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Yin K, Zhang T, Huang J, Hao B. Nup358 and Nup153 Facilitate nuclear import of BmNPV nucleocapsids in Bombyx mori cells. J Invertebr Pathol 2025; 211:108318. [PMID: 40120667 DOI: 10.1016/j.jip.2025.108318] [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: 07/26/2024] [Revised: 03/17/2025] [Accepted: 03/18/2025] [Indexed: 03/25/2025]
Abstract
Nuclear pore complexes (NPCs) are crucial for the nuclear import of viral genomes, serving as gateways for many viruses to deliver their genetic material into host cell nuclei. However, the role of NPCs in the entry of Bombyx mori nucleopolyhedrovirus (BmNPV) remains poorly understood. This study investigates the involvement of specific nucleoporins in the nuclear import of BmNPV nucleocapsids, a crucial step for viral replication in host cells. Using a combination of Importazole, wheat germ agglutinin (WGA), and small interfering RNAs (siRNAs), we demonstrate that BmNPV entry into BmN cells is mediated through the NPC. Importazole and WGA treatment significantly inhibited viral infection, highlighting the essential role of nucleoporins in BmNPV entry. Additionally, siRNA-mediated knockdown of Nup358 and Nup153 resulted in a marked accumulation of nucleocapsids in the cytoplasm. Overexpression of the N-terminal fragment of Nup358 (1-1127) enhanced nucleocapsid entry efficiency, whereas deletion of the phenylalanine-glycine (FG) repeats in Nup153 (Nup153ΔFG) reduced viral entry. These results confirm the pivotal roles of Nup358 and Nup153 in the nuclear import of BmNPV nucleocapsids. Our findings provide new insights into the molecular interactions between BmNPV and host NPCs, offering potential targets for controlling BmNPV infections in sericulture.
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Affiliation(s)
- Kangping Yin
- Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
| | - Tianran Zhang
- Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
| | - Jinshan Huang
- Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China; Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang 212100, China
| | - Bifang Hao
- Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China; Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang 212100, China.
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2
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Hou Z, Shen Y, Fronik S, Shen J, Shi J, Xu J, Chen L, Hardenbrook N, Thompson C, Neumann S, Engelman AN, Aiken C, Zhang P. Correlative In Situ Cryo-ET Reveals Cellular and Viral Remodeling Associated with Selective HIV-1 Core Nuclear Import. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.04.641496. [PMID: 40093063 PMCID: PMC11908238 DOI: 10.1101/2025.03.04.641496] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Lentiviruses like HIV-1 infect non-dividing cells by traversing the nuclear pore, but studying this process has been challenging due to its scarcity and dynamic nature in infected cells. Here, we developed a robust cell-permeabilization system that recapitulates HIV-1 nuclear import and established an integrated cryo-correlative workflow combining cryo-CLEM, cryo-FIB, and cryo-ET for targeted imaging of this process. These advancements enabled the successful capture of 1,899 HIV-1 cores at various stages of nuclear import. Statistical and structural analyses of native wild-type and mutant cores revealed that HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability, as well as nuclear factors such as CPSF6. Brittle cores fail to enter the nuclear pore complex (NPC), while CPSF6-binding-deficient cores stall inside the NPC, resulting in impaired nuclear import. Intriguingly, nuclear pores function as selective filters favoring the import of smaller, tube-shaped cores. Our study opens new avenues for dissecting the biochemistry and structural biology of HIV-1 nuclear import as well as downstream events including core uncoating and potentially integration, with unprecedented detail.
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Affiliation(s)
- Zhen Hou
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Yao Shen
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Stanley Fronik
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Juan Shen
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Jiong Shi
- Department of Pathology, Microbiology and Immunology and Vanderbitl Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jialu Xu
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Long Chen
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Nathan Hardenbrook
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Christopher Thompson
- Materials & Structural Analysis, Thermo Fisher Scientific, Eindhoven, The Netherlands
| | - Sarah Neumann
- Materials & Structural Analysis, Thermo Fisher Scientific, Eindhoven, The Netherlands
| | - Alan N. Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA, USA
| | - Christopher Aiken
- Department of Pathology, Microbiology and Immunology and Vanderbitl Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Peijun Zhang
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
- Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK
- Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK
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3
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Zhang X, Lim K, Qiu Y, Hazawa M, Wong RW. Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways. Viruses 2025; 17:151. [PMID: 40006906 PMCID: PMC11860923 DOI: 10.3390/v17020151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Revised: 01/20/2025] [Accepted: 01/21/2025] [Indexed: 02/27/2025] Open
Abstract
Viruses frequently exploit the host's nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins and other components, they strategically target host nuclear transport receptors (NTRs) and nucleoporins within the spiderweb-like inner channel of the nuclear pore complex (NPC), enabling efficient access to the host nucleus. This review explores the intricate mechanisms governing the nuclear import and export of viral components, with a focus on the interplay between viral factors and host determinants that are essential for these processes. Given the pivotal role of nucleocytoplasmic shuttling in the viral life cycle, we also examine therapeutic strategies aimed at disrupting the host's nuclear transport pathways. This includes evaluating the efficacy of pharmacological inhibitors in impairing viral replication and assessing their potential as antiviral treatments. Furthermore, we emphasize the need for continued research to develop targeted therapies that leverage vulnerabilities in nucleocytoplasmic trafficking. Emerging high-resolution techniques, such as advanced imaging and computational modeling, are transforming our understanding of the dynamic interactions between viruses and the NPC. These cutting-edge tools are driving progress in identifying novel therapeutic opportunities and uncovering deeper insights into viral pathogenesis. This review highlights the importance of these advancements in paving the way for innovative antiviral strategies.
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Affiliation(s)
- Xin Zhang
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
| | - Keesiang Lim
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
| | - Yujia Qiu
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
| | - Masaharu Hazawa
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
| | - Richard W. Wong
- Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan; (X.Z.); (Y.Q.)
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan;
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
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4
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Ringlander J, Rydell GE, Kann M. From the Cytoplasm into the Nucleus-Hepatitis B Virus Travel and Genome Repair. Microorganisms 2025; 13:157. [PMID: 39858925 PMCID: PMC11767736 DOI: 10.3390/microorganisms13010157] [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: 12/18/2024] [Revised: 01/03/2025] [Accepted: 01/08/2025] [Indexed: 01/27/2025] Open
Abstract
Hepatitis B virus (HBV) is a major global health concern, affecting millions of people worldwide. HBV is part of the hepadnaviridae family and one of the primary causes of acute and chronic liver infections, leading to conditions such as cirrhosis and hepatocellular carcinoma (HCC). Understanding the intracellular transport and genome repair mechanisms of HBV is crucial for developing new drugs, which-in combination with immune modulators-may contribute to potential cures. This review will explore the current knowledge of HBV intracytoplasmic and nuclear transport, as well as genome repair processes, while drawing comparisons to other viruses with nuclear replication.
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Affiliation(s)
- Johan Ringlander
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
- Department of Clinical Microbiology, Region Västra Götaland, Sahlgrenska University Hospital, 41346 Gothenburg, Sweden
| | - Gustaf E. Rydell
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
| | - Michael Kann
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 41346 Gothenburg, Sweden; (J.R.); (G.E.R.)
- Department of Clinical Microbiology, Region Västra Götaland, Sahlgrenska University Hospital, 41346 Gothenburg, Sweden
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5
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Chuang YC, Ou JHJ. Hepatitis B virus entry, assembly, and egress. Microbiol Mol Biol Rev 2024; 88:e0001424. [PMID: 39440957 DOI: 10.1128/mmbr.00014-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2024] Open
Abstract
SUMMARYHepatitis B virus (HBV) is an important human pathogen that chronically infects approximately 250 million people in the world, resulting in ~1 million deaths annually. This virus is a hepatotropic virus and can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. The entry of HBV into hepatocytes is initiated by the interaction of its envelope proteins with its receptors. This is followed by the delivery of the viral nucleocapsid to the nucleus for the release of its genomic DNA and the transcription of viral RNAs. The assembly of the viral capsid particles may then take place in the nucleus or the cytoplasm and may involve cellular membranes. This is followed by the egress of the virus from infected cells. In recent years, significant research progresses had been made toward understanding the entry, the assembly, and the egress of HBV particles. In this review, we discuss the molecular pathways of these processes and compare them with those used by hepatitis delta virus and hepatitis C virus , two other hepatotropic viruses that are also enveloped. The understanding of these processes will help us to understand how HBV replicates and causes diseases, which will help to improve the treatments for HBV patients.
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Affiliation(s)
- Yu-Chen Chuang
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
| | - J-H James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
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Lei Z, Wang L, Gao H, Guo S, Kang X, Yuan J, Lv Z, Jiang Y, Yi J, Chen Z, Wang G. Mechanisms underlying the compromised clinical efficacy of interferon in clearing HBV. Virol J 2024; 21:314. [PMID: 39633459 PMCID: PMC11619119 DOI: 10.1186/s12985-024-02589-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2024] [Accepted: 11/26/2024] [Indexed: 12/07/2024] Open
Abstract
Hepatitis B virus (HBV) is a hepatotropic DNA virus that can cause acute or chronic hepatitis, representing a significant global health concern. By 2019, approximately 296 million individuals were chronically infected with HBV, with 1.5 million new cases annually and 820,000 deaths due to HBV-related cirrhosis and liver cancer. Current treatments for chronic hepatitis B include nucleotide analogs (NAs) and interferons (IFNs), particularly IFN-α. NAs, such as entecavir and tenofovir, inhibit viral reverse transcription, while IFN-α exerts antiviral effects by directly suppressing viral replication, modulating viral genome epigenetics, degrading cccDNA, and activating immune responses. Despite its potential, IFN-α shows limited clinical efficacy, partly due to HBV's interference with the IFN signaling pathway. HBV encodes proteins like HBc, Pol, HBsAg, and HBx that disrupt IFN-α function. For example, HBV Pol inhibits STAT1 phosphorylation, HBsAg suppresses STAT3 phosphorylation, and HBx interferes with IFN-α efficacy through multiple mechanisms. Additionally, HBV downregulates key genes in the IFN signaling pathway, further diminishing IFN-α's antiviral effects. Understanding these interactions is crucial for improving IFN-α-based therapies. Future research may focus on overcoming HBV resistance by targeting viral proteins or optimizing IFN-α delivery. In summary, HBV's ability to resist IFN-α limits its therapeutic effectiveness, highlighting the need for new strategies to enhance treatment outcomes.
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Affiliation(s)
- Zhuoyan Lei
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Luye Wang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Hanlin Gao
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Shubian Guo
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Xinjian Kang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Jiajun Yuan
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Ziying Lv
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Yuxin Jiang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China
| | - Jinping Yi
- Department of Clinical Laboratory, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Zhi Chen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, National Clinical Research Center for Infectious Diseases, Zhejiang University, Hangzhou, China
| | - Gang Wang
- Key Laboratory of Artificial Organs and Computational Medicine of Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, 8 Shuren St, Gongshu District, Hangzhou, 310015, Zhejiang Province, China.
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7
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Feng Q, Saladin M, Wu C, Cao E, Zheng W, Zhang A, Bhardwaj P, Li X, Shen Q, Kapinos LE, Kozai T, Mariappan M, Lusk CP, Xiong Y, Lim RYH, Lin C. Channel width modulates the permeability of DNA origami-based nuclear pore mimics. SCIENCE ADVANCES 2024; 10:eadq8773. [PMID: 39536094 PMCID: PMC11559598 DOI: 10.1126/sciadv.adq8773] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/04/2024] [Accepted: 10/09/2024] [Indexed: 11/16/2024]
Abstract
Nucleoporins (nups) in the nuclear pore complex (NPC) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptor (NTR)-bound cargos. Recent studies have shown that the NPC may dilate and constrict, but how altering the NPC diameter affects its selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangements to model the constricted and dilated NPCs. We find that Nup62 proteins form a dynamic cross-channel barrier impermeable to hepatitis B virus (HBV) capsids when grafted inside 60-nm-wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importin-β1 substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through the 60-nm NPC mimics containing Nup62 and Nup153. Our study shows that transport channel width is critical to the permeability of nup barriers and underscores NTRs' role in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.
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Affiliation(s)
- Qingzhou Feng
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
| | | | - Chunxiang Wu
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Eason Cao
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
| | - Wei Zheng
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Amy Zhang
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
| | - Pushpanjali Bhardwaj
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Xia Li
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
| | - Qi Shen
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | | | - Toshiya Kozai
- Biozentrum, University of Basel, Basel, Switzerland
- Swiss Nanoscience Institute, University of Basel, Basel, Switzerland
| | - Malaiyalam Mariappan
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
| | - C. Patrick Lusk
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
| | - Yong Xiong
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Roderick Y. H. Lim
- Biozentrum, University of Basel, Basel, Switzerland
- Swiss Nanoscience Institute, University of Basel, Basel, Switzerland
| | - Chenxiang Lin
- Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA
- Nanobiology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
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8
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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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9
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Bendahmane S, Follo M, Zhang F, Linhardt RJ. Towards Cell-Permeable Hepatitis B Virus Core Protein Variants as Potential Antiviral Agents. Microorganisms 2024; 12:1776. [PMID: 39338451 PMCID: PMC11434381 DOI: 10.3390/microorganisms12091776] [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: 06/11/2024] [Revised: 07/17/2024] [Accepted: 08/01/2024] [Indexed: 09/30/2024] Open
Abstract
Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile.
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Affiliation(s)
- Sanaa Bendahmane
- Private Faculty of Health Professions and Technologies, Private University of Marrakech, Marrakech 42312, Morocco
| | - Marie Follo
- Department of Medicine I, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, 79085 Freiburg, Germany
| | - Fuming Zhang
- Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
| | - Robert J Linhardt
- Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
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10
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Park IW, Fiadjoe HK, Chaudhary P. Impact of Annexin A2 on virus life cycles. Virus Res 2024; 345:199384. [PMID: 38702018 PMCID: PMC11091703 DOI: 10.1016/j.virusres.2024.199384] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2024] [Revised: 04/29/2024] [Accepted: 04/30/2024] [Indexed: 05/06/2024]
Abstract
Due to the limited size of viral genomes, hijacking host machinery by the viruses taking place throughout the virus life cycle is inevitable for the survival and proliferation of the virus in the infected hosts. Recent reports indicated that Annexin A2 (AnxA2), a calcium- and lipid-binding cellular protein, plays an important role as a critical regulator in various steps of the virus life cycle. The multifarious AnxA2 functions in cells, such as adhesion, adsorption, endocytosis, exocytosis, cell proliferation and division, inflammation, cancer metastasis, angiogenesis, etc., are intimately related to the various clinical courses of viral infection. Ubiquitous expression of AnxA2 across multiple cell types indicates the broad range of susceptibility of diverse species of the virus to induce disparate viral disease in various tissues, and intracellular expression of AnxA2 in the cytoplasmic membrane, cytosol, and nucleus suggests the involvement of AnxA2 in the regulation of the different stages of various virus life cycles within host cells. However, it is yet unclear as to the molecular processes on how AnxA2 and the infected virus interplay to regulate virus life cycles and thereby the virus-associated disease courses, and hence elucidation of the molecular mechanisms on AnxA2-mediated virus life cycle will provide essential clues to develop therapeutics deterring viral disease.
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Affiliation(s)
- In-Woo Park
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States.
| | - Hope K Fiadjoe
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States
| | - Pankaj Chaudhary
- Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, United States.
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11
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Feng Q, Saladin M, Wu C, Cao E, Zheng W, Zhang A, Bhardwaj P, Li X, Shen Q, Kapinos LE, Mariappan M, Lusk CP, Xiong Y, Lim RYH, Lin C. Channel width modulates the permeability of DNA origami based nuclear pore mimics. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.09.593438. [PMID: 38766144 PMCID: PMC11100828 DOI: 10.1101/2024.05.09.593438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2024]
Abstract
Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importing substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.
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Affiliation(s)
- Qingzhou Feng
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
| | | | - Chunxiang Wu
- Department of Biophysics and Biochemistry, Yale University, USA
| | - Eason Cao
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
| | - Wei Zheng
- Department of Biophysics and Biochemistry, Yale University, USA
| | - Amy Zhang
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
| | | | - Xia Li
- Department of Cell Biology, Yale School of Medicine, USA
| | - Qi Shen
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
- Department of Biophysics and Biochemistry, Yale University, USA
| | | | - Malaiyalam Mariappan
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
| | | | - Yong Xiong
- Department of Biophysics and Biochemistry, Yale University, USA
| | - Roderick Y. H. Lim
- Biozentrum, University of Basel, Switzerland
- Swiss Nanoscience Institute, University of Basel, Switzerland
| | - Chenxiang Lin
- Department of Cell Biology, Yale School of Medicine, USA
- Nanobiology Institute, Yale University, USA
- Department of Biomedical Engineering, Yale University, USA
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12
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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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13
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Yip RPH, Kwok DCY, Lai LTF, Ho SM, Wong ICK, Chan CP, Lau WCY, Ngo JCK. SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction. PLoS Pathog 2024; 20:e1011978. [PMID: 38324561 PMCID: PMC10878513 DOI: 10.1371/journal.ppat.1011978] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 02/20/2024] [Accepted: 01/17/2024] [Indexed: 02/09/2024] Open
Abstract
Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.
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Affiliation(s)
- Ryan Pak Hong Yip
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
| | - Doris Ching Ying Kwok
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
| | - Louis Tung Faat Lai
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
| | - Siu-Ming Ho
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
- State Key Laboratory of Liver Research (The University of Hong Kong), Pokfulam, Hong Kong, Hong Kong SAR, China
| | - Ivan Chun Kit Wong
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
| | - Chi-Ping Chan
- School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China
- State Key Laboratory of Liver Research (The University of Hong Kong), Pokfulam, Hong Kong, Hong Kong SAR, China
| | - Wilson Chun Yu Lau
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
- Center for Soybean Research of the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
| | - Jacky Chi Ki Ngo
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China
- Center for Soybean Research of the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
- Center for Novel Biomaterials, The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
- Center for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
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14
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Yang R, Ko YH, Li F, Lokareddy RK, Hou CFD, Kim C, Klein S, Antolínez S, Marín JF, Pérez-Segura C, Jarrold MF, Zlotnick A, Hadden-Perilla JA, Cingolani G. Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core. SCIENCE ADVANCES 2024; 10:eadi7606. [PMID: 38198557 PMCID: PMC10780889 DOI: 10.1126/sciadv.adi7606] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Accepted: 12/08/2023] [Indexed: 01/12/2024]
Abstract
Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/β1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.
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Affiliation(s)
- Ruoyu Yang
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA
| | - Ying-Hui Ko
- Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, 1825 University Blvd, Birmingham, AL 35294, USA
| | - Fenglin Li
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA
| | - Ravi K. Lokareddy
- Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, 1825 University Blvd, Birmingham, AL 35294, USA
| | - Chun-Feng David Hou
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA
| | - Christine Kim
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, IN 47405, USA
| | - Shelby Klein
- Department of Chemistry, Indiana University, Bloomington, Indiana, IN 47405, USA
| | - Santiago Antolínez
- Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
| | - Juan F. Marín
- Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
| | - Carolina Pérez-Segura
- Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
| | - Martin F. Jarrold
- Department of Chemistry, Indiana University, Bloomington, Indiana, IN 47405, USA
| | - Adam Zlotnick
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, IN 47405, USA
| | | | - Gino Cingolani
- Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, 1825 University Blvd, Birmingham, AL 35294, USA
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15
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Cross EM, Marin O, Ariawan D, Aragão D, Cozza G, Di Iorio E, Forwood JK, Alvisi G. Structural determinants of phosphorylation-dependent nuclear transport of HCMV DNA polymerase processivity factor UL44. FEBS Lett 2024; 598:199-209. [PMID: 38158756 DOI: 10.1002/1873-3468.14797] [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: 10/30/2023] [Revised: 12/08/2023] [Accepted: 12/11/2023] [Indexed: 01/03/2024]
Abstract
Human cytomegalovirus DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/β through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2-mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue by studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10-fold, and a comparative structural analysis of UL44 NLS phosphorylated and non-phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation-dependent inhibition of UL44 nuclear import.
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Affiliation(s)
- Emily M Cross
- School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, Australia
- Diamond Light Source, Didcot, UK
| | - Oriano Marin
- Department of Biomedical Sciences, University of Padova, Italy
| | - Daryl Ariawan
- Dementia Research Centre, Macquarie University, Sydney, Australia
| | | | - Giorgio Cozza
- Department of Molecular Medicine, University of Padua, Italy
| | - Enzo Di Iorio
- Department of Molecular Medicine, University of Padua, Italy
| | - Jade K Forwood
- School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, Australia
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16
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Li X, Nakashima K, Ito M, Matsuda M, Chida T, Sekihara K, Takahashi H, Kato T, Sawasaki T, Suzuki T. SRPKIN-1 as an inhibitor against hepatitis B virus blocking the viral particle formation and the early step of the viral infection. Antiviral Res 2023; 220:105756. [PMID: 37992764 DOI: 10.1016/j.antiviral.2023.105756] [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: 04/24/2023] [Revised: 11/13/2023] [Accepted: 11/13/2023] [Indexed: 11/24/2023]
Abstract
New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.
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Affiliation(s)
- Xiaofang Li
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kenji Nakashima
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masahiko Ito
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Mami Matsuda
- Department of Virology II, National Institute of Infectious Diseases, Musashi-murayama, Japan
| | - Takeshi Chida
- Department of Internal Medicine II, Hamamatsu University School of Medicine, Shizuoka, Japan; Department of Regional Medical Care Support, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kazumasa Sekihara
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Hirotaka Takahashi
- Division of Cell-Free Science, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tatsuya Sawasaki
- Division of Cell-Free Science, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan
| | - Tetsuro Suzuki
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan.
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17
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Nayak S, Gowda J, Abbas SA, Kim H, Han SB. Recent Advances in the Development of Sulfamoyl-Based Hepatitis B Virus Nucleocapsid Assembly Modulators. Viruses 2023; 15:2367. [PMID: 38140607 PMCID: PMC10747759 DOI: 10.3390/v15122367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2023] [Revised: 11/24/2023] [Accepted: 11/27/2023] [Indexed: 12/24/2023] Open
Abstract
Hepatitis B virus (HBV) is the primary contributor to severe liver ailments, encompassing conditions such as cirrhosis and hepatocellular carcinoma. Globally, 257 million people are affected by HBV annually and 887,000 deaths are attributed to it, representing a substantial health burden. Regrettably, none of the existing therapies for chronic hepatitis B (CHB) have achieved satisfactory clinical cure rates. This issue stems from the existence of covalently closed circular DNA (cccDNA), which is difficult to eliminate from the nucleus of infected hepatocytes. HBV genetic material is composed of partially double-stranded DNA that forms complexes with viral polymerase inside an icosahedral capsid composed of a dimeric core protein. The HBV core protein, consisting of 183 to 185 amino acids, plays integral roles in multiple essential functions within the HBV replication process. In this review, we describe the effects of sulfamoyl-based carboxamide capsid assembly modulators (CAMs) on capsid assembly, which can suppress HBV replication and disrupt the production of new cccDNA. We present research on classical, first-generation sulfamoyl benzocarboxamide CAMs, elucidating their structural composition and antiviral efficacy. Additionally, we explore newly identified sulfamoyl-based CAMs, including sulfamoyl bicyclic carboxamides, sulfamoyl aromatic heterocyclic carboxamides, sulfamoyl aliphatic heterocyclic carboxamides, cyclic sulfonamides, and non-carboxamide sulfomoyl-based CAMs. We believe that certain molecules derived from sulfamoyl groups have the potential to be developed into essential components of a well-suited combination therapy, ultimately yielding superior clinical efficacy outcomes in the future.
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Affiliation(s)
- Sandesha Nayak
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Jayaraj Gowda
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Syed Azeem Abbas
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
| | - Hyejin Kim
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
| | - Soo Bong Han
- Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
- Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
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18
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Kim C, Schlicksup CJ, Pérez-Segura C, Hadden-Perilla JA, Wang JCY, Zlotnick A. Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit. J Biol Chem 2023; 299:105104. [PMID: 37517693 PMCID: PMC10463254 DOI: 10.1016/j.jbc.2023.105104] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2023] [Revised: 07/08/2023] [Accepted: 07/11/2023] [Indexed: 08/01/2023] Open
Abstract
Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/β complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impβ as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impβ-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.
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Affiliation(s)
- Christine Kim
- Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA
| | | | - Carolina Pérez-Segura
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Jodi A Hadden-Perilla
- Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
| | - Joseph Che-Yen Wang
- Department of Microbiology & Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Adam Zlotnick
- Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA.
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19
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Kwon H, Kim J, Song C, Sajjad MA, Ha J, Jung J, Park S, Shin HJ, Kim K. Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication. Front Cell Infect Microbiol 2023; 13:1195063. [PMID: 37404723 PMCID: PMC10315659 DOI: 10.3389/fcimb.2023.1195063] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2023] [Accepted: 06/01/2023] [Indexed: 07/06/2023] Open
Abstract
Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.
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Affiliation(s)
- Hyeonjoong Kwon
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jumi Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Chanho Song
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Muhammad Azhar Sajjad
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jiseon Ha
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Jaesung Jung
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Sun Park
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Ho-Joon Shin
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
| | - Kyongmin Kim
- Department of Microbiology, Ajou University School of Medicine, Suwon, Republic of Korea
- Department of Biomedical Science, Graduate School of Ajou University, Suwon, Republic of Korea
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20
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Hofmann S, Plank V, Groitl P, Skvorc N, Hofmann K, Luther J, Ko C, Zimmerman P, Bruss V, Stadler D, Carpentier A, Rezk S, Nassal M, Protzer U, Schreiner S. SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA. Microbiol Spectr 2023; 11:e0044623. [PMID: 37199632 PMCID: PMC10269885 DOI: 10.1128/spectrum.00446-23] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2023] [Accepted: 04/24/2023] [Indexed: 05/19/2023] Open
Abstract
Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.
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Affiliation(s)
- Samuel Hofmann
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Verena Plank
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
| | - Peter Groitl
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
| | - Nathalie Skvorc
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
| | - Katharina Hofmann
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Julius Luther
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Chunkyu Ko
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Helmholtz Zentrum München, Munich, Germany
| | - Peter Zimmerman
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
| | - Volker Bruss
- Institute of Virology, Helmholtz Zentrum München, Munich, Germany
| | - Daniela Stadler
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Helmholtz Zentrum München, Munich, Germany
| | | | - Shahinda Rezk
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Medical Research Institute, Department of Molecular and Diagnostic Microbiology, Alexandria University, Alexandria, Egypt
| | - Michael Nassal
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
| | - Ulrike Protzer
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Helmholtz Zentrum München, Munich, Germany
- German Center for Infection Research, Munich, Germany
| | - Sabrina Schreiner
- Institute of Virology, School of Medicine, Technical University of Munich, Germany
- Institute of Virology, Hannover Medical School, Hannover, Germany
- Institute of Virology, Helmholtz Zentrum München, Munich, Germany
- German Center for Infection Research, Munich, Germany
- Cluster of Excellence RESIST (Resolving Infection Susceptibility; EXC 2155), Hannover Medical School, Hannover, Germany
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21
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Abstract
Cacao swollen shoot virus causes cacao swollen shoot disease of Theobroma cacao (cacao) plants. At least six cacao-infecting Badnavirus species-Cacao swollen shoot Togo A virus, Cacao swollen shoot Togo B virus (previously known as Cacao swollen shoot virus), Cacao swollen shoot CE virus, Cacao swollen shoot Ghana M virus, Cacao swollen shoot Ghana N virus, and Cacao swollen shoot Ghana Q virus-are responsible for the swollen shoot disease of cacao in Ghana. Each of these species consists of a multiplicity of strains. The New Juaben strain, the most virulent cacao swollen shoot virus strain in Ghana, belongs to the Cacao swollen shoot Togo B virus species, and is a commonly used strain in laboratory transmission assays. Infection of cacao trees with multiple strains of the virus is common and new evidence suggests that these coinfections may have resulted in the emergence of recombinant strains of the virus. The impact of these emerging recombinant strains on disease severity is uncertain. This review focuses largely on the discovery of cacao swollen shoot virus in Ghana, diversity of the virus strains, molecular characterization, propagation of virus infection in cacao plants, emergence of recombinant virus strains, vector-mediated transmission of the virus, and the management of the cacao swollen shoot disease in Ghana. It also contains sections on the botany and origin of the cacao tree, its introduction to Ghana, the role of cacao swollen shoot disease in facilitating Ghana's independence from Britain, and a brief history of chocolate.
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Affiliation(s)
| | - Owusu Domfeh
- Plant Pathology Division, Cocoa Research Institute of Ghana, New Tafo, Akim, Ghana
| | - George Akumfi Ameyaw
- Plant Pathology Division, Cocoa Research Institute of Ghana, New Tafo, Akim, Ghana
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22
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Pfister S, Rabl J, Wiegand T, Mattei S, Malär AA, Lecoq L, Seitz S, Bartenschlager R, Böckmann A, Nassal M, Boehringer D, Meier BH. Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style. Nat Commun 2023; 14:1574. [PMID: 36949039 PMCID: PMC10033635 DOI: 10.1038/s41467-023-37068-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Accepted: 03/02/2023] [Indexed: 03/24/2023] Open
Abstract
The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.
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Affiliation(s)
- Sara Pfister
- Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland
| | - Julius Rabl
- Cryo-EM Knowledge hub, ETH Zurich, 8093, Zurich, Switzerland
| | - Thomas Wiegand
- Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland
- Max-Planck-Institute for Chemical Energy Conversion, Stiftstr. 34-36, 45470, Mülheim an der Ruhr, Germany
- Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, 52074, Aachen, Germany
| | - Simone Mattei
- EMBL Imaging Centre, European Molecular Biology Laboratory, EMBL Heidelberg, 69117, Heidelberg, Germany
| | | | - Lauriane Lecoq
- Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon, 69367, Lyon, France
| | - Stefan Seitz
- Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120, Heidelberg, Germany
| | - Ralf Bartenschlager
- Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120, Heidelberg, Germany
| | - Anja Böckmann
- Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon, 69367, Lyon, France.
| | - Michael Nassal
- Department of Medicine II / Molecular Biology, University of Freiburg, Freiburg im Breisgau, Germany.
| | | | - Beat H Meier
- Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
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23
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Petrovic S, Mobbs GW, Bley CJ, Nie S, Patke A, Hoelz A. Structure and Function of the Nuclear Pore Complex. Cold Spring Harb Perspect Biol 2022; 14:a041264. [PMID: 36096637 PMCID: PMC9732903 DOI: 10.1101/cshperspect.a041264] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The nucleus, a genome-containing organelle eponymous of eukaryotes, is enclosed by a double membrane continuous with the endoplasmic reticulum. The nuclear pore complex (NPC) is an ∼110-MDa, ∼1000-protein channel that selectively transports macromolecules across the nuclear envelope and thus plays a central role in the regulated flow of genetic information from transcription to translation. Its size, complexity, and flexibility have hindered determination of atomistic structures of intact NPCs. Recent studies have overcome these hurdles by combining biochemical reconstitution and docking of high-resolution structures of NPC subcomplexes into cryo-electron tomographic reconstructions with biochemical and physiological validation. Here, we provide an overview of the near-atomic composite structure of the human NPC, a milestone toward unlocking a molecular understanding of mRNA export, NPC-associated diseases, and viral host-pathogen interactions, serving as a paradigm for studying similarly large complexes.
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Affiliation(s)
- Stefan Petrovic
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - George W Mobbs
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Christopher J Bley
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Si Nie
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - Alina Patke
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA
| | - André Hoelz
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
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24
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Callon M, Malär AA, Lecoq L, Dujardin M, Fogeron M, Wang S, Schledorn M, Bauer T, Nassal M, Böckmann A, Meier BH. Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis B Virus Capsid C-Terminal Domain. Angew Chem Int Ed Engl 2022; 61:e202201083. [PMID: 35653505 PMCID: PMC9400876 DOI: 10.1002/anie.202201083] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Indexed: 11/08/2022]
Abstract
Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.
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Affiliation(s)
| | | | - Lauriane Lecoq
- Molecular Microbiology and Structural Biochemistry (MMSB) UMR 5086 CNRS/Université de Lyon, Labex Ecofect7 passage du Vercors69367LyonFrance
| | - Marie Dujardin
- Molecular Microbiology and Structural Biochemistry (MMSB) UMR 5086 CNRS/Université de Lyon, Labex Ecofect7 passage du Vercors69367LyonFrance
| | - Marie‐Laure Fogeron
- Molecular Microbiology and Structural Biochemistry (MMSB) UMR 5086 CNRS/Université de Lyon, Labex Ecofect7 passage du Vercors69367LyonFrance
| | - Shishan Wang
- Molecular Microbiology and Structural Biochemistry (MMSB) UMR 5086 CNRS/Université de Lyon, Labex Ecofect7 passage du Vercors69367LyonFrance
| | | | - Thomas Bauer
- Physical Chemistry, ETH Zürich8093ZürichSwitzerland
| | - Michael Nassal
- University Hospital FreiburgDept. of Medicine II/Molecular BiologyMedical CenterUniversity of FreiburgFreiburgGermany
| | - Anja Böckmann
- Molecular Microbiology and Structural Biochemistry (MMSB) UMR 5086 CNRS/Université de Lyon, Labex Ecofect7 passage du Vercors69367LyonFrance
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25
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Callon M, Malär AA, Lecoq L, Dujardin M, Fogeron ML, Wang S, Schledorn M, Bauer T, Nassal M, Böckmann A, Meier BH. Fast Magic‐Angle‐Spinning NMR Reveals the Evasive Hepatitis B Virus Capsid C‐Terminal Domain. Angew Chem Int Ed Engl 2022. [DOI: 10.1002/ange.202201083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Morgane Callon
- ETH Zurich D-CHAB: Eidgenossische Technische Hochschule Zurich Departement Chemie und Angewandte Biowissenschaften D-CHAB SWITZERLAND
| | - Alexander A. Malär
- ETH Zurich D-CHAB: Eidgenossische Technische Hochschule Zurich Departement Chemie und Angewandte Biowissenschaften D-CHAB SWITZERLAND
| | | | | | | | | | - Maarten Schledorn
- ETH Zurich D-CHAB: Eidgenossische Technische Hochschule Zurich Departement Chemie und Angewandte Biowissenschaften D-CHAB SWITZERLAND
| | - Thomas Bauer
- ETH Zurich D-CHAB: Eidgenossische Technische Hochschule Zurich Departement Chemie und Angewandte Biowissenschaften D-CHAB SWITZERLAND
| | - Michael Nassal
- University of Freiburg Hospital: Universitatsklinikum Freiburg Molecular Biology GERMANY
| | | | - Beat H Meier
- ETH Zurich D-CHAB: Eidgenossische Technische Hochschule Zurich Departement Chemie und Angewandte Biowissenschaften Department of Physical Chemistry Wolfgang-Pauli-Strasse 10 8093 Zürich SWITZERLAND
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26
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Harati Taji Z, Bielytskyi P, Shein M, Sani MA, Seitz S, Schütz AK. Transient RNA Interactions Leave a Covalent Imprint on a Viral Capsid Protein. J Am Chem Soc 2022; 144:8536-8550. [PMID: 35512333 PMCID: PMC9121876 DOI: 10.1021/jacs.1c12439] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The hepatitis B virus (HBV) is the leading cause of persistent liver infections. Its DNA-based genome is synthesized through reverse transcription of an RNA template inside the assembled capsid shell. In addition to the structured assembly domain, the capsid protein harbors a C-terminal extension that mediates both the enclosure of RNA during capsid assembly and the nuclear entry of the capsid during infection. The arginine-rich motifs within this extension, though common to many viruses, have largely escaped atomic-scale investigation. Here, we leverage solution and solid-state nuclear magnetic resonance spectroscopy at ambient and cryogenic temperatures, under dynamic nuclear polarization signal enhancement, to investigate the organization of the genome within the capsid. Transient interactions with phosphate groups of the RNA backbone confine the arginine-rich motifs to the interior capsid space. While no secondary structure is induced in the C-terminal extension, interactions with RNA counteract the formation of a disulfide bond, which covalently tethers this peptide arm onto the inner capsid surface. Electrostatic and covalent contributions thus compete in the spatial regulation of capsid architecture. This disulfide switch represents a coupling mechanism between the structured assembly domain of the capsid and the enclosed nucleic acids. In particular, it enables the redox-dependent regulation of the exposure of the C-terminal extension on the capsid surface, which is required for nuclear uptake of the capsid. Phylogenetic analysis of capsid proteins from hepadnaviruses points toward a function of this switch in the persistence of HBV infections.
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Affiliation(s)
- Zahra Harati Taji
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Garching 85748, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany
| | - Pavlo Bielytskyi
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Garching 85748, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany
| | - Mikhail Shein
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Garching 85748, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany
| | - Marc-Antoine Sani
- School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, Victoria 3010, Australia
| | - Stefan Seitz
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg 69120, Germany.,Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg 69120, Germany
| | - Anne K Schütz
- Bavarian NMR Center, Department of Chemistry, Technical University of Munich, Garching 85748, Germany.,Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany
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27
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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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28
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Abstract
Hepatitis B virus (HBV) is a hepatotropic virus and an important human pathogen. There are an estimated 296 million people in the world that are chronically infected by this virus, and many of them will develop severe liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBV is a small DNA virus that replicates via the reverse transcription pathway. In this review, we summarize the molecular pathways that govern the replication of HBV and its interactions with host cells. We also discuss viral and non-viral factors that are associated with HBV-induced carcinogenesis and pathogenesis, as well as the role of host immune responses in HBV persistence and liver pathogenesis.
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Affiliation(s)
- Yu-Chen Chuang
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA
| | - Kuen-Nan Tsai
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA
| | - Jing-Hsiung James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA
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29
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Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt HBV Capsids. J Virol 2021; 96:e0139521. [PMID: 34705562 DOI: 10.1128/jvi.01395-21] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Impβ) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.
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30
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Van Damme E, Vanhove J, Severyn B, Verschueren L, Pauwels F. The Hepatitis B Virus Interactome: A Comprehensive Overview. Front Microbiol 2021; 12:724877. [PMID: 34603251 PMCID: PMC8482013 DOI: 10.3389/fmicb.2021.724877] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Accepted: 08/17/2021] [Indexed: 12/19/2022] Open
Abstract
Despite the availability of a prophylactic vaccine, chronic hepatitis B (CHB) caused by the hepatitis B virus (HBV) is a major health problem affecting an estimated 292 million people globally. Current therapeutic goals are to achieve functional cure characterized by HBsAg seroclearance and the absence of HBV-DNA after treatment cessation. However, at present, functional cure is thought to be complicated due to the presence of covalently closed circular DNA (cccDNA) and integrated HBV-DNA. Even if the episomal cccDNA is silenced or eliminated, it remains unclear how important the high level of HBsAg that is expressed from integrated HBV DNA is for the pathology. To identify therapies that could bring about high rates of functional cure, in-depth knowledge of the virus' biology is imperative to pinpoint mechanisms for novel therapeutic targets. The viral proteins and the episomal cccDNA are considered integral for the control and maintenance of the HBV life cycle and through direct interaction with the host proteome they help create the most optimal environment for the virus whilst avoiding immune detection. New HBV-host protein interactions are continuously being identified. Unfortunately, a compendium of the most recent information is lacking and an interactome is unavailable. This article provides a comprehensive review of the virus-host relationship from viral entry to release, as well as an interactome of cccDNA, HBc, and HBx.
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Affiliation(s)
- Ellen Van Damme
- Janssen Research & Development, Janssen Pharmaceutical Companies, Beerse, Belgium
| | - Jolien Vanhove
- Janssen Research & Development, Janssen Pharmaceutical Companies, Beerse, Belgium.,Early Discovery Biology, Charles River Laboratories, Beerse, Belgium
| | - Bryan Severyn
- Janssen Research & Development, Janssen Pharmaceutical Companies, Springhouse, PA, United States
| | - Lore Verschueren
- Janssen Research & Development, Janssen Pharmaceutical Companies, Beerse, Belgium
| | - Frederik Pauwels
- Janssen Research & Development, Janssen Pharmaceutical Companies, Beerse, Belgium
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31
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Wei L, Ploss A. Mechanism of Hepatitis B Virus cccDNA Formation. Viruses 2021; 13:v13081463. [PMID: 34452329 PMCID: PMC8402782 DOI: 10.3390/v13081463] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 07/14/2021] [Accepted: 07/21/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.
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32
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Shen Q, Wang YE, Palazzo AF. Crosstalk between nucleocytoplasmic trafficking and the innate immune response to viral infection. J Biol Chem 2021; 297:100856. [PMID: 34097873 PMCID: PMC8254040 DOI: 10.1016/j.jbc.2021.100856] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2021] [Revised: 05/24/2021] [Accepted: 06/03/2021] [Indexed: 12/16/2022] Open
Abstract
The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.
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Affiliation(s)
- Qingtang Shen
- School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
| | - Yifan E Wang
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
| | - Alexander F Palazzo
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
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Blanco-Rodriguez G, Di Nunzio F. The Viral Capsid: A Master Key to Access the Host Nucleus. Viruses 2021; 13:v13061178. [PMID: 34203080 PMCID: PMC8234750 DOI: 10.3390/v13061178] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2021] [Revised: 06/02/2021] [Accepted: 06/16/2021] [Indexed: 12/15/2022] Open
Abstract
Viruses are pathogens that have evolved to hijack the cellular machinery to replicate themselves and spread to new cells. During the course of evolution, viruses developed different strategies to overcome the cellular defenses and create new progeny. Among them, some RNA and many DNA viruses require access to the nucleus to replicate their genome. In non-dividing cells, viruses can only access the nucleus through the nuclear pore complex (NPC). Therefore, viruses have developed strategies to usurp the nuclear transport machinery and gain access to the nucleus. The majority of these viruses use the capsid to manipulate the nuclear import machinery. However, the particular tactics employed by each virus to reach the host chromatin compartment are very different. Nevertheless, they all require some degree of capsid remodeling. Recent notions on the interplay between the viral capsid and cellular factors shine new light on the quest for the nuclear entry step and for the fate of these viruses. In this review, we describe the main components and function of nuclear transport machinery. Next, we discuss selected examples of RNA and DNA viruses (HBV, HSV, adenovirus, and HIV) that remodel their capsid as part of their strategies to access the nucleus and to replicate.
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Affiliation(s)
- Guillermo Blanco-Rodriguez
- Advanced Molecular Virology and Retroviral Dynamics Group, Department of Virology, Pasteur Institute, 75015 Paris, France;
- Immunity and Cancer Department, Curie Institute, PSL Research University, INSERM U932, 75005 Paris, France
| | - Francesca Di Nunzio
- Advanced Molecular Virology and Retroviral Dynamics Group, Department of Virology, Pasteur Institute, 75015 Paris, France;
- Correspondence:
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34
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Sajidah ES, Lim K, Wong RW. How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System. Cells 2021; 10:1424. [PMID: 34200500 PMCID: PMC8230057 DOI: 10.3390/cells10061424] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2021] [Revised: 05/31/2021] [Accepted: 06/03/2021] [Indexed: 12/14/2022] Open
Abstract
The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.
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Affiliation(s)
- Elma Sakinatus Sajidah
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
| | - Keesiang Lim
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
| | - Richard W. Wong
- Division of Nano Life Science in the Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
- WPI-Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan
- Cell-Bionomics Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
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35
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Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation. Viruses 2021; 13:v13050757. [PMID: 33925977 PMCID: PMC8145197 DOI: 10.3390/v13050757] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) remains a major public health concern, with more than 250 million chronically infected people who are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma. Although antiviral treatments efficiently control virus replication and improve liver function, they cannot cure HBV infection. Viral persistence is due to the maintenance of the viral circular episomal DNA, called covalently closed circular DNA (cccDNA), in the nuclei of infected cells. cccDNA not only resists antiviral therapies, but also escapes innate antiviral surveillance. This viral DNA intermediate plays a central role in HBV replication, as cccDNA is the template for the transcription of all viral RNAs, including pregenomic RNA (pgRNA), which in turn feeds the formation of cccDNA through a step of reverse transcription. The establishment and/or expression of cccDNA is thus a prime target for the eradication of HBV. In this review, we provide an update on the current knowledge on the initial steps of HBV infection, from the nuclear import of the nucleocapsid to the formation of the cccDNA.
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36
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Paci G, Caria J, Lemke EA. Cargo transport through the nuclear pore complex at a glance. J Cell Sci 2021; 134:237315. [PMID: 33495357 DOI: 10.1242/jcs.247874] [Citation(s) in RCA: 53] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Bidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a 'cargo perspective'. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties.
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Affiliation(s)
- Giulia Paci
- Biocentre, Johannes Gutenberg-University Mainz, Hans-Dieter-Hüsch-Weg 15, 555128 Mainz, Germany.,Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany.,European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
| | - Joana Caria
- Biocentre, Johannes Gutenberg-University Mainz, Hans-Dieter-Hüsch-Weg 15, 555128 Mainz, Germany.,Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany.,European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
| | - Edward A Lemke
- Biocentre, Johannes Gutenberg-University Mainz, Hans-Dieter-Hüsch-Weg 15, 555128 Mainz, Germany .,Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany.,European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
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37
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Xi J, Luckenbaugh L, Hu J. Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication. PLoS Pathog 2021; 17:e1009230. [PMID: 33493210 PMCID: PMC7861550 DOI: 10.1371/journal.ppat.1009230] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2020] [Revised: 02/04/2021] [Accepted: 12/08/2020] [Indexed: 12/18/2022] Open
Abstract
Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication. Hepatitis B virus (HBV) causes acute and chronic viral hepatitis, liver fibrosis, cirrhosis and cancer. The dynamic phosphorylation and dephosphorylation of the viral capsid protein (HBc), which are controlled by host cell protein kinases and phosphatases, play a critical role in regulating multiple stages of HBV replication. While a number of cellular kinases have been identified that mediate HBc phosphorylation, there is little information on cellular phosphatases that mediate its dephosphorylation. Herein we have identified a consensus binding motif in HBc for one of the major cellular phosphatases, the protein phosphatase 2A (PP2A). Genetic analysis of this motif revealed that it played multiple roles in regulating CTD phosphorylation state, as well as viral RNA packaging, reverse transcription, virion secretion, and formation of the nuclear HBV episome responsible for viral persistence. Furthermore, pharmacological inhibition of PP2A decreased HBc dephosphorylation and increased the nuclear episome, further supporting a role of PP2A in HBc dephosphorylation and HBV persistence. These results thus suggest that HBc recruits PP2A, among other host factors, to regulate HBc phosphorylation and dephosphorylation dynamics and HBV replication and persistence.
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Affiliation(s)
- Ji Xi
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Laura Luckenbaugh
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- * E-mail:
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38
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Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy. Viruses 2021; 13:v13020167. [PMID: 33499411 PMCID: PMC7911914 DOI: 10.3390/v13020167] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Revised: 01/21/2021] [Accepted: 01/21/2021] [Indexed: 12/15/2022] Open
Abstract
Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.
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39
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Molenberghs F, Bogers JJ, De Vos WH. Confined no more: Viral mechanisms of nuclear entry and egress. Int J Biochem Cell Biol 2020; 129:105875. [PMID: 33157236 DOI: 10.1016/j.biocel.2020.105875] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 10/27/2020] [Accepted: 10/29/2020] [Indexed: 12/14/2022]
Abstract
Viruses are obligatory intracellular parasites. For their efficient replication, many require access to the nuclear interior. Yet, only few viral particles are small enough to passively diffuse through the nuclear pore complexes, calling for alternative strategies to bypass the nuclear envelope barrier. Some viruses will await mitotic nuclear envelope breakdown to gain access, whereas others will exploit more active means, for instance by hijacking nuclear pore transport or by directly targeting constituents of the nuclear envelope so as to remodel and temporarily perturb its integrity. After replication, newly produced viral DNA complexes need to cross the same barrier to exit the nucleus and enter the cytoplasm, where the final stages of virion maturation take place. There are also different flavours to the feat of nuclear egress that vary in delicacy and intensity. In this review, we define the major entry and egress strategies that are exploited by different viruses and describe the molecular details thereof. Ultimately, a deeper understanding of these pathways may help identifying molecular targets for blocking viral reproduction or spreading.
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Affiliation(s)
- Freya Molenberghs
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences/Medicine and Health Sciences, University of Antwerp, Belgium
| | - Johannes J Bogers
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences/Medicine and Health Sciences, University of Antwerp, Belgium
| | - Winnok H De Vos
- Laboratory of Cell Biology and Histology, Department of Veterinary Sciences/Medicine and Health Sciences, University of Antwerp, Belgium.
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40
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Chakraborty A, Ko C, Henning C, Lucko A, Harris JM, Chen F, Zhuang X, Wettengel JM, Roessler S, Protzer U, McKeating JA. Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle. Cell Microbiol 2020; 22:e13250. [PMID: 32799415 PMCID: PMC7611726 DOI: 10.1111/cmi.13250] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Revised: 07/15/2020] [Accepted: 07/27/2020] [Indexed: 12/11/2022]
Abstract
Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.
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Affiliation(s)
- Anindita Chakraborty
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany.,Technical University of Munich, Institute for Advanced Study, Munich, Germany
| | - Chunkyu Ko
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany
| | - Christin Henning
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany
| | - Aaron Lucko
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany
| | - James M Harris
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Fuwang Chen
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany
| | - Xiaodong Zhuang
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Jochen M Wettengel
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany
| | - Stephanie Roessler
- Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany
| | - Ulrike Protzer
- Institute of Virology, Technical University of Munich, School of Medicine/Helmholtz Zentrum München, Munich, Germany.,Technical University of Munich, Institute for Advanced Study, Munich, Germany.,German Center for Infection Research (DZIF), Munich, Germany
| | - Jane A McKeating
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
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41
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Intracellular Trafficking of HBV Particles. Cells 2020; 9:cells9092023. [PMID: 32887393 PMCID: PMC7563130 DOI: 10.3390/cells9092023] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 08/31/2020] [Accepted: 09/02/2020] [Indexed: 12/15/2022] Open
Abstract
The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.
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42
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Tsukuda S, Watashi K. Hepatitis B virus biology and life cycle. Antiviral Res 2020; 182:104925. [PMID: 32866519 DOI: 10.1016/j.antiviral.2020.104925] [Citation(s) in RCA: 206] [Impact Index Per Article: 41.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Revised: 07/24/2020] [Accepted: 08/26/2020] [Indexed: 12/12/2022]
Abstract
Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. The HBV life cycle is unique in that the genomic DNA (relaxed-circular partially double-stranded DNA: rcDNA) is converted to a molecular template DNA (covalently closed circular DNA: cccDNA) to amplify a viral RNA intermediate, which is then reverse-transcribed back to viral DNA. The highly stable characteristics of cccDNA result in chronic infection and a poor rate of cure. This complex life cycle of HBV offers a variety of targets to develop antiviral agents. We provide here an update on the current knowledge of HBV biology and its life cycle, which may help to identify new antiviral targets.
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Affiliation(s)
- Senko Tsukuda
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Department of Applied Biological Science, Tokyo University of Science, Noda, Japan; Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; MIRAI, JST, Saitama, Japan.
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43
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Patterson A, Zhao Z, Waymire E, Zlotnick A, Bothner B. Dynamics of Hepatitis B Virus Capsid Protein Dimer Regulate Assembly through an Allosteric Network. ACS Chem Biol 2020; 15:2273-2280. [PMID: 32662972 DOI: 10.1021/acschembio.0c00481] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
While there is an effective vaccine for Human Hepatitis B Virus (HBV), 257 million people have chronic infections for which there is no cure. The assembly process for the viral capsid is a potential therapeutic target. In order to understand the capsid assembly process, we investigated the dimeric building blocks of the capsid. To understand what blocks assembly, we took advantage of an assembly incompetent mutant dimer, Cp149-Y132A, located in the interdimer interface. This mutation leads to changes in protein dynamics throughout the structure of the dimer as measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). To further understand how the HBV capsid assembles, the homologue woodchuck HBV (WHV) capsid protein dimer (Cp) was used. WHV is more stable than HBV in HDX-MS and native mass spectrometry experiments. Because the WHV Cp assembles more rapidly into viral capsids than HBV, it was suspected that an increase in stability of the intradimer interface and/or in the contact region leads to increased assembly rates. The differences in dynamics when comparing HBV and human Cp149-Y132A as well as the differences in dynamics when comparing the HBV and WHV Cps allowed us to map an allosteric network within the HBV dimer. Through a careful comparison of structure, stability, and dynamics using four different capsid protein dimers, we conclude that protein subunit dynamics regulate HBV capsid assembly.
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Affiliation(s)
- Angela Patterson
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States
| | - Zhongchao Zhao
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Elizabeth Waymire
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States
| | - Adam Zlotnick
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Brian Bothner
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States
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44
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Paci G, Zheng T, Caria J, Zilman A, Lemke EA. Molecular determinants of large cargo transport into the nucleus. eLife 2020; 9:e55963. [PMID: 32692309 PMCID: PMC7375812 DOI: 10.7554/elife.55963] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Accepted: 06/18/2020] [Indexed: 01/03/2023] Open
Abstract
Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex (NPC). Among the thousands of molecules that cross the NPC, even very large (>15 nm) cargoes such as pathogens, mRNAs and pre-ribosomes can pass the NPC intact. For these cargoes, there is little quantitative understanding of the requirements for their nuclear import, especially the role of multivalent binding to transport receptors via nuclear localisation sequences (NLSs) and the effect of size on import efficiency. Here, we assayed nuclear import kinetics of 30 large cargo models based on four capsid-like particles in the size range of 17-36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for nuclear transport can be recapitulated by a simple two-parameter biophysical model that correlates the import flux with the energetics of large cargo transport through the NPC. Together, our results reveal key molecular determinants of large cargo import in cells.
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Affiliation(s)
- Giulia Paci
- Biocentre, Johannes Gutenberg-University MainzMainzGermany
- Institute of Molecular BiologyMainzGermany
- European Molecular Biology LaboratoryHeidelbergGermany
| | - Tiantian Zheng
- Department of Physics, University of TorontoTorontoCanada
| | - Joana Caria
- Biocentre, Johannes Gutenberg-University MainzMainzGermany
- Institute of Molecular BiologyMainzGermany
- European Molecular Biology LaboratoryHeidelbergGermany
| | - Anton Zilman
- Department of Physics, University of TorontoTorontoCanada
- Institute for Biomaterials and Biomedical Engineering (IBBME), University of TorontoTorontoCanada
| | - Edward A Lemke
- Biocentre, Johannes Gutenberg-University MainzMainzGermany
- Institute of Molecular BiologyMainzGermany
- European Molecular Biology LaboratoryHeidelbergGermany
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45
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Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid. Viruses 2020; 12:v12070738. [PMID: 32650547 PMCID: PMC7412469 DOI: 10.3390/v12070738] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 07/02/2020] [Accepted: 07/03/2020] [Indexed: 02/08/2023] Open
Abstract
The morphogenesis of Hepatitis B Virus (HBV) viral particles is nucleated by the oligomerization of HBc protein molecules, resulting in the formation of an icosahedral capsid shell containing the replication-competent nucleoprotein complex made of the viral polymerase and the pre-genomic RNA (pgRNA). HBc is a phospho-protein containing two distinct domains acting together throughout the viral replication cycle. The N-terminal domain, (residues 1–140), shown to self-assemble, is linked by a short flexible domain to the basic C-terminal domain (residues 150–183) that interacts with nucleic acids (NAs). In addition, the C-terminal domain contains a series of phospho-acceptor residues that undergo partial phosphorylation and de-phosphorylation during virus replication. This highly dynamic process governs the homeostatic charge that is essential for capsid stability, pgRNA packaging and to expose the C-terminal domain at the surface of the particles for cell trafficking. In this review, we discuss the roles of the N-terminal and C-terminal domains of HBc protein during HBV morphogenesis, focusing on how the C-terminal domain phosphorylation dynamics regulate its interaction with nucleic acids throughout the assembly and maturation of HBV particles.
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Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context. J Mol Biol 2020; 432:3802-3819. [PMID: 32371046 DOI: 10.1016/j.jmb.2020.04.026] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 04/25/2020] [Accepted: 04/28/2020] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.
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Yuan X, Qin B, Yin H, Shi Y, Jiang M, Luo L, Luo Z, Zhang J, Li X, Zhu C, Du Y, You J. Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway. ACS CENTRAL SCIENCE 2020; 6:174-188. [PMID: 32123735 PMCID: PMC7047280 DOI: 10.1021/acscentsci.9b01052] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Indexed: 05/11/2023]
Abstract
Gene vectors play a critical role in gene therapy. To achieve efficient transfection, we developed a novel nonvirus cationic liposome (Lipo-Par), which was bound covalently with the cationic polypeptide pardaxin (Par). Interestingly, the Lipo-Pars exhibited highly enhanced gene transfection efficiency in various cell lines compared to that of the non-Par-bonded liposomes (Lipo-Nons). As a result, the internalization and intracellular transport mechanisms of the Lipo-Pars were investigated, and the findings indicated their ability to actively target the endoplasmic reticulum (ER) by moving along the cell cytoskeleton after undergoing caveolin-mediated endocytosis. This intracellular transport process is similar to that of some viruses. It was also found that ER stress and calcium level disturbances can affect the Lipo-Par-mediated expression of certain exogenous genes. A possible, yet non-negligible explanation for the high transfection efficiency of the Lipo-Par is its virus-like intracellular behavior and the intimate relationship between the ER membrane and the nuclear envelope.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | - Jian You
- . Phone: 086-571-88208443. Fax: 086-571-88208439
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Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery. J Virol 2020; 94:JVI.01532-19. [PMID: 31748386 DOI: 10.1128/jvi.01532-19] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Accepted: 11/16/2019] [Indexed: 02/06/2023] Open
Abstract
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin β-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin β-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin β-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.
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EGFR-c-Src-Mediated HDAC3 Phosphorylation Exacerbates Invasion of Breast Cancer Cells. Cells 2019; 8:cells8080930. [PMID: 31430896 PMCID: PMC6721651 DOI: 10.3390/cells8080930] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 08/08/2019] [Accepted: 08/14/2019] [Indexed: 01/09/2023] Open
Abstract
Breast cancer is one of the leading causes of morbidity and mortality among women. Epidermal growth factor receptor (EGFR) and proto-oncogene tyrosine-protein kinase Src (c-Src) are critical components of the signaling pathways that are associated with breast cancer. However, the regulatory mechanism of histone deacetylase 3 (HDAC3) in these pathways remains unclear. Using the Net Phos 3.1 program for the analysis of kinase consensus motifs, we found two c-Src-mediated putative phosphorylation sites, tyrosine (Tyr, Y)-328 and Y331 on HDAC3, and generated a phospho-specific HDAC3 antibody against these sites. c-Src-mediated phosphorylation was observed in the cells expressing wild-type HDAC3 (HDAC3WT), but not in cells overexpressing phosphorylation-defective HDAC3 (HDAC3Y328/331A). Phosphorylated HDAC3 showed relatively higher deacetylase activity, and PP2, which is a c-Src inhibitor, blocked HDAC3 phosphorylation and reduced its enzymatic activity. EGF treatment resulted in HDAC3 phosphorylation in both MDA-MB-231 and EGFR-overexpressing MCF7 (MCF7-EGFR) cells, but not in MCF7 cells. Total internal reflection fluorescence analysis showed that HDAC3 was recruited to the plasma membrane following EGF stimulation. HDAC3 inhibition with either c-Src knockdown or PP2 treatment significantly ameliorated the invasiveness of breast cancer cells. Altogether, our findings reveal an EGF signaling cascade involving EGFR, c-Src, and HDAC3 in breast cancer cells.
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Mitra B, Wang J, Kim ES, Mao R, Dong M, Liu Y, Zhang J, Guo H. Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation. J Virol 2019; 93:e00196-19. [PMID: 31019054 PMCID: PMC6580977 DOI: 10.1128/jvi.00196-19] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Accepted: 04/18/2019] [Indexed: 02/07/2023] Open
Abstract
Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg-) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg- patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy.IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.
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Affiliation(s)
- Bidisha Mitra
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Jinyu Wang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Elena S Kim
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Richeng Mao
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Medical Molecular Virology of the Ministry of Health and Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Minhui Dong
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
| | - Yuanjie Liu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
| | - Jiming Zhang
- Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Medical Molecular Virology of the Ministry of Health and Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA
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