Chemistry, University of Wisconsin-Madison College of Letters and Sciences, Madison, Wisconsin, 53706, United States
| | - Nathan M Sherer
- McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, 53705, United States
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States
| | - Lloyd M Smith
- Department of Chemistry, University of Wisconsin-Madison College of Letters and Sciences, Madison, Wisconsin, 53706, United States
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Chang CH, Shih C. Significance of hepatitis B virus capsid dephosphorylation via polymerase. J Biomed Sci 2024; 31:34. [PMID: 38561844 PMCID: PMC10983652 DOI: 10.1186/s12929-024-01022-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Accepted: 03/19/2024] [Indexed: 04/04/2024] Open
Abstract
BACKGROUND It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.
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Affiliation(s)
- Chih-Hsu Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 112, Taiwan
| | - Chiaho Shih
- Institute of Biomedical Sciences, Academia Sinica, Taipei, 112, Taiwan.
- Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.
- Graduate Institute of Cell Biology, China Medical University, Taichung, 406, Taiwan.
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Kim GW, Siddiqui A. Analysis of N6-Methyladenosine Modification of HBV RNA by Methylated RNA Immunoprecipitation. Methods Mol Biol 2024; 2837:59-66. [PMID: 39044075 DOI: 10.1007/978-1-0716-4027-2_6] [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
Of all the chemical modifications of RNAs, the N6-methyladenosine (m6A) modification is the most prevalent and well-characterized RNA modification that is functionally implicated in a wide range of biological processes. The m6A modification occurs in hepatitis B virus (HBV) RNAs and this modification regulates the HBV life cycle in several ways. Thus, understanding the mechanisms underlying m6A modification of HBV RNAs is crucial in understanding HBV infectious process and associated pathogenesis. Here, we describe the currently utilized method in the detection and characterization of m6A-methylated RNAs during viral infection.
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Affiliation(s)
- Geon-Woo Kim
- Divsion of Infectious Diseases and Global Public Health, Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
- Department of Microbiology and Molecular Biology, Chungnam National University, Yuseong-gu, Daejeon, Republic of Korea.
| | - Aleem Siddiqui
- Divsion of Infectious Diseases and Global Public Health, Department of Medicine, University of California, San Diego, La Jolla, CA, USA
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Deng W, Chen F, Zhao Y, Zhou M, Guo M. Anti-hepatitis B virus activities of natural products and their antiviral mechanisms. Chin J Nat Med 2023; 21:803-811. [PMID: 38035936 DOI: 10.1016/s1875-5364(23)60505-9] [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] [Received: 04/12/2023] [Indexed: 12/02/2023]
Abstract
Chronic hepatitis B (CHB) infections caused by the hepatitis B virus (HBV) continue to pose a significant global public health challenge. Currently, the approved treatments for CHB are limited to interferon and nucleos(t)ide analogs, both of which have their limitations, and achieving a complete cure remains an elusive goal. Therefore, the identification of new therapeutic targets and the development of novel antiviral strategies are of utmost importance. Natural products (NPs) constitute a class of substances known for their diverse chemical structures, wide-ranging biological activities, and low toxicity profiles. They have shown promise as potential candidates for combating various diseases, with a substantial number demonstrating anti-HBV properties. This comprehensive review focuses on the current applications of NPs in the fight against HBV and provides a summary of their antiviral mechanisms, considering their impact on the viral life cycle and host hepatocytes. By offering insights into the world of anti-HBV NPs, this review aims to furnish valuable information to support the future development of antiviral drugs.
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Affiliation(s)
- Wanyu Deng
- College of Life Science, Shangrao Normal University, Shangrao 334001, China
| | - Fu Chen
- College of Life Science, Shangrao Normal University, Shangrao 334001, China
| | - Yue Zhao
- State Key Laboratory of Natural Medicines, School of Life Science&Technology, China Pharmaceutical University, Nanjing 211198, China
| | - Ming Zhou
- BGI-Shenzhen, Shenzhen 518000, China; Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518110, China; Liver-biotechnology (Shenzhen) Co., Ltd., Shenzhen 518110, China.
| | - Min Guo
- State Key Laboratory of Natural Medicines, School of Life Science&Technology, China Pharmaceutical University, Nanjing 211198, China.
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8
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Zhang T, Zheng H, Lu D, Guan G, Li D, Zhang J, Liu S, Zhao J, Guo JT, Lu F, Chen X. RNA binding protein TIAR modulates HBV replication by tipping the balance of pgRNA translation. Signal Transduct Target Ther 2023; 8:346. [PMID: 37699883 PMCID: PMC10497612 DOI: 10.1038/s41392-023-01573-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 06/20/2023] [Accepted: 07/23/2023] [Indexed: 09/14/2023] Open
Abstract
The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves not only as a bicistronic message RNA to translate core protein (Cp) and DNA polymerase (Pol), but also as the template for reverse transcriptional replication of viral DNA upon packaging into nucleocapsid. Although it is well known that pgRNA translates much more Cp than Pol, the molecular mechanism underlying the regulation of Cp and Pol translation efficiency from pgRNA remains elusive. In this study, we systematically profiled HBV nucleocapsid- and pgRNA-associated cellular proteins by proteomic analysis and identified TIA-1-related protein (TIAR) as a novel cellular protein that binds pgRNA and promotes HBV DNA replication. Interestingly, loss- and gain-of-function genetic analyses showed that manipulation of TIAR expression did not alter the levels of HBV transcripts nor the secretion of HBsAg and HBeAg in human hepatoma cells supporting HBV replication. However, Ribo-seq and PRM-based mass spectrometry analyses demonstrated that TIAR increased the translation of Pol but decreased the translation of Cp from pgRNA. RNA immunoprecipitation (RIP) and pulldown assays further revealed that TIAR directly binds pgRNA at the 5' stem-loop (ε). Moreover, HBV replication or Cp expression induced the increased expression and redistribution of TIAR from the nucleus to the cytoplasm of hepatocytes. Our results thus imply that TIAR is a novel cellular factor that regulates HBV replication by binding to the 5' ε structure of pgRNA to tip the balance of Cp and Pol translation. Through induction of TIAR translocation from the nucleus to the cytoplasm, Cp indirectly regulates the Pol translation and balances Cp and Pol expression levels in infected hepatocytes to ensure efficient viral replication.
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Affiliation(s)
- Ting Zhang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Huiling Zheng
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Danjuan Lu
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Guiwen Guan
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Deyao Li
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Jing Zhang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China
| | - Shuhong Liu
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, 100039, China
| | - Jingmin Zhao
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, 100039, China
| | - Ju-Tao Guo
- Department of Experimental Therapeutics, Baruch S. Blumberg Institute, Doylestown, PA, 18902, USA.
| | - Fengmin Lu
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China.
- Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Peking University Hepatology Institute, Peking University People's Hospital, Beijing, 100044, China.
| | - Xiangmei Chen
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China.
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Olenginski LT, Attionu SK, Henninger EN, LeBlanc RM, Longhini AP, Dayie TK. Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target. Viruses 2023; 15:1913. [PMID: 37766319 PMCID: PMC10534774 DOI: 10.3390/v15091913] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Revised: 09/01/2023] [Accepted: 09/05/2023] [Indexed: 09/29/2023] Open
Abstract
Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε-P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.
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Affiliation(s)
- Lukasz T. Olenginski
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Department of Biochemistry, University of Colorado, Boulder, CO 80309, USA
| | - Solomon K. Attionu
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Erica N. Henninger
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Regan M. LeBlanc
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Andrew P. Longhini
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Theodore K. Dayie
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
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10
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Wu S, Zhao Y, Wang D, Chen Z. Mode of Action of Heat Shock Protein (HSP) Inhibitors against Viruses through Host HSP and Virus Interactions. Genes (Basel) 2023; 14:genes14040792. [PMID: 37107550 PMCID: PMC10138296 DOI: 10.3390/genes14040792] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2023] [Revised: 03/17/2023] [Accepted: 03/20/2023] [Indexed: 03/29/2023] Open
Abstract
Misfolded proteins after stress-induced denaturation can regain their functions through correct re-folding with the aid of molecular chaperones. As a molecular chaperone, heat shock proteins (HSPs) can help client proteins fold correctly. During viral infection, HSPs are involved with replication, movement, assembly, disassembly, subcellular localization, and transport of the virus via the formation of macromolecular protein complexes, such as the viral replicase complex. Recent studies have indicated that HSP inhibitors can inhibit viral replication by interfering with the interaction of the virus with the HSP. In this review, we describe the function and classification of HSPs, the transcriptional mechanism of HSPs promoted by heat shock factors (HSFs), discuss the interaction between HSPs and viruses, and the mode of action of HSP inhibitors at two aspects of inhibiting the expression of HSPs and targeting the HSPs, and elaborate their potential use as antiviral agents.
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11
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Olenginski LT, Kasprzak WK, Attionu SK, Shapiro BA, Dayie TK. Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target. Molecules 2023; 28:1803. [PMID: 36838792 PMCID: PMC9963113 DOI: 10.3390/molecules28041803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/07/2023] [Accepted: 02/11/2023] [Indexed: 02/17/2023] Open
Abstract
The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.
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Affiliation(s)
- Lukasz T. Olenginski
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Wojciech K. Kasprzak
- Bioinformatics and Computational Science Program, Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
| | - Solomon K. Attionu
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Bruce A. Shapiro
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA
| | - Theodore K. Dayie
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
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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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Luo M, Hou J, Mai H, Chen J, Chen H, Zhou B, Hou J, Jiang DK. TRIM26 inhibits hepatitis B virus replication by promoting HBx degradation and TRIM26 genetic polymorphism predicts PegIFNα treatment response of HBeAg-positive chronic hepatitis B Patients. Aliment Pharmacol Ther 2022; 56:878-889. [PMID: 35872575 DOI: 10.1111/apt.17124] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 01/11/2022] [Accepted: 06/23/2022] [Indexed: 12/13/2022]
Abstract
BACKGROUND Hepatitis B virus (HBV) infection is a serious global health burden. TRIM26 has been reported to affect hepatitis C virus replication. AIMS To manifest the role of TRIM26 on HBV replication and explore if there are single-nucleotide polymorphisms (SNPs) in TRIM26 associated with response to pegylated interferon-alpha (PegIFNα) treatment in patients with chronic hepatitis B (CHB). METHODS We investigated the effect and mechanism of TRIM26 on HBV replication in vitro. The association between SNPs in TRIM26 and PegIFNα treatment response was evaluated in two independent cohorts including 238 and 707 patients with HBeAg-positive CHB. RESULTS Knockdown of TRIM26 increased, while overexpression of TRIM26 inhibited, HBV replication. Co-immunoprecipitation assays and immunofluorescence showed that TRIM26 interacted and co-localised with HBx. Co-transfection of HBx-HIS and TRIM26-FLAG plasmids in Huh7 cells showed that TRIM26 inhibited the expression of HBx. Furthermore, TRIM26 inhibited HBV replication by mediating HBx ubiquitination degradation, and TRIM26 SPRY domain was responsible for the interaction and degradation of HBx. Besides, IFN increased TRIM26 expression. TRIM26 rs116806878 was associated with response to PegIFNα in two CHB cohorts. Moreover, a polygenic score integrating TRIM26 rs116806878, STAT4 rs7574865 and CFB rs12614 (previously reported to be associated with response to PegIFNα) was related to response to PegIFNα in CHB. CONCLUSIONS TRIM26 inhibits HBV replication; IFN promotes TRIM26 expression. TRIM26 exerts an inhibitory effect on HBx by promoting ubiquitin-mediated degradation of HBx. Furthermore, TRIM26 rs116806878 is a potential predictive biomarker of response to PegIFNα in patients with CHB.
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Affiliation(s)
- Mengqi Luo
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
- The Key Laboratory of Molecular Pathology (Hepatic Diseases) of Guangxi, Department of Pathology, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China
| | - Jia Hou
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haoming Mai
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Jiaxuan Chen
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Haitao Chen
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
- School of Public Health (Shenzhen), Sun Yat-sen University, Shenzhen, China
| | - Bin Zhou
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Jinlin Hou
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - De-Ke Jiang
- State Key Laboratory of Organ Failure Research, Guangdong Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Institutes of Liver Diseases Research of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
- The Key Laboratory of Molecular Pathology (Hepatic Diseases) of Guangxi, Department of Pathology, The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China
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14
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Olenginski LT, Kasprzak WK, Bergonzo C, Shapiro BA, Dayie TK. Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication. J Mol Biol 2022; 434:167633. [PMID: 35595167 DOI: 10.1016/j.jmb.2022.167633] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Revised: 04/28/2022] [Accepted: 05/07/2022] [Indexed: 12/30/2022]
Abstract
Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5'-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and μs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.
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Affiliation(s)
- Lukasz T Olenginski
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Wojciech K Kasprzak
- Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
| | - Christina Bergonzo
- Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and University of Maryland, Rockville, MD 20850, USA
| | - Bruce A Shapiro
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA
| | - Theodore K Dayie
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
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15
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CRM1-spike-mediated nuclear export of hepatitis B virus encapsidated viral RNA. Cell Rep 2022; 38:110472. [PMID: 35263598 DOI: 10.1016/j.celrep.2022.110472] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 01/23/2022] [Accepted: 02/10/2022] [Indexed: 11/20/2022] Open
Abstract
Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.
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16
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N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein. Proc Natl Acad Sci U S A 2022; 119:2120485119. [PMID: 35135882 PMCID: PMC8851549 DOI: 10.1073/pnas.2120485119] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/17/2022] [Indexed: 12/19/2022] Open
Abstract
HBV infections are the leading cause of chronic hepatitis and carry the risk of liver cirrhosis and cancer. The HBV life cycle is perpetuated by an RNA intermediate termed pregenomic RNA (pgRNA), which is encapsidated by the viral core protein. The pgRNA packaging process is an essential step in viral replication. Here, we investigated the role of N6-methyladenosine (m6A) modification in the recognition of pgRNA by the core protein during encapsidation. m6A modification of 5′ epsilon structural motifs serves as the recognition signal for the core protein interaction, as evidenced by the failure of 5′ epsilon m6A mutant to encapsidate pgRNA. This study identifies the structural role of m6A modification in pgRNA encapsidation and provides an avenue in RNA–protein complex interactions. Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5′ end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)–modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5′ ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5′ epsilon, we present evidence that m6A methylation of 5′ epsilon is necessary for its encapsidation. The m6A modification of 5′ epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3′ epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5′ epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5′ epsilon m6A site–mutated pgRNA failed to interact. HBV polymerase interaction with 5′ epsilon was independent of m6A modification of 5′ epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA–protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.
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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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Abstract
Hepatitis B virus (HBV) is a hepatotropic, partially double-stranded DNA virus that replicates by reverse transcription and is a major cause of chronic liver disease and hepatocellular carcinoma. Reverse transcription is catalyzed by the four-domain multifunctional HBV polymerase (P) protein that has protein-priming, RNA- and DNA-dependent DNA synthesis (i.e., reverse transcriptase), and ribonuclease H activities. P also likely promotes the three strand transfers that occur during reverse transcription, and it may participate in immune evasion by HBV. Reverse transcription is primed by a tyrosine residue in the amino-terminal domain of P, and P remains covalently attached to the product DNA throughout reverse transcription. The reverse transcriptase activity of P is the target for the nucleos(t)ide analog drugs that dominate HBV treatment, and P is the target of ongoing efforts to develop new drugs against both the reverse transcriptase and ribonuclease H activities. Despite the unusual reverse transcription pathway catalyzed by P and the importance of P to HBV therapy, understanding the enzymology and structure of HBV P severely lags that of the retroviral reverse transcriptases due to substantial technical challenges to studying the enzyme. Obtaining a better understanding of P will broaden our appreciation of the diversity among reverse transcribing elements in nature, and will help improve treatment for people chronically infected with HBV.
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Affiliation(s)
- Daniel N Clark
- Department of Microbiology, Weber State University, Ogden, UT, United States
| | - Razia Tajwar
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, United States
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, United States
| | - John E Tavis
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, United States.
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19
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Zhang H, Tu T. Approaches to quantifying Hepatitis B Virus covalently closed circular (ccc)DNA. Clin Mol Hepatol 2021; 28:135-149. [PMID: 34674513 PMCID: PMC9013611 DOI: 10.3350/cmh.2021.0283] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 10/21/2021] [Indexed: 11/19/2022] Open
Abstract
Chronic hepatitis B is a major cause of liver disease worldwide and is currently incurable. Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is a key form of the virus responsible for its persistence and is the transcriptional template for all viral transcripts. The field is focussed on methods to clear HBV cccDNA but this been limited by technical difficulties in its quantification due to: identical sequence to other forms of HBV DNA; low copy number per cell; and high resistance to denaturation by heat, leading to difficulty using polymerase chain reaction or hybridization methods for detection. A number of assays have been developed in order to overcome these hurdles either directly or detecting cccDNA levels indirectly via its transcriptional products. In this review, we summarize the approaches to cccDNA quantification that are currently used, and outline key open questions in the cccDNA biology field which remain to be answered due to the limitations of current methods.
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Affiliation(s)
- Henrik Zhang
- Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia
| | - Thomas Tu
- Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.,Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney at Westmead Hospital, Westmead NSW 2145, Australia
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20
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Ibrahim MK, Abdelhafez TH, Takeuchi JS, Wakae K, Sugiyama M, Tsuge M, Ito M, Watashi K, El Kassas M, Kato T, Murayama A, Suzuki T, Chayama K, Shimotohno K, Muramatsu M, Aly HH, Wakita T. MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter. J Virol 2021; 95:e0076721. [PMID: 33980595 PMCID: PMC8274605 DOI: 10.1128/jvi.00767-21] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 05/06/2021] [Indexed: 01/04/2023] Open
Abstract
Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (NL) reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. IMPORTANCE HBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Therefore, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis and developing anti-HBV strategies. MafF was shown to suppress transcription from the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1β and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.
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Affiliation(s)
- Marwa K. Ibrahim
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Microbial Biotechnology, Division of Genetic Engineering and Biotechnology Research, National Research Centre, Giza, Egypt
| | - Tawfeek H. Abdelhafez
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Microbial Biotechnology, Division of Genetic Engineering and Biotechnology Research, National Research Centre, Giza, Egypt
| | - Junko S. Takeuchi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
- Organization for the Strategic Coordination of Research and Intellectual Properties, Meiji University, Kawasaki, Japan
| | - Kosho Wakae
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Masaya Sugiyama
- Genome Medical Sciences Project, National Center for Global Health and Medicine, Ichikawa, Japan
| | - Masataka Tsuge
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
| | - Masahiko Ito
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Mohamed El Kassas
- Endemic Medicine Department, Faculty of Medicine, Helwan University, Cairo, Egypt
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Kazuaki Chayama
- Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kunitada Shimotohno
- Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hussein H. Aly
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
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21
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LeBlanc RM, Kasprzak WK, Longhini AP, Olenginski LT, Abulwerdi F, Ginocchio S, Shields B, Nyman J, Svirydava M, Del Vecchio C, Ivanic J, Schneekloth JS, Shapiro BA, Dayie TK, Le Grice SFJ. Structural insights of the conserved "priming loop" of hepatitis B virus pre-genomic RNA. J Biomol Struct Dyn 2021; 40:9761-9773. [PMID: 34155954 PMCID: PMC10167916 DOI: 10.1080/07391102.2021.1934544] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 05/20/2021] [Indexed: 12/16/2022]
Abstract
Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral polymerase with a cis-acting regulatory signal, designated epsilon (ε), located at the 5'-end of its pre-genomic RNA (pgRNA). Binding of polymerase to ε is also necessary for pgRNA encapsidation. While the mechanistic basis of this interaction remains elusive, mutagenesis studies suggest its internal 6-nt "priming loop" provides an important structural contribution. ε might therefore be considered a promising target for small molecule interventions to complement current nucleoside-analog based anti-HBV therapies. An ideal prerequisite to any RNA-directed small molecule strategy would be a detailed structural description of this important element. Herein, we present a solution NMR structure for HBV ε which, in combination with molecular dynamics and docking simulations, reports on a flexible ligand "pocket", reminiscent of those observed in proteins. We also demonstrate the binding of the selective estrogen receptor modulators (SERMs) Raloxifene, Bazedoxifene, and a de novo derivative to the priming loop.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Regan M. LeBlanc
- Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
- Vertex Pharmaceuticals, Boston, MA, USA
| | - Wojciech K. Kasprzak
- Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Andrew P. Longhini
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA
- Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA
| | - Lukasz T. Olenginski
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA
| | - Fardokht Abulwerdi
- Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Stefano Ginocchio
- Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Brigit Shields
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Julie Nyman
- Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Maryia Svirydava
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA
| | | | - Joseph Ivanic
- Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | | | - Bruce A. Shapiro
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD, USA
| | - Theodore Kwaku Dayie
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA
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22
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The RNA Binding Proteins YTHDC1 and FMRP Regulate the Nuclear Export of N6-Methyladenosine-Modified Hepatitis B Virus Transcripts and Affect the Viral Life Cycle. J Virol 2021; 95:e0009721. [PMID: 33883220 DOI: 10.1128/jvi.00097-21] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A)-modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A methylated, and this modification regulates the viral life cycle. HBV is particularly interesting, as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it positively affects pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the virus life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP- or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate in the nucleus to reduce reverse transcription in HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.
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Olenginski LT, Dayie TK. Quantifying the effects of long-range 13C- 13C dipolar coupling on measured relaxation rates in RNA. JOURNAL OF BIOMOLECULAR NMR 2021; 75:203-211. [PMID: 33914223 PMCID: PMC8131303 DOI: 10.1007/s10858-021-00368-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Accepted: 04/05/2021] [Indexed: 06/12/2023]
Abstract
Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that complicate 13C relaxation analyses. While these phenomena are well documented for sites with adjacent 13C nuclei (e.g. ribose C1'), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å) 13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible 13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1 measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range 13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1 rates in terms of motional models for large RNAs.
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Affiliation(s)
- Lukasz T Olenginski
- Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD, 20742, USA
| | - Theodore K Dayie
- Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD, 20742, USA.
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Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B. J Hepatol 2021; 74:783-793. [PMID: 33188902 DOI: 10.1016/j.jhep.2020.10.034] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 10/05/2020] [Accepted: 10/28/2020] [Indexed: 12/30/2022]
Abstract
BACKGROUND & AIMS In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.
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Abstract
Hepatitis B virus (HBV), which was discovered in 1965, is a threat to global public health. HBV infects human hepatocytes and leads to acute and chronic liver diseases, and there is no cure. In cells infected by HBV, viral DNA can be integrated into the cellular genome. HBV DNA integration is a complicated process during the HBV life cycle. Although HBV integration normally results in replication-incompetent transcripts, it can still act as a template for viral protein expression. Of note, it is a primary driver of hepatocellular carcinoma (HCC). Recently, with the development of detection methods and research models, the molecular biology and the pathogenicity of HBV DNA integration have been better revealed. Here, we review the advances in the research of HBV DNA integration, including molecular mechanisms, detection methods, research models, the effects on host and viral gene expression, the role of HBV integrations in the pathogenesis of HCC, and potential treatment strategies. Finally, we discuss possible future research prospects of HBV DNA integration.
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Affiliation(s)
- Kaitao Zhao
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
| | - Andrew Liu
- Laboratory of Molecular Cardiology, National Heart Lung Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA
| | - Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
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Li X, Liu H, Cheng W, Wang J, Zhang H, Lu F, Chen X, Lin W. Junceellolide B, a novel inhibitor of Hepatitis B virus. Bioorg Med Chem 2020; 28:115603. [PMID: 32690259 DOI: 10.1016/j.bmc.2020.115603] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Revised: 06/17/2020] [Accepted: 06/19/2020] [Indexed: 02/08/2023]
Abstract
HBV infection is a common cause of liver disease with a high burden worldwide. Current therapeutic strategy relies on interferon and nucleos(t)ide-type drugs with the limitation of functional cure. In this study, a structure-based screening of marine natural products from an in-house library was performed to hit HBV inhibitors, and the gorgonian-derived briarane-type diterpenoids showed inhibitory effects against HBV DNA replication in HepAD38 cells. Preliminary analyses of structure-activity relationship demonstrated that a briarane-based scaffold with an 3E,5(16)-diene and a chlorine-substitution at C-6 is required for the anti-HBV activity. Junceellolide B is one of the potent HBV inhibitors exhibiting efficient reduction of HBsAg and HBeAg production in HBV infected HepG2-NTCP cells with a dose-dependent manner (p < 0.001). It also significantly reduced the secreted HBV DNA, HBV RNA, and HBeAg in HepAD38 cells with the EC50 values of 0.83, 2.87 and 7.75 μM, respectively. Mechanistically, junceellolide B potently inhibited HBV RNA transcription without promoting HBV RNA degradation. RNA-seq analysis indicated that junceellolide B significantly decreased HBV cccDNA-transcripted products accompanying stable down-regulation of the expression of RNA polymerase II related host transcription factors (ZBED6 and ZBTB7B). These findings suggest junceellolide B to be a transcription inhibitor of cccDNA and a promising lead for the development of new anti-HBV agent.
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Affiliation(s)
- Xiaodan Li
- State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China
| | - Hui Liu
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China
| | - Wei Cheng
- State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China
| | - Jie Wang
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China
| | - He Zhang
- State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China
| | - Fengmin Lu
- State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China
| | - Xiangmei Chen
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China.
| | - Wenhan Lin
- State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China; Institute of Ocean Research, Peking University, Beijing 100875, PR China.
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Liu XQ, Ohsaki E, Ueda K. Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase. Genes Cells 2020; 25:523-537. [PMID: 32415897 PMCID: PMC7496097 DOI: 10.1111/gtc.12778] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Revised: 04/14/2020] [Accepted: 05/08/2020] [Indexed: 12/18/2022]
Abstract
Although several nucleo(s)tide analogs are available for treatment of HBV infection, long‐term treatment with these drugs can lead to the emergence of drug‐resistant viruses. Recent HIV‐1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non‐nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI‐resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)‐reverse transcriptase (RT) (TP‐RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP‐RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA‐binding assay system. Then, we used TP‐RT in cell‐free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6‐hydroxy‐DL‐DOPA and N‐oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell‐based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP‐RT domain to treat HBV infection.
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Affiliation(s)
- Xiao-Quan Liu
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
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Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target. Viruses 2020; 12:v12050570. [PMID: 32455999 PMCID: PMC7291194 DOI: 10.3390/v12050570] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 05/17/2020] [Accepted: 05/19/2020] [Indexed: 12/15/2022] Open
Abstract
Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure.
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Toyoda T, Wang Y, Wen Y, Tanaka Y. Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity. Anal Biochem 2020; 597:113642. [PMID: 32171777 DOI: 10.1016/j.ab.2020.113642] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 02/04/2020] [Accepted: 02/18/2020] [Indexed: 12/14/2022]
Abstract
Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with the success of adefovir, tenofovir, and entecavir. However, the polymerization rate was lower than that of human immunodeficiency virus RT. All HBV genotypes displayed similar RT activity, except for genotype B, which demonstrated increased elongation activity.
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Affiliation(s)
- Tetsuya Toyoda
- Choju Medical Institute, Fukushimura Hospital, 19-14 Azayamanaka, Noyori-Cho, Toyohashi, Aichi, 441-8124, Japan.
| | - Yongxiang Wang
- Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China
| | - Yumei Wen
- Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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Elalfy H, Besheer T, Elhammady D, Mesery AE, Shaltout SW, El-Maksoud MA, Amin AI, Bekhit AN, Aziz MAE, El-Bendary M. Pathological characterization of occult hepatitis B virus infection in hepatitis C virus-associated or non-alcoholic steatohepatitis-related hepatocellular carcinoma. World J Meta-Anal 2020; 8:67-77. [DOI: 10.13105/wjma.v8.i2.67] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 01/08/2020] [Accepted: 04/09/2020] [Indexed: 02/06/2023] Open
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Oropeza CE, Tarnow G, Sridhar A, Taha TY, Shalaby RE, McLachlan A. The Regulation of HBV Transcription and Replication. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1179:39-69. [PMID: 31741333 DOI: 10.1007/978-981-13-9151-4_3] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Hepatitis B virus (HBV) is a major human pathogen lacking a reliable curative therapy. Current therapeutics target the viral reverse transcriptase/DNA polymerase to inhibit viral replication but generally fail to resolve chronic HBV infections. Due to the limited coding potential of the HBV genome, alternative approaches for the treatment of chronic infections are desperately needed. An alternative approach to the development of antiviral therapeutics is to target cellular gene products that are critical to the viral life cycle. As transcription of the viral genome is an essential step in the viral life cycle, the selective inhibition of viral RNA synthesis is a possible approach for the development of additional therapeutic modalities that might be used in combination with currently available therapies. To address this possibility, a molecular understanding of the relationship between viral transcription and replication is required. The first step is to identify the transcription factors that are the most critical in controlling the levels of HBV RNA synthesis and to determine their in vivo role in viral biosynthesis. Mapping studies in cell culture utilizing reporter gene constructs permitted the identification of both ubiquitous and liver-enriched transcription factors capable of modulating transcription from the four HBV promoters. However, it was challenging to determine their relative importance for viral biosynthesis in the available human hepatoma replication systems. This technical limitation was addressed, in part, by the development of non-hepatoma HBV replication systems where viral biosynthesis was dependent on complementation with exogenously expressed transcription factors. These systems revealed the importance of specific nuclear receptors and hepatocyte nuclear factor 3 (HNF3)/forkhead box A (FoxA) transcription factors for HBV biosynthesis. Furthermore, using the HBV transgenic mouse model of chronic viral infection, the importance of various nuclear receptors and FoxA isoforms could be established in vivo. The availability of this combination of systems now permits a rational approach toward the development of selective host transcription factor inhibitors. This might permit the development of a new class of therapeutics to aid in the treatment and resolution of chronic HBV infections, which currently affects approximately 1 in 30 individuals worldwide and kills up to a million people annually.
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Affiliation(s)
- Claudia E Oropeza
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA
| | - Grant Tarnow
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA
| | - Abhayavarshini Sridhar
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA
| | - Taha Y Taha
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA
| | - Rasha E Shalaby
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.,Department of Microbiology and Immunology, Faculty of Medicine, Tanta University, Egypt, Egypt
| | - Alan McLachlan
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.
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Liu T, Sun Q, Liu Y, Cen S, Zhang Q. The MOV10 helicase restricts hepatitis B virus replication by inhibiting viral reverse transcription. J Biol Chem 2019; 294:19804-19813. [PMID: 31722967 DOI: 10.1074/jbc.ra119.009435] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Revised: 11/02/2019] [Indexed: 01/05/2023] Open
Abstract
Interferons inhibit viruses by inducing antiviral protein expression. One of the interferon-induced antiviral proteins, human Moloney leukemia virus 10 (MOV10), a superfamily 1 RNA helicase, has been shown to inhibit retroviruses and several RNA viruses. However, it remains undetermined whether MOV10 also inhibits DNA viruses, including hepatitis B virus (HBV). Here, we report that MOV10 dramatically reduces the levels of intracellular HBV DNA, resulting in significant inhibition of both the HBV experimental strain and the clinical isolates. Mechanistic experiments revealed that MOV10 interacts with HBV RNA and blocks the early step of viral reverse transcription, thereby impairing viral DNA synthesis, without affecting viral gene expression and pregenomic RNA encapsidation. Moreover, mutation of the helicase domain of MOV10 caused loss of binding to HBV RNA and of the anti-HBV activity. Together, our results indicate that MOV10 restricts HBV replication, insights that may open new avenues to the development of anti-HBV therapeutics.
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Affiliation(s)
- Tingting Liu
- Department of Transfusion Medicine, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008 China
| | - Qingsong Sun
- Department of Emergency, The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University, Huai'an 223301, China
| | - Yong Liu
- Department of Laboratory Medicine, Nanjing Drum Tower Hospital and Jiangsu Key Laboratory for Molecular Medicine, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008 China
| | - Shan Cen
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing 100050 China
| | - Quan Zhang
- Department of Laboratory Medicine, Nanjing Drum Tower Hospital and Jiangsu Key Laboratory for Molecular Medicine, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008 China .,Department of Infectious Diseases, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing Medical University, Nanjing 210008 China
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Birkus G, Snyder C, Jordan R, Kobayashi T, Dick R, Puscau V, Li L, Ramirez R, Willkom M, Morikawa Y, Delaney Iv WE, Schmitz U. Anti-HBV activity of retinoid drugs in vitro versus in vivo. Antiviral Res 2019; 169:104538. [PMID: 31226346 DOI: 10.1016/j.antiviral.2019.104538] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 05/20/2019] [Accepted: 06/17/2019] [Indexed: 02/06/2023]
Abstract
We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC50 values ranging from 0.4 to 2.6 μM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.
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Affiliation(s)
- Gabriel Birkus
- IOCB, Flemingovo nám. 542/2, 160 00, Praha 6, Czech Republic
| | - Chelsea Snyder
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Robert Jordan
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | | | - Ryan Dick
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Vlad Puscau
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Li Li
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Ricardo Ramirez
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | | | - Yoshida Morikawa
- Phoenix Bio, 3-4-1, Kagamiyama, Higashi-Hiroshima City, 739-0046, Japan
| | | | - Uli Schmitz
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA.
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Seo HW, Seo JP, Cho Y, Ko E, Kim YJ, Jung G. Cetylpyridinium chloride interaction with the hepatitis B virus core protein inhibits capsid assembly. Virus Res 2019; 263:102-111. [DOI: 10.1016/j.virusres.2019.01.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 01/08/2019] [Accepted: 01/09/2019] [Indexed: 01/11/2023]
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RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element. J Virol 2019; 93:JVI.02161-18. [PMID: 30626666 DOI: 10.1128/jvi.02161-18] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2018] [Accepted: 12/18/2018] [Indexed: 12/14/2022] Open
Abstract
Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.
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Krieger J, Stifter K, Riedl P, Schirmbeck R. Cationic domains in particle-forming and assembly-deficient HBV core antigens capture mammalian RNA that stimulates Th1-biased antibody responses by DNA vaccination. Sci Rep 2018; 8:14660. [PMID: 30279478 PMCID: PMC6168482 DOI: 10.1038/s41598-018-32971-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 09/19/2018] [Indexed: 12/21/2022] Open
Abstract
The HBV core protein self-assembles into particles and encapsidates immune-stimulatory bacterial RNA through a cationic COOH-terminal (C150-183) domain. To investigate if different cationic domains have an impact on the endogenous RNA-binding of HBV-C antigens in mammalian cells, we developed a strep-tag (st) based expression/purification system for HBV-C/RNA antigens in vector-transfected HEK-293 cells. We showed that HBV-stC but not HBV-stC149 particles (lacking the cationic domain) capture low amounts of mammalian RNA. Prevention of specific phosphorylation in cationic domains, either by exchanging the serine residues S155, S162 and S170 with alanines (HBV-stCAAA) or by exchanging the entire cationic domain with a HIV-tat48-57-like sequence (HBV-stC149tat) enhanced the encapsidation of RNA into mutant core particles. Particle-bound mammalian RNA functioned as TLR-7 ligand and induced a Th1-biased humoral immunity in B6 but not in TLR-7-/- mice by exogenous (protein) and endogenous (DNA) vaccines. Compared to core particles, binding of mammalian RNA to freely exposed cationic domains in assembly-deficient antigens was enhanced. However, RNA bound to non-particulate antigens unleash its Th1-stimulating adjuvant activity by DNA- but not protein-based vaccination. Mammalian RNAs targeted by an endogenously expressed antigen thus function as a natural adjuvant in the host that facilitates priming of Th1-biased immune responses by DNA-based immunization.
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Affiliation(s)
- Jana Krieger
- Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany
| | - Katja Stifter
- Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany
| | - Petra Riedl
- Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany
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Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions. J Virol 2018; 92:JVI.00272-18. [PMID: 29743374 DOI: 10.1128/jvi.00272-18] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Accepted: 05/04/2018] [Indexed: 02/06/2023] Open
Abstract
During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.
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Abstract
An estimated 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). Despite readily available vaccination, HBV infections remain highly prevalent. As established HBV infections constitute a strong risk factor for developing hepatocellular carcinoma their treatment is a major task for the health system. Unfortunately, HBV is not curable with today's medicine. Approximately 15 million HBV patients have developed a hepatitis delta (HDV) infection on top of their HBV infection. The patients superinfected with this satellite virus suffer from a more severe disease development. The knowledge of the viruses, their classifications, clinical implications, treatment options and efforts to increase the drug variety are compiled in this review. The current standard therapies include nucleosidic reverse transcriptase inhibitors and interferon. As the known treatments fail to cure HBV and HDV, targeted treatment is highly warranted. The focus of this review is set on the drugs currently under clinical investigation. Furthermore, strategies for the development of targeted treatment, and compounds with novel mode of action are described.
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39
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Seo HW, Seo JP, Kim YJ, Jung G. WITHDRAWN: Cetylpyridinium chloride as a novel inhibitor of hepatitis B viral capsid assembly. Biochem Biophys Res Commun 2018:S0006-291X(18)30103-7. [PMID: 29353039 DOI: 10.1016/j.bbrc.2018.01.088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2017] [Accepted: 01/12/2018] [Indexed: 10/18/2022]
Abstract
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
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Affiliation(s)
- Hyun Wook Seo
- Department of Biological Sciences, College of Natural Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, South Korea
| | - Joon Pyung Seo
- Department of Biological Sciences, College of Natural Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, South Korea
| | - Yoon Jun Kim
- Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, 110-744, South Korea
| | - Guhung Jung
- Department of Biological Sciences, College of Natural Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, South Korea
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40
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Lim KH, Park ES, Kim DH, Cho KC, Kim KP, Park YK, Ahn SH, Park SH, Kim KH, Kim CW, Kang HS, Lee AR, Park S, Sim H, Won J, Seok K, You JS, Lee JH, Yi NJ, Lee KW, Suh KS, Seong BL, Kim KH. Suppression of interferon-mediated anti-HBV response by single CpG methylation in the 5'-UTR of TRIM22. Gut 2018; 67:166-178. [PMID: 28341749 DOI: 10.1136/gutjnl-2016-312742] [Citation(s) in RCA: 59] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2016] [Revised: 02/17/2017] [Accepted: 02/21/2017] [Indexed: 12/18/2022]
Abstract
OBJECTIVE Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections. DESIGN We explored the cellular targets of HBV in response to IFNs using proteome-wide screening. RESULTS Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, including TRIM22, which is known as an antiretroviral protein. We demonstrated that HBx suppresses the transcription of TRIM22 through a single CpG methylation in its 5'-UTR, which further reduces the IFN regulatory factor-1 binding affinity, thereby suppressing the IFN-stimulated induction of TRIM22. CONCLUSIONS We verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.
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Affiliation(s)
- Keo-Heun Lim
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Eun-Sook Park
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Doo Hyun Kim
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Kyung Cho Cho
- Department of Applied Chemistry, Kyung Hee University, Yongin, Gyeonggi, Korea
| | - Kwang Pyo Kim
- Department of Applied Chemistry, Kyung Hee University, Yongin, Gyeonggi, Korea
| | - Yong Kwang Park
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Sung Hyun Ahn
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Seung Hwa Park
- Department of Anatomy, School of Medicine, Konkuk University, Seoul, Korea
| | - Kee-Hwan Kim
- Department of Surgery, Uijeongbu St Mary's Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea
| | - Chang Wook Kim
- Department of Internal Medicine, Uijeongbu St Mary's Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea
| | - Hong Seok Kang
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Ah Ram Lee
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Soree Park
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Heewoo Sim
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Juhee Won
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Kieun Seok
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea
| | - Jueng Soo You
- Department of Biochemistry, School of Medicine, Konkuk University, Seoul, Korea
| | - Jeong-Hoon Lee
- Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea
| | - Nam-Joon Yi
- Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
| | - Kwang-Woong Lee
- Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
| | - Kyung-Suk Suh
- Department of Surgery, Seoul National University College of Medicine, Seoul, Korea
| | - Baik L Seong
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea
| | - Kyun-Hwan Kim
- Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea.,KU Open Innovation Center, Konkuk University, Seoul, Korea.,Research Institute of Medical Sciences, Konkuk University, Seoul, Korea
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41
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Mak LY, Wong DKH, Seto WK, Lai CL, Yuen MF. Hepatitis B core protein as a therapeutic target. Expert Opin Ther Targets 2017; 21:1153-1159. [PMID: 29065733 DOI: 10.1080/14728222.2017.1397134] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Lung-Yi Mak
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
| | - Danny Ka-Ho Wong
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
- State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
| | - Wai-Kay Seto
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
- State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
| | - Ching-Lung Lai
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
- State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
| | - Man Fung Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China
- State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
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42
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Patel N, White SJ, Thompson RF, Bingham R, Weiß EU, Maskell DP, Zlotnick A, Dykeman E, Tuma R, Twarock R, Ranson NA, Stockley PG. HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly. Nat Microbiol 2017; 2:17098. [PMID: 28628133 PMCID: PMC5495169 DOI: 10.1038/nmicrobiol.2017.98] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Accepted: 05/17/2017] [Indexed: 12/20/2022]
Abstract
Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein-RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.
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Affiliation(s)
- Nikesh Patel
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Simon J White
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Rebecca F Thompson
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Richard Bingham
- Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York, YO10 5DD, UK
| | - Eva U Weiß
- Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York, YO10 5DD, UK
| | - Daniel P Maskell
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Adam Zlotnick
- Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
| | - Eric Dykeman
- Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York, YO10 5DD, UK
| | - Roman Tuma
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Reidun Twarock
- Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York, YO10 5DD, UK
| | - Neil A Ranson
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Peter G Stockley
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
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43
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Menéndez-Arias L, Sebastián-Martín A, Álvarez M. Viral reverse transcriptases. Virus Res 2017; 234:153-176. [PMID: 28043823 DOI: 10.1016/j.virusres.2016.12.019] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2016] [Revised: 12/19/2016] [Accepted: 12/24/2016] [Indexed: 12/11/2022]
Abstract
Reverse transcriptases (RTs) play a major role in the replication of Retroviridae, Metaviridae, Pseudoviridae, Hepadnaviridae and Caulimoviridae. RTs are enzymes that are able to synthesize DNA using RNA or DNA as templates (DNA polymerase activity), and degrade RNA when forming RNA/DNA hybrids (ribonuclease H activity). In retroviruses and LTR retrotransposons (Metaviridae and Pseudoviridae), the coordinated action of both enzymatic activities converts single-stranded RNA into a double-stranded DNA that is flanked by identical sequences known as long terminal repeats (LTRs). RTs of retroviruses and LTR retrotransposons are active as monomers (e.g. murine leukemia virus RT), homodimers (e.g. Ty3 RT) or heterodimers (e.g. human immunodeficiency virus type 1 (HIV-1) RT). RTs lack proofreading activity and display high intrinsic error rates. Besides, high recombination rates observed in retroviruses are promoted by poor processivity that causes template switching, a hallmark of reverse transcription. HIV-1 RT inhibitors acting on its polymerase activity constitute the backbone of current antiretroviral therapies, although novel drugs, including ribonuclease H inhibitors, are still necessary to fight HIV infections. In Hepadnaviridae and Caulimoviridae, reverse transcription leads to the formation of nicked circular DNAs that will be converted into episomal DNA in the host cell nucleus. Structural and biochemical information on their polymerases is limited, although several drugs inhibiting HIV-1 RT are known to be effective against the human hepatitis B virus polymerase. In this review, we summarize current knowledge on reverse transcription in the five virus families and discuss available biochemical and structural information on RTs, including their biosynthesis, enzymatic activities, and potential inhibition.
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Affiliation(s)
- Luis Menéndez-Arias
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain.
| | - Alba Sebastián-Martín
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain
| | - Mar Álvarez
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain
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44
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Castelain S, Descamps V, Brochot E, Helle F, Duverlie G, Nguyen-Khac E, François C. High association of T1858-G1896 precore mutations with impaired base pairing and high hepatitis B virus DNA levels in HBeAg-negative chronically infected patients. Arch Virol 2017; 162:1913-1920. [PMID: 28289975 DOI: 10.1007/s00705-017-3312-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 02/21/2017] [Indexed: 12/20/2022]
Abstract
The progression of liver disease in hepatitis B virus (HBV) infection is fostered by active virus replication. Mutations in the basal core promoter (BCP) and precore (PC) regions of the HBV genome are known to have an impact on viral replication. The aim of the present study was to assess the correlation of mutation profiles in the BCP and PC regions with the viral load in HBeAg-negative chronically infected patients. The HBV genotype, BCP/PC mutations, serum HBV DNA levels, and associated serological markers were analyzed in 92 HBeAg-negative chronically infected patients. Sequence analysis of the BCP and PC regions revealed variability of 19% and 24.1%, respectively. This variability was primarily associated with five critical positions (1753, 1762, 1764, 1896 and 1899). An elevated HBV viral load (>20,000 IU/ml) was classically correlated with F2-F4 liver fibrosis, elevated serum alanine aminotransferase levels, 1762/1764 and 1753 combination mutations, and surprisingly, with an 1858T-1896G double mutation that impairs base pairing at the base of the bulge in the ε encapsidation signal. An analysis of covariance confirmed the independent nature of the relationship between the 1858T-1896G double mutation and the HBV viral load. In conclusion, independently of conventional parameters, this study demonstrates that a high serum HBV DNA level was also associated with PC 1858-1896 mutations. These BCP/PC mutations may have important clinical implications as predictive factors for HBV DNA increase.
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Affiliation(s)
- Sandrine Castelain
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France. .,EA4294, Université de Picardie Jules Verne, Amiens, France.
| | - Véronique Descamps
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Etienne Brochot
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - François Helle
- EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Gilles Duverlie
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
| | - Eric Nguyen-Khac
- Hepatology Department, Centre Hospitalo-Universitaire Amiens Picardie, Amiens, France
| | - Catherine François
- Virology Department, Centre de Biologie Humaine, Centre Hospitalo-Universitaire Amiens Picardie, 80054, Amiens Cedex, France.,EA4294, Université de Picardie Jules Verne, Amiens, France
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45
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Nishitsuji H, Yamamoto H, Shiina R, Harada K, Ujino S, Shimotohno K. Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle. J Vis Exp 2017. [PMID: 28190076 DOI: 10.3791/54849] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.
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Affiliation(s)
- Hironori Nishitsuji
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine;
| | - Hiromi Yamamoto
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine
| | - Ritsuko Shiina
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine
| | - Keisuke Harada
- Central Pharmaceutical Research Institute, Japan Tobacco Inc
| | - Saneyuki Ujino
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine
| | - Kunitada Shimotohno
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine
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46
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HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids. Sci Rep 2016; 6:38959. [PMID: 27958343 PMCID: PMC5154190 DOI: 10.1038/srep38959] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2016] [Accepted: 11/14/2016] [Indexed: 12/22/2022] Open
Abstract
Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature.
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47
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Bertoletti A, Ferrari C. Adaptive immunity in HBV infection. J Hepatol 2016; 64:S71-S83. [PMID: 27084039 DOI: 10.1016/j.jhep.2016.01.026] [Citation(s) in RCA: 352] [Impact Index Per Article: 39.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Revised: 01/12/2016] [Accepted: 01/25/2016] [Indexed: 02/06/2023]
Abstract
During hepatitis B virus (HBV) infection, the presence of HBV-specific antibody producing B cells and functional HBV-specific T cells (with helper or cytotoxic effects) ultimately determines HBV infection outcome. In this review, in addition to summarizing the present state of knowledge of HBV-adaptive immunity, we will highlight controversies and uncertainties concerning the HBV-specific B and T lymphocyte response, and propose future directions for research aimed at the generation of more efficient immunotherapeutic strategies.
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Affiliation(s)
- Antonio Bertoletti
- Emerging Infectious Diseases (EID) Program, Duke-NUS Medical School, Singapore; Viral Hepatitis Laboratory, Singapore Institute for Clinical Sciences, Agency of Science Technology and Research (A*STAR), Singapore.
| | - Carlo Ferrari
- Divisione Malattie Infettive, Ospdale Maggiore Parma, Parma, Italy
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48
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Wang H, Liu K, Fang BAM, Wu H, Li F, Xiang X, Tang W, Zhao G, Lin L, Bao S, Xie Q. Identification of acetyltransferase genes (HAT1 and KAT8) regulating HBV replication by RNAi screening. Cell Biosci 2015; 5:66. [PMID: 26640654 PMCID: PMC4669656 DOI: 10.1186/s13578-015-0059-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2015] [Accepted: 11/23/2015] [Indexed: 12/31/2022] Open
Abstract
Background The initiation of hepatitis B virus (HBV) replication involves the formation of covalently closed circular DNA (cccDNA) and its transcription into pregenomic RNA (pgRNA) in hepatocyte nuclei. The regulatory mechanism of HBV replication by acetyltransferase is thus far not well understood, but human acetyltransferase has been reported as being involved in the regulation of HBV replication. Results Depletion of KAT8 or HAT1 via RNA interference (RNAi) markedly down-regulated HBV-DNA and pgRNA levels in HepG2.2.15 cells, with KAT8 knockdown reducing both HBsAg and HBeAg more than HAT1 knockdown. Consistent with these observations, HBV replication regulators hepatocyte nuclear factor-4-α (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator- (PPARGC-) 1-α were decreased following knockdown of HAT1 or KAT8. Conclusions These data suggest that KAT8 or HAT1 regulate HBV replication and may be potential drug targets of anti-HBV therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13578-015-0059-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Hui Wang
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - KeHui Liu
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Bernard A M Fang
- Discipline of Pathology, School of Medical Sciences and The Bosch Institute, Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006 Australia.,Central Clinical School, Sydney Medical School, The University of Sydney, Sydney, NSW 2006 Australia
| | - HaiQing Wu
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,Department of Cardiology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - FengDi Li
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - XiaoGang Xiang
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - WeiLiang Tang
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - GangDe Zhao
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - LanYi Lin
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Shisan Bao
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,Discipline of Pathology, School of Medical Sciences and The Bosch Institute, Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006 Australia
| | - Qing Xie
- Department of Infectious Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation. J Virol 2015. [PMID: 26202253 DOI: 10.1128/jvi.01481-15] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.
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50
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Hu J, Seeger C. Hepadnavirus Genome Replication and Persistence. Cold Spring Harb Perspect Med 2015; 5:a021386. [PMID: 26134841 DOI: 10.1101/cshperspect.a021386] [Citation(s) in RCA: 104] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Hallmarks of the hepadnavirus replication cycle are the formation of covalently closed circular DNA (cccDNA) and the reverse transcription of a pregenomic RNA (pgRNA) in core particles leading to synthesis of the relaxed circular DNA (rcDNA) genome. cccDNA, the template for viral RNA transcription, is the basis for the persistence of these viruses in infected hepatocytes. In this review, we summarize the current state of knowledge on the mechanisms of hepadnavirus reverse transcription and the biochemical and structural properties of the viral reverse transcriptase (RT). We highlight important gaps in knowledge regarding cccDNA biosynthesis and stability. In addition, we discuss the impact of current antiviral therapies on viral persistence, particularly on cccDNA.
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Affiliation(s)
- Jianming Hu
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, Pennsylvania 17033
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