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Diesterbeck US, Muslinkina LA, Gittis AG, Singh K, Moss B, Garboczi DN. The 2.3 Å Structure of A21, a Protein Component of the Conserved Poxvirus Entry-Fusion Complex. J Mol Biol 2025; 437:169097. [PMID: 40118206 PMCID: PMC12040579 DOI: 10.1016/j.jmb.2025.169097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2025] [Revised: 03/13/2025] [Accepted: 03/15/2025] [Indexed: 03/23/2025]
Abstract
Poxviruses are exceptional in having an entry-fusion complex (EFC) consisting of eleven conserved proteins embedded in the membrane of mature virions. With the goal of understanding the function of the EFC, extensive efforts have been made to determine the structures and roles of its components, and to date, structures have been determined for nine of the eleven proteins. Here, we report the crystal structure of A21, the 10th EFC protein, comprising two α-helices clasping a twisted antiparallel β-sheet stabilized by two conserved disulfide bonds. The stability of each of the three A21 loops is provided by hydrogen bonds between main-chain atoms and several highly conserved residues, making the overall fold of A21 and its orthologs resilient to evolutionary change. Based on AlphaFold modeling and phylogenetic analysis of A21, we suggest that its highly conserved N-terminal transmembrane domain and C-terminal α-helix enable A21 integration into EFC, where it primarily interacts with the G3/L5 subcomplex and the smallest of EFC components, the O3 protein.
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Affiliation(s)
- Ulrike S Diesterbeck
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; Ceva Innovation Center GmbH, Am Pharmapark, 06861 Dessau-Rosslau, Germany
| | - Liya A Muslinkina
- Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Apostolos G Gittis
- Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Kavita Singh
- Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20903, USA
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
| | - David N Garboczi
- Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
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2
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Yu H, Resch W, Moss B. Poxvirus structural biology for application to vaccine design. Trends Immunol 2025:S1471-4906(25)00094-8. [PMID: 40340168 DOI: 10.1016/j.it.2025.04.002] [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: 02/17/2025] [Revised: 04/06/2025] [Accepted: 04/10/2025] [Indexed: 05/10/2025]
Abstract
The upsurge of mpox (formerly known as monkeypox) in Africa and its global spread highlight the need for improved vaccines. The development of new recombinant vaccines, including mRNA and protein nanoparticles, depends on understanding the biology of poxviruses and selecting the most protective immunogens. Animal studies demonstrate that vaccines need to target the antigens of both infectious forms - the mature virion and the enveloped virion - which display surface proteins responsible for cell entry and cell-to-cell spread, respectively. Although some of these proteins have been shown to induce protective antibodies, others including most of those that are essential for membrane fusion remain to be tested. We review the structures of orthopoxvirus surface proteins as a guide to the selection of optimal antigens for recombinant vaccines.
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Affiliation(s)
- Huibin Yu
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA
| | - Wolfgang Resch
- Center for Information Technology, NIH, Bethesda, MD, USA
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA.
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3
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Deng Y, Navarro-Forero S, Yang Z. Temporal expression classes and functions of vaccinia virus and mpox (monkeypox) virus genes. mBio 2025; 16:e0380924. [PMID: 40111027 PMCID: PMC11980589 DOI: 10.1128/mbio.03809-24] [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: 03/22/2025] Open
Abstract
Poxviruses comprise pathogens that are highly pathogenic to humans and animals, causing diseases such as smallpox and mpox (formerly monkeypox). The family also contains members developed as vaccine vectors and oncolytic agents to fight other diseases. Vaccinia virus is the prototype poxvirus and the vaccine used to eradicate smallpox. Poxvirus genes follow a cascade temporal expression pattern, categorized into early, intermediate, and late stages using distinct transcription factors. This review comprehensively summarized the temporal expression classification of over 200 vaccinia virus genes. The relationships between expression classes and functions, as well as different branches of immune responses, were discussed. Based on the vaccinia virus orthologs, we classified the temporal expression classes of all the mpox virus genes, including a few that were not previously annotated with orthologs in vaccinia viruses. Additionally, we reviewed the functions of all vaccinia virus genes based on the up-to-date published papers. This review provides a readily usable resource for researchers working on poxvirus biology, medical countermeasures, and poxvirus utility development.
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Affiliation(s)
- Yining Deng
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Santiago Navarro-Forero
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Zhilong Yang
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
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Nikitin VN, Merkuleva IA, Shcherbakov DN. Monoclonal Antibodies in Light of Mpox Outbreak: Current Research, Therapeutic Targets, and Animal Models. Antibodies (Basel) 2025; 14:20. [PMID: 40136469 PMCID: PMC11939467 DOI: 10.3390/antib14010020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2024] [Revised: 02/19/2025] [Accepted: 02/21/2025] [Indexed: 03/27/2025] Open
Abstract
The rapid rise in monkeypox virus infections among humans from 2022 to 2024 has captured the attention of the global healthcare community. In light of the lack of mandatory vaccination and limited data on next-generation vaccines for monkeypox prevention, the urgent development of therapeutic agents has become a priority. One promising approach involves the use of neutralizing monoclonal antibodies. This review highlights significant advancements in the search for antibodies against human pathogenic orthopoxviruses, particularly focusing on their potential application against the monkeypox virus. We also analyze viral proteins that serve as targets for identifying therapeutic antibodies capable of neutralizing a wide range of viruses. Finally, we deemed it essential to address the challenges associated with selecting an animal model that can adequately reflect the infectious process of each orthopoxvirus species in humans.
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Affiliation(s)
| | - Iuliia A. Merkuleva
- State Research Center of Virology and Biotechnology Vector, Rospotrebnadzor, Koltsovo 630559, Russia; (V.N.N.); (D.N.S.)
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Radaelli A, Zanotto C, Brambilla C, Adami T, De Giuli Morghen C. Enhanced Expression of the L1R Gene of Vaccinia Virus by the tPA Signal Sequence Inserted in a Fowlpox-Based Recombinant Vaccine. Vaccines (Basel) 2024; 12:1115. [PMID: 39460282 PMCID: PMC11511345 DOI: 10.3390/vaccines12101115] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2024] [Revised: 09/19/2024] [Accepted: 09/23/2024] [Indexed: 10/28/2024] Open
Abstract
The use of Vaccinia virus (VACV) as a preventive vaccine against variola, the etiological agent of smallpox, led to the eradication of smallpox as a human disease. The L1 protein, a myristylated transmembrane protein present on the surface of mature virions, plays a significant role in infection and morphogenesis, is well-conserved in all orthopoxviruses, and is the target of neutralizing antibodies. DNA recombinant vaccines expressing this protein were successfully used, but they showed lower efficacy in non-human and human primates when used alone, and viral-vectored fowlpox vaccines were already proved to increase immunogenicity when used as a boost. Here, we constructed a novel fowlpox-based recombinant (FPtPA-L1R), in which the tissue plasminogen activator signal sequence was linked to the 5' end of the L1R gene to drive the L1 protein into the cellular secretion pathway. FPtPA-L1R expresses a functional heterologous protein that can be immunoprecipitated by hyperimmune rabbit serum. The protein shows cytoplasmic and membrane subcellular localizations and long-lasting expression in CEF, non-human primate Vero and human MRC-5 cells. The tissue plasminogen activator signal sequence can thus contribute significantly to the expression of the L1 protein and may enhance the immunogenicity of a putative DNA/FP prime-boost vaccine.
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Affiliation(s)
- Antonia Radaelli
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy; (A.R.); (C.B.); (T.A.)
- Department of Pharmacy, Faculty of Pharmacy, Catholic University “Our Lady of Good Counsel”, Rr. Dritan Hoxha, 123, 1001 Tirana, Albania;
| | - Carlo Zanotto
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy; (A.R.); (C.B.); (T.A.)
| | - Chiara Brambilla
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy; (A.R.); (C.B.); (T.A.)
| | - Tommaso Adami
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy; (A.R.); (C.B.); (T.A.)
| | - Carlo De Giuli Morghen
- Department of Pharmacy, Faculty of Pharmacy, Catholic University “Our Lady of Good Counsel”, Rr. Dritan Hoxha, 123, 1001 Tirana, Albania;
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Li J, Li X, Dong J, Wei J, Guo X, Wang G, Xu M, Zhao A. Enhanced Immune Responses in Mice by Combining the Mpox Virus B6R-Protein and Aluminum Hydroxide-CpG Vaccine Adjuvants. Vaccines (Basel) 2024; 12:776. [PMID: 39066415 PMCID: PMC11281346 DOI: 10.3390/vaccines12070776] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2024] [Revised: 07/12/2024] [Accepted: 07/12/2024] [Indexed: 07/28/2024] Open
Abstract
Novel adjuvants and innovative combinations of adjuvants (Adjuvant Systems) have facilitated the development of enhanced and new vaccines against re-emerging and challenging pathogenic microorganisms. Nonetheless, the efficacy of adjuvants is influenced by various factors, and the same adjuvant may generate entirely different immune responses when paired with different antigens. Herein, we combined the MPXV-B6R antigen with BC02, a novel adjuvant with proprietary technology, to assess its capability to induce both cellular and humoral immunity in mouse models. Mice received two intramuscular injections of B6R-BC02, which resulted in the production of MPXV-specific IgG, IgG1, and IgG2a antibodies. Additionally, it elicited strong MPXV-specific Th1-oriented cellular immunity and persistent effector memory B-cell responses. The advantages of BC02 were further validated, including rapid initiation of the immune response, robust recall memory, and sustained immune response induction. Although the potential of immunized mice to produce serum-neutralizing antibodies against the vaccinia virus requires further improvement, the exceptional performance of BC02 as an adjuvant for the MPXV-B6R antigen has been consistently demonstrated.
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Affiliation(s)
- Junli Li
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Xiaochi Li
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Jiaxin Dong
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Jiazheng Wei
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- College of Life Sciences and Biopharmaceuticals, Shenyang Pharmaceutical University, Shenyang 117004, China
| | - Xiaonan Guo
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Guozhi Wang
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Miao Xu
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
| | - Aihua Zhao
- Division of Tuberculosis Vaccine and Allergen Products, Institute of Biological Product Control, National Institutes for Food and Drug Control, Beijing 102629, China; (J.L.); (X.L.); (J.D.); (J.W.); (X.G.); (G.W.); (M.X.)
- Key Laboratory for Quality Research and Evaluation of Biological Products, National Medical Products Administration (NMPA), Beijing 102629, China
- Key Laboratory of Research on Quality and Standardization of Biotech Products, National Health Commission (NHC), Beijing 102629, China
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Sagdat K, Batyrkhan A, Kanayeva D. Exploring monkeypox virus proteins and rapid detection techniques. Front Cell Infect Microbiol 2024; 14:1414224. [PMID: 38863833 PMCID: PMC11165096 DOI: 10.3389/fcimb.2024.1414224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2024] [Accepted: 05/03/2024] [Indexed: 06/13/2024] Open
Abstract
Monkeypox (mpox) is an infectious disease caused by the mpox virus and can potentially lead to fatal outcomes. It resembles infections caused by viruses from other families, challenging identification. The pathogenesis, transmission, and clinical manifestations of mpox and other Orthopoxvirus species are similar due to their closely related genetic material. This review provides a comprehensive discussion of the roles of various proteins, including extracellular enveloped virus (EEV), intracellular mature virus (IMV), and profilin-like proteins of mpox. It also highlights recent diagnostic techniques based on these proteins to detect this infection rapidly.
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Affiliation(s)
| | | | - Damira Kanayeva
- Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan
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8
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Hernandez-Gonzalez M, Calcraft T, Nans A, Rosenthal PB, Way M. Palisade structure in intact vaccinia virions. mBio 2024; 15:e0313423. [PMID: 38171004 PMCID: PMC10865856 DOI: 10.1128/mbio.03134-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 11/30/2023] [Indexed: 01/05/2024] Open
Abstract
Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.
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Affiliation(s)
- Miguel Hernandez-Gonzalez
- Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Thomas Calcraft
- Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Andrea Nans
- Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom
| | - Peter B. Rosenthal
- Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Michael Way
- Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, London, United Kingdom
- Department of Infectious Disease, Imperial College, London, United Kingdom
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Aggarwal T, Kondabagil K. Assembly and Evolution of Poxviruses. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2024; 1451:35-54. [PMID: 38801570 DOI: 10.1007/978-3-031-57165-7_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).
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Affiliation(s)
- Tanvi Aggarwal
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra, 400076, India
| | - Kiran Kondabagil
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra, 400076, India.
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Mirzakhanyan Y, Jankevics A, Scheltema RA, Gershon PD. Combination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion. mBio 2023; 14:e0113523. [PMID: 37646531 PMCID: PMC10653903 DOI: 10.1128/mbio.01135-23] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Accepted: 07/11/2023] [Indexed: 09/01/2023] Open
Abstract
IMPORTANCE An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.
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Affiliation(s)
- Yeva Mirzakhanyan
- Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, California, USA
| | - Andris Jankevics
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands
| | - Richard A. Scheltema
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, the Netherlands
| | - Paul David Gershon
- Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, California, USA
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Wang Y, Yang K, Zhou H. Immunogenic proteins and potential delivery platforms for mpox virus vaccine development: A rapid review. Int J Biol Macromol 2023; 245:125515. [PMID: 37353117 PMCID: PMC10284459 DOI: 10.1016/j.ijbiomac.2023.125515] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 06/15/2023] [Accepted: 06/20/2023] [Indexed: 06/25/2023]
Abstract
Since May 2022, the mpox virus (MPXV) has spread worldwide and become a potential threat to global public health. Vaccines are important tools for preventing MPXV transmission and infection in the population. However, there are still no available potent and applicable vaccines specifically for MPXV. Herein, we highlight several potential vaccine targets for MPVX and emphasize potent immunogens, such as M1R, E8L, H3L, A29L, A35R, and B6R proteins. These proteins can be integrated into diverse vaccine platforms to elicit powerful B-cell and T-cell responses, thereby providing protective immunity against MPXV infection. Overall, research on the MPXV vaccine targets would provide valuable information for developing timely effective MPXV-specific vaccines.
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Affiliation(s)
- Yang Wang
- College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu 610000, China
| | - Kaiwen Yang
- College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu 610000, China
| | - Hao Zhou
- College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu 610000, China.
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12
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Hernandez-Gonzalez M, Calcraft T, Nans A, Rosenthal PB, Way M. A succession of two viral lattices drives vaccinia virus assembly. PLoS Biol 2023; 21:e3002005. [PMID: 36862727 PMCID: PMC10013923 DOI: 10.1371/journal.pbio.3002005] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Revised: 03/14/2023] [Accepted: 01/19/2023] [Indexed: 03/03/2023] Open
Abstract
During its cytoplasmic replication, vaccinia virus assembles non-infectious spherical immature virions (IV) coated by a viral D13 lattice. Subsequently, IV mature into infectious brick-shaped intracellular mature virions (IMV) that lack D13. Here, we performed cryo-electron tomography (cryo-ET) of frozen-hydrated vaccinia-infected cells to structurally characterise the maturation process in situ. During IMV formation, a new viral core forms inside IV with a wall consisting of trimeric pillars arranged in a new pseudohexagonal lattice. This lattice appears as a palisade in cross-section. As maturation occurs, which involves a 50% reduction in particle volume, the viral membrane becomes corrugated as it adapts to the newly formed viral core in a process that does not appear to require membrane removal. Our study suggests that the length of this core is determined by the D13 lattice and that the consecutive D13 and palisade lattices control virion shape and dimensions during vaccinia assembly and maturation.
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Affiliation(s)
- Miguel Hernandez-Gonzalez
- Cellular signalling and cytoskeletal function laboratory, The Francis Crick Institute, London, United Kingdom
| | - Thomas Calcraft
- Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Andrea Nans
- Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom
| | - Peter B Rosenthal
- Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Michael Way
- Cellular signalling and cytoskeletal function laboratory, The Francis Crick Institute, London, United Kingdom
- Department of Infectious Disease, Imperial College, London, United Kingdom
- * E-mail:
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13
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AC81 Is a Putative Disulfide Isomerase Involved in Baculoviral Disulfide Bond Formation. J Virol 2022; 96:e0116722. [PMID: 36468861 PMCID: PMC9769380 DOI: 10.1128/jvi.01167-22] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.
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14
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Park ES, Dezhbord M, Lee AR, Park BB, Kim KH. Dysregulation of Liver Regeneration by Hepatitis B Virus Infection: Impact on Development of Hepatocellular Carcinoma. Cancers (Basel) 2022; 14:cancers14153566. [PMID: 35892823 PMCID: PMC9329784 DOI: 10.3390/cancers14153566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 07/19/2022] [Accepted: 07/21/2022] [Indexed: 02/04/2023] Open
Abstract
The liver is unique in its ability to regenerate in response to damage. The complex process of liver regeneration consists of multiple interactive pathways. About 2 billion people worldwide have been infected with hepatitis B virus (HBV), and HBV causes 686,000 deaths each year due to its complications. Long-term infection with HBV, which causes chronic inflammation, leads to serious liver-related diseases, including cirrhosis and hepatocellular carcinoma. HBV infection has been reported to interfere with the critical mechanisms required for liver regeneration. In this review, the studies on liver tissue characteristics and liver regeneration mechanisms are summarized. Moreover, the inhibitory mechanisms of HBV infection in liver regeneration are investigated. Finally, the association between interrupted liver regeneration and hepatocarcinogenesis, which are both triggered by HBV infection, is outlined. Understanding the fundamental and complex liver regeneration process is expected to provide significant therapeutic advantages for HBV-associated hepatocellular carcinoma.
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Affiliation(s)
- Eun-Sook Park
- Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seoul 05029, Korea; (E.-S.P.); (B.B.P.)
| | - Mehrangiz Dezhbord
- Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon 16419, Korea; (M.D.); (A.R.L.)
| | - Ah Ram Lee
- Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon 16419, Korea; (M.D.); (A.R.L.)
| | - Bo Bae Park
- Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seoul 05029, Korea; (E.-S.P.); (B.B.P.)
| | - Kyun-Hwan Kim
- Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon 16419, Korea; (M.D.); (A.R.L.)
- Correspondence: ; Tel.: +82-31-299-6126
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15
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Bidgood SR, Samolej J, Novy K, Collopy A, Albrecht D, Krause M, Burden JJ, Wollscheid B, Mercer J. Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response. PLoS Pathog 2022; 18:e1010614. [PMID: 35834477 PMCID: PMC9282662 DOI: 10.1371/journal.ppat.1010614] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2021] [Accepted: 05/24/2022] [Indexed: 01/23/2023] Open
Abstract
All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.
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Affiliation(s)
- Susanna R. Bidgood
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
| | - Jerzy Samolej
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
| | - Karel Novy
- Swiss Federal Institute of Technology (ETH Zürich), Department of Health Sciences and Technology (D-HEST), Institute of Translational Medicine (ITM), Zürich, Switzerland
| | - Abigail Collopy
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
| | - David Albrecht
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
| | - Melanie Krause
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
| | - Jemima J. Burden
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
| | - Bernd Wollscheid
- Swiss Federal Institute of Technology (ETH Zürich), Department of Health Sciences and Technology (D-HEST), Institute of Translational Medicine (ITM), Zürich, Switzerland
- Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland
| | - Jason Mercer
- MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
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16
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Peñaflor-Téllez Y, Chávez-Munguía B, Lagunes-Guillén A, Salazar-Villatoro L, Gutiérrez-Escolano AL. The Feline Calicivirus Leader of the Capsid Protein Has the Functional Characteristics of a Viroporin. Viruses 2022; 14:v14030635. [PMID: 35337042 PMCID: PMC8955107 DOI: 10.3390/v14030635] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 02/21/2022] [Accepted: 02/22/2022] [Indexed: 12/27/2022] Open
Abstract
The leader of the capsid (LC) protein is exclusive to the Vesivirus genus, and it is needed for successful feline calicivirus (FCV) replication, as well as an efficient apoptosis induction through the mitochondrial pathway. In this work, we aimed to determine if the LC protein from the FCV is a viroporin. Although lacking in a transmembrane domain or an amphipathic helix, the LC protein from the FCV is toxic when expressed in bacteria and it oligomerizes through disulfide bonds, which are both key characteristics of viroporins. An electron microscopy analysis of LC-expressing E. coli cells suggest that the protein induces osmotic stress. Moreover, we found that the previously studied C40A LC mutant, that fails to induce apoptosis and that hinders the replication cycle, also oligomerizes but it has a reduced toxicity and fails to induce osmotic stress in bacteria. We propose that the LC protein is a viroporin that acts as a disulfide bond-dependent antimicrobial peptide, similar to the Ebola virus delta peptide.
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17
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Cuesta-Geijo MÁ, García-Dorival I, del Puerto A, Urquiza J, Galindo I, Barrado-Gil L, Lasala F, Cayuela A, Sorzano COS, Gil C, Delgado R, Alonso C. New insights into the role of endosomal proteins for African swine fever virus infection. PLoS Pathog 2022; 18:e1009784. [PMID: 35081156 PMCID: PMC8820605 DOI: 10.1371/journal.ppat.1009784] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 02/07/2022] [Accepted: 01/11/2022] [Indexed: 01/01/2023] Open
Abstract
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection. African swine fever virus (ASFV) causes a deadly disease of pigs and wild boars that was endemic in Africa but has spread in recent years to Europe, Asia and Oceania with a high socioeconomic impact. ASFV enters the cell by endocytosis and has adapted to the endosomal conditions to acquire infectivity. Fusion of the internal viral membrane with the endosomal membrane is required for the exit of viral DNA into the cytoplasm to start replication. We have found that ASF virion internal membrane proteins E248R and E199L interact with the endosomal proteins Niemann Pick C1 (NPC1) and lysosomal membrane proteins (Lamp)-1 and -2. And, appear to be required for endosomal trafficking of ASF virions endosomal traffic and exit to the cytoplasm in the cell entry process. These molecules act regulating cholesterol flux from the endosome to the endoplasmic reticulum, and appear to be important for the viral infection cycle. In silenced and knockout cells, ASFV infection was affected at early and later stages. In null cells, virion entry and progression through the endosomal pathway at entry was arrested and several viral cores were retained at late endosomes without entering the fusion phase for cytoplasmic exit. These results provide new insights into the role of endosomal proteins for ASFV infection.
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Affiliation(s)
- Miguel Ángel Cuesta-Geijo
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Isabel García-Dorival
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Ana del Puerto
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Jesús Urquiza
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Inmaculada Galindo
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Lucía Barrado-Gil
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
| | - Fátima Lasala
- Instituto de Investigación Hospital 12 de Octubre Imas12, Madrid, Spain
| | - Ana Cayuela
- Centro Nacional de Biotecnología CSIC, Madrid, Spain
| | | | - Carmen Gil
- Centro de Investigaciones Biológicas Margarita Salas CSIC, Madrid, Spain
| | - Rafael Delgado
- Instituto de Investigación Hospital 12 de Octubre Imas12, Madrid, Spain
- Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain
| | - Covadonga Alonso
- Departmento de Biotecnología, INIA-CSIC, Centro Nacional Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
- * E-mail:
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18
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Structural basis of the complete poxvirus transcription initiation process. Nat Struct Mol Biol 2021; 28:779-788. [PMID: 34556871 DOI: 10.1038/s41594-021-00655-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Accepted: 07/28/2021] [Indexed: 01/19/2023]
Abstract
Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.
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19
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Guo F, Shi Y, Yang M, Guo Y, Shen Z, Li M, Chen Y, Liang R, Yang Y, Chen H, Peng G. The structural basis of African swine fever virus core shell protein p15 binding to DNA. FASEB J 2021; 35:e21350. [PMID: 33629764 DOI: 10.1096/fj.202002145r] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Revised: 12/09/2020] [Accepted: 12/22/2020] [Indexed: 11/11/2022]
Abstract
African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV). The mortality rate of acute infection up to 100% have posed an unprecedented challenge of the swine industry. Currently no commercial antiviral drug is available for the control and treatment of ASFV. The structural resolution of ASFV virions reveals the details of ASFV morphogenesis, providing a new perspective for the research and promotion of the development of ASFV vaccines. Although the architecture of ASFV have been solved via cryo-EM, the structural details of four of the five viral layers remain unclear (except the outer capsid). In this study, we resolved the crystal structure of the ASFV core shell protein p15. The secondary structural elements of a protomer include four α-helix structures and six antiparallel β-strands. Further analysis revealed that ASFV p15 forms disulfide-linked trimers between the Cys9 from one protomer and Cys30 from other protomer. Additionally, the nucleic acid-binding property was characterized by electrophoretic mobility shift assay. Two critical amino acid Lys10 and Lys39 have been identified which is essential to the nucleic acid-binding affinity of ASFV p15. Together, these findings may provide new insight into antiviral drug development.
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Affiliation(s)
- Fenglin Guo
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Yuejun Shi
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Mengfang Yang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Yilin Guo
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Zhou Shen
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Mengxia Li
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Yixi Chen
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Rui Liang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Yilin Yang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Huanchun Chen
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
| | - Guiqing Peng
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People's Republic of China
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20
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African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration. mBio 2020; 11:mBio.00789-20. [PMID: 32788374 PMCID: PMC7439464 DOI: 10.1128/mbio.00789-20] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies. African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication.
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21
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Per Os Infectivity Factor 5 Identified as a Substrate of P33 in the Baculoviral Disulfide Bond Formation Pathway. J Virol 2020; 94:JVI.00615-20. [PMID: 32434885 DOI: 10.1128/jvi.00615-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Accepted: 05/08/2020] [Indexed: 01/19/2023] Open
Abstract
Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection.IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulfide-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.
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22
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Moss B. Investigating Viruses During the Transformation of Molecular Biology: Part II. Annu Rev Virol 2020; 7:15-36. [PMID: 32392458 DOI: 10.1146/annurev-virology-021020-100558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
My scientific career started at an extraordinary time, shortly after the discoveries of the helical structure of DNA, the central dogma of DNA to RNA to protein, and the genetic code. Part I of this series emphasizes my education and early studies highlighted by the isolation and characterization of numerous vaccinia virus enzymes, determination of the cap structure of messenger RNA, and development of poxviruses as gene expression vectors for use as recombinant vaccines. Here I describe a shift in my research focus to combine molecular biology and genetics for a comprehensive understanding of poxvirus biology. The dominant paradigm during the early years was to select a function, isolate the responsible proteins, and locate the corresponding gene, whereas later the common paradigm was to select a gene, make a mutation, and determine the altered function. Motivations, behind-the-scenes insights, importance of new technologies, and the vital roles of trainees and coworkers are emphasized.
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA;
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23
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Ceh-Pavia E, Tang X, Liu Y, Heyes DJ, Zhao B, Xiao P, Lu H. Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria. FEBS J 2019; 287:2281-2291. [PMID: 31713999 PMCID: PMC7318334 DOI: 10.1111/febs.15136] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2019] [Revised: 10/08/2019] [Accepted: 11/10/2019] [Indexed: 11/29/2022]
Abstract
The mitochondrial import and assembly (MIA) pathway plays a vitally important role in import and oxidative folding of mitochondrial proteins. Erv1, a member of the FAD-dependent Erv1/ALR disulphide bond generating enzyme family, is a key player of the MIA pathway. Although considerable progress has been made, the molecular mechanism of electron transfer within Erv1 is still not fully understood. The reduction potentials of the three redox centres were previously determined to be -320 mV for the shuttle disulphide, -150 mV for the active-site disulphide and -215 mV for FAD cofactor. However, it is unknown why FAD of Erv1 has such a low potential compared with other sulfhydryl oxidases, and why the shuttle disulphide has a potential as low as many of the stable structural disulphides of the substrates of MIA pathway. In this study, the three reduction potentials of Erv1 were reassessed using the wild-type and inactive mutants of Erv1 under anaerobic conditions. Our results show that the standard potentials for the shuttle and active-site disulphides are approximately -250 mV and -215 ~ -260 mV, respectively, and the potential for FAD cofactor is -148 mV. Our results support a model that both disulphide bonds are redox-active, and electron flow in Erv1 is thermodynamically favourable. Furthermore, the redox behaviour of Erv1 was confirmed, for the first time using Mia40, the physiological electron donor of Erv1. Together with previous studies on proteins of MIA pathway, we conclude that electron flow in the MIA pathway is a thermodynamically favourable, smoothly downhill process for all steps. DATABASE: Erv1: EC 1.8.3.2.
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Affiliation(s)
- Efrain Ceh-Pavia
- Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, UK
| | - Xiaofan Tang
- Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, UK.,School of Materials, University of Manchester, UK
| | - Yawen Liu
- State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, China
| | - Derren J Heyes
- Manchester Institute of Biotechnology, University of Manchester, UK
| | - Bing Zhao
- State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, China
| | - Ping Xiao
- School of Materials, University of Manchester, UK
| | - Hui Lu
- Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, UK
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24
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Abstract
The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. Escherichia coli played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host E. coli.
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Affiliation(s)
| | - Dana Boyd
- Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115
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Koonin EV, Yutin N. Evolution of the Large Nucleocytoplasmic DNA Viruses of Eukaryotes and Convergent Origins of Viral Gigantism. Adv Virus Res 2019; 103:167-202. [PMID: 30635076 DOI: 10.1016/bs.aivir.2018.09.002] [Citation(s) in RCA: 117] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The Nucleocytoplasmic Large DNA Viruses (NCLDV) of eukaryotes (proposed order "Megavirales") comprise an expansive group of eukaryotic viruses that consists of the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, and Mimiviridae, as well as Pandoraviruses, Molliviruses, and Faustoviruses that so far remain unaccounted by the official virus taxonomy. All these viruses have double-stranded DNA genomes that range in size from about 100 kilobases (kb) to more than 2.5 megabases. The viruses with genomes larger than 500kb are informally considered "giant," and the largest giant viruses surpass numerous bacteria and archaea in both particle and genome size. The discovery of giant viruses has been highly unexpected and has changed the perception of viral size and complexity, and even, arguably, the entire concept of a virus. Given that giant viruses encode multiple proteins that are universal among cellular life forms and are components of the translation system, the quintessential cellular molecular machinery, attempts have been made to incorporate these viruses in the evolutionary tree of cellular life. Moreover, evolutionary scenarios of the origin of giant viruses from a fourth, supposedly extinct domain of cellular life have been proposed. However, despite all the differences in the genome size and gene repertoire, the NCLDV can be confidently defined as monophyletic group, on the strength of the presence of about 40 genes that can be traced back to their last common ancestor. Using several most strongly conserved genes from this ancestral set, a well-resolved phylogenetic tree of the NCLDV was built and employed as the scaffold to reconstruct the history of gene gain and loss throughout the course of the evolution of this group of viruses. This reconstruction reveals extremely dynamic evolution that involved extensive gene gain and loss in many groups of viruses and indicates that giant viruses emerged independently in several clades of the NCLDV. Thus, these giants of the virus world evolved repeatedly from smaller and simpler viruses, rather than from a fourth domain of cellular life, and captured numerous genes, including those for translation system components, from eukaryotes, along with some bacterial genes. Even deeper evolutionary reconstructions reveal apparent links between the NCLDV and smaller viruses of eukaryotes, such as adenoviruses, and ultimately, derive all these viruses from tailless bacteriophages.
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Affiliation(s)
- Eugene V Koonin
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, United States.
| | - Natalya Yutin
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, United States
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26
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
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Novy K, Kilcher S, Omasits U, Bleck CKE, Beerli C, Vowinckel J, Martin CK, Syedbasha M, Maiolica A, White I, Mercer J, Wollscheid B. Proteotype profiling unmasks a viral signalling network essential for poxvirus assembly and transcriptional competence. Nat Microbiol 2018; 3:588-599. [DOI: 10.1038/s41564-018-0142-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 03/07/2018] [Indexed: 11/09/2022]
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Karki M, Kumar A, Venkatesan G, Arya S, Pandey AB. Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2018; 58:224-231. [PMID: 29306003 DOI: 10.1016/j.meegid.2018.01.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2017] [Revised: 12/27/2017] [Accepted: 01/01/2018] [Indexed: 10/18/2022]
Abstract
Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.
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Affiliation(s)
- Monu Karki
- Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar 263 138, Nainital, Uttarakhand, India
| | - Amit Kumar
- Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar 263 138, Nainital, Uttarakhand, India
| | - Gnanavel Venkatesan
- Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar 263 138, Nainital, Uttarakhand, India.
| | - Sargam Arya
- Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar 263 138, Nainital, Uttarakhand, India
| | - A B Pandey
- Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar 263 138, Nainital, Uttarakhand, India
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Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function. J Virol 2017; 91:JVI.01158-17. [PMID: 28904203 PMCID: PMC5686738 DOI: 10.1128/jvi.01158-17] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Accepted: 08/30/2017] [Indexed: 01/07/2023] Open
Abstract
Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.
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Combined Proteomics/Genomics Approach Reveals Proteomic Changes of Mature Virions as a Novel Poxvirus Adaptation Mechanism. Viruses 2017; 9:v9110337. [PMID: 29125539 PMCID: PMC5707544 DOI: 10.3390/v9110337] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2017] [Revised: 11/06/2017] [Accepted: 11/07/2017] [Indexed: 12/16/2022] Open
Abstract
DNA viruses, like poxviruses, possess a highly stable genome, suggesting that adaptation of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses are zoonotic poxviruses with an extraordinarily broad host range, demonstrating their adaptive potential in vivo. To elucidate adaptation mechanisms of poxviruses, we isolated cowpox virus particles from a rat and passaged them five times in a human and a rat cell line. Subsequently, we analyzed the proteome and genome of the non-passaged virions and each passage. While the overall viral genome sequence was stable during passaging, proteomics revealed multiple changes in the virion composition. Interestingly, an increased viral fitness in human cells was observed in the presence of increased immunomodulatory protein amounts. As the only minor variant with increasing frequency during passaging was located in a viral RNA polymerase subunit and, moreover, most minor variants were found in transcription-associated genes, protein amounts were presumably regulated at transcription level. This study is the first comparative proteome analysis of virus particles before and after cell culture propagation, revealing proteomic changes as a novel poxvirus adaptation mechanism.
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Bechtel TJ, Weerapana E. From structure to redox: The diverse functional roles of disulfides and implications in disease. Proteomics 2017; 17. [PMID: 28044432 DOI: 10.1002/pmic.201600391] [Citation(s) in RCA: 105] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2016] [Revised: 12/02/2016] [Accepted: 12/28/2016] [Indexed: 12/16/2022]
Abstract
This review provides a comprehensive overview of the functional roles of disulfide bonds and their relevance to human disease. The critical roles of disulfide bonds in protein structure stabilization and redox regulation of protein activity are addressed. Disulfide bonds are essential to the structural stability of many proteins within the secretory pathway and can exist as intramolecular or inter-domain disulfides. The proper formation of these bonds often relies on folding chaperones and oxidases such as members of the protein disulfide isomerase (PDI) family. Many of the PDI family members catalyze disulfide-bond formation, reduction, and isomerization through redox-active disulfides and perturbed PDI activity is characteristic of carcinomas and neurodegenerative diseases. In addition to catalytic function in oxidoreductases, redox-active disulfides are also found on a diverse array of cellular proteins and act to regulate protein activity and localization in response to oxidative changes in the local environment. These redox-active disulfides are either dynamic intramolecular protein disulfides or mixed disulfides with small-molecule thiols generating glutathionylation and cysteinylation adducts. The oxidation and reduction of redox-active disulfides are mediated by cellular reactive oxygen species and activity of reductases, such as glutaredoxin and thioredoxin. Dysregulation of cellular redox conditions and resulting changes in mixed disulfide formation are directly linked to diseases such as cardiovascular disease and Parkinson's disease.
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Affiliation(s)
- Tyler J Bechtel
- Department of Chemistry, Boston College, Chestnut Hill, MA, USA
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Segovia D, Haouz A, Porley D, Olivero N, Martínez M, Mariadassou M, Berois M, André-Leroux G, Villarino A. OH1 from Orf Virus: A New Tyrosine Phosphatase that Displays Distinct Structural Features and Triple Substrate Specificity. J Mol Biol 2017; 429:2816-2824. [PMID: 28754374 DOI: 10.1016/j.jmb.2017.07.017] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2017] [Revised: 07/21/2017] [Accepted: 07/21/2017] [Indexed: 10/19/2022]
Abstract
Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.
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Affiliation(s)
- Danilo Segovia
- Sección Bioquímica y Biología Molecular, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay
| | - Ahmed Haouz
- Institut Pasteur, Plate-forme de Cristallographie, CNRS-UMR 3528, 75724 Paris, France
| | - Darío Porley
- Sección Bioquímica y Biología Molecular, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay; Sección Virología, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay
| | - Natalia Olivero
- Sección Virología, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay
| | - Mariano Martínez
- Institut Pasteur, UMS, CNRS-UMR 3528 and Université Paris Diderot, 75724 Paris, France
| | | | - Mabel Berois
- Sección Virología, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay.
| | | | - Andrea Villarino
- Sección Bioquímica y Biología Molecular, Facultad de Ciencias, UdelaR, 11400 Montevideo, Uruguay.
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Abstract
Cysteine thiols are among the most reactive functional groups in proteins, and their pairing in disulfide linkages is a common post-translational modification in proteins entering the secretory pathway. This modest amino acid alteration, the mere removal of a pair of hydrogen atoms from juxtaposed cysteine residues, contrasts with the substantial changes that characterize most other post-translational reactions. However, the wide variety of proteins that contain disulfides, the profound impact of cross-linking on the behavior of the protein polymer, the numerous and diverse players in intracellular pathways for disulfide formation, and the distinct biological settings in which disulfide bond formation can take place belie the simplicity of the process. Here we lay the groundwork for appreciating the mechanisms and consequences of disulfide bond formation in vivo by reviewing chemical principles underlying cysteine pairing and oxidation. We then show how enzymes tune redox-active cofactors and recruit oxidants to improve the specificity and efficiency of disulfide formation. Finally, we discuss disulfide bond formation in a cellular context and identify important principles that contribute to productive thiol oxidation in complex, crowded, dynamic environments.
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Affiliation(s)
- Deborah Fass
- Department of Structural Biology, Weizmann Institute of Science , Rehovot 7610001, Israel
| | - Colin Thorpe
- Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States
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Abstract
This Reflections article describes my early work on viral enzymes and the discovery of mRNA capping, how my training in medicine and biochemistry merged as I evolved into a virologist, the development of viruses as vaccine vectors, and how scientific and technological developments during the 1970s and beyond set the stage for the interrogation of nearly every step in the reproductive cycle of vaccinia virus (VACV), a large DNA virus with about 200 genes. The reader may view this article as a work in progress, because I remain actively engaged in research at the National Institutes of Health (NIH) notwithstanding 50 memorable years there.
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Affiliation(s)
- Bernard Moss
- From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892
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A bioinformatics pipeline to search functional motifs within whole-proteome data: a case study of poxviruses. Virus Genes 2016; 53:173-178. [PMID: 28000080 PMCID: PMC5357487 DOI: 10.1007/s11262-016-1416-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 12/01/2016] [Indexed: 12/19/2022]
Abstract
Proteins harbor domains or short linear motifs, which facilitate their functions and interactions. Finding functional motifs in protein sequences could predict the putative cellular roles or characteristics of hypothetical proteins. In this study, we present Shetti-Motif, which is an interactive tool to (i) map UniProt and PROSITE flat files, (ii) search for multiple pre-defined consensus patterns or experimentally validated functional motifs in large datasets protein sequences (proteome-wide), (iii) search for motifs containing repeated residues (low-complexity regions, e.g., Leu-, SR-, PEST-rich motifs, etc.). As proof of principle, using this comparative proteomics pipeline, eleven proteomes encoded by member of Poxviridae family were searched against about 100 experimentally validated functional motifs. The closely related viruses and viruses infect the same host cells (e.g. vaccinia and variola viruses) show similar motif-containing proteins profile. The motifs encoded by these viruses are correlated, which explains why poxviruses are able to interact with wide range of host cells. In conclusion, this in silico analysis is useful to establish a dataset(s) or potential proteins for further investigation or compare between species.
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Wu X, Liu G, Mu M, Peng Y, Li X, Deng L, Zhang Z, Chen M, You S, Kong X. Augmenter of Liver Regeneration Gene Therapy Using a Novel Minicircle DNA Vector Alleviates Liver Fibrosis in Rats. Hum Gene Ther 2016; 27:880-891. [PMID: 27136973 DOI: 10.1089/hum.2016.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Affiliation(s)
- Xin Wu
- Institute of Life Science and Bio-Pharmaceutics, Shenyang Pharmaceutical University, Shenyang, China
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Guangze Liu
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Mao Mu
- Institute of Life Science and Bio-Pharmaceutics, Shenyang Pharmaceutical University, Shenyang, China
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Yuting Peng
- Institute of Life Science and Bio-Pharmaceutics, Shenyang Pharmaceutical University, Shenyang, China
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Xiumei Li
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Lisi Deng
- The Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai, China
| | - Zhenwei Zhang
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Meijuan Chen
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
| | - Song You
- Institute of Life Science and Bio-Pharmaceutics, Shenyang Pharmaceutical University, Shenyang, China
| | - Xiangping Kong
- Key Laboratory of Liver Disease, Centre of Infectious Diseases, 458th Hospital of PLA, Guangzhou, China
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Protein Primary Structure of the Vaccinia Virion at Increased Resolution. J Virol 2016; 90:9905-9919. [PMID: 27558425 DOI: 10.1128/jvi.01042-16] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Accepted: 08/17/2016] [Indexed: 01/16/2023] Open
Abstract
Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins. IMPORTANCE Poxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.
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Moss B. Membrane fusion during poxvirus entry. Semin Cell Dev Biol 2016; 60:89-96. [PMID: 27423915 DOI: 10.1016/j.semcdb.2016.07.015] [Citation(s) in RCA: 96] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 07/11/2016] [Accepted: 07/12/2016] [Indexed: 12/23/2022]
Abstract
Poxviruses comprise a large family of enveloped DNA viruses that infect vertebrates and invertebrates. Poxviruses, unlike most DNA viruses, replicate in the cytoplasm and encode enzymes and other proteins that enable entry, gene expression, genome replication, virion assembly and resistance to host defenses. Entry of vaccinia virus, the prototype member of the family, can occur at the plasma membrane or following endocytosis. Whereas many viruses encode one or two proteins for attachment and membrane fusion, vaccinia virus encodes four proteins for attachment and eleven more for membrane fusion and core entry. The entry-fusion proteins are conserved in all poxviruses and form a complex, known as the Entry Fusion Complex (EFC), which is embedded in the membrane of the mature virion. An additional membrane that encloses the mature virion and is discarded prior to entry is present on an extracellular form of the virus. The EFC is held together by multiple interactions that depend on nine of the eleven proteins. The entry process can be divided into attachment, hemifusion and core entry. All eleven EFC proteins are required for core entry and at least eight for hemifusion. To mediate fusion the virus particle is activated by low pH, which removes one or more fusion repressors that interact with EFC components. Additional EFC-interacting fusion repressors insert into cell membranes and prevent secondary infection. The absence of detailed structural information, except for two attachment proteins and one EFC protein, is delaying efforts to determine the fusion mechanism.
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae. J Virol 2015; 89:9348-67. [PMID: 26136578 PMCID: PMC4542343 DOI: 10.1128/jvi.01174-15] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2015] [Accepted: 06/23/2015] [Indexed: 11/20/2022] Open
Abstract
Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures. IMPORTANCE Aquaculture is an increasingly important global source of high-quality food. To sustain the growth in aquaculture, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wildlife is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases that they cause. There is a possibility that viruses with enhanced virulence may spread to new species, as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.
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Moussatche N, Condit RC. Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization. Virology 2014; 475:204-18. [PMID: 25486587 DOI: 10.1016/j.virol.2014.11.020] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Accepted: 11/18/2014] [Indexed: 10/24/2022]
Abstract
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.
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Affiliation(s)
- Nissin Moussatche
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA.
| | - Richard C Condit
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA
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Leão TL, da Fonseca FG. Subversion of cellular stress responses by poxviruses. World J Clin Infect Dis 2014; 4:27-40. [DOI: 10.5495/wjcid.v4.i4.27] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2014] [Revised: 07/26/2014] [Accepted: 09/10/2014] [Indexed: 02/06/2023] Open
Abstract
Cellular stress responses are powerful mechanisms that prevent and cope with the accumulation of macromolecular damage in the cells and also boost host defenses against pathogens. Cells can initiate either protective or destructive stress responses depending, to a large extent, on the nature and duration of the stressing stimulus as well as the cell type. The productive replication of a virus within a given cell places inordinate stress on the metabolism machinery of the host and, to assure the continuity of its replication, many viruses have developed ways to modulate the cell stress responses. Poxviruses are among the viruses that have evolved a large number of strategies to manipulate host stress responses in order to control cell fate and enhance their replicative success. Remarkably, nearly every step of the stress responses that is mounted during infection can be targeted by virally encoded functions. The fine-tuned interactions between poxviruses and the host stress responses has aided virologists to understand specific aspects of viral replication; has helped cell biologists to evaluate the role of stress signaling in the uninfected cell; and has tipped immunologists on how these signals contribute to alert the cells against pathogen invasion and boost subsequent immune responses. This review discusses the diverse strategies that poxviruses use to subvert host cell stress responses.
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Mazzon M, Castro C, Roberts LD, Griffin JL, Smith GL. A role for vaccinia virus protein C16 in reprogramming cellular energy metabolism. J Gen Virol 2014; 96:395-407. [PMID: 25351724 PMCID: PMC4298679 DOI: 10.1099/vir.0.069591-0] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vΔC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vΔC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α.
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Affiliation(s)
- Michela Mazzon
- Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, UK
| | - Cecilia Castro
- Department of Biochemistry and Cambridge Systems Biology Centre, Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK
| | - Lee D Roberts
- Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulborn Road, Cambridge CB1 9NL, UK.,Department of Biochemistry and Cambridge Systems Biology Centre, Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK
| | - Julian L Griffin
- Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulborn Road, Cambridge CB1 9NL, UK.,Department of Biochemistry and Cambridge Systems Biology Centre, Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK
| | - Geoffrey L Smith
- Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, UK
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The putative herpes simplex virus 1 chaperone protein UL32 modulates disulfide bond formation during infection. J Virol 2014; 89:443-53. [PMID: 25320327 DOI: 10.1128/jvi.01913-14] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation. IMPORTANCE Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
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Li SN, Wang JY, Yuan MJ, Yang K. Disruption of the baculovirus core gene ac78 results in decreased production of multiple nucleocapsid-enveloped occlusion-derived virions and the failure of primary infection in vivo. Virus Res 2014; 191:70-82. [PMID: 25087880 DOI: 10.1016/j.virusres.2014.07.019] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2014] [Revised: 07/19/2014] [Accepted: 07/21/2014] [Indexed: 02/07/2023]
Abstract
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac78 gene is one of the baculovirus core genes. Recent studies showed that ac78 is essential for budded virion (BV) production and the embedding of occlusion-derived virion (ODV) into occlusion body during the AcMNPV life cycle. Here, we report that an ac78-knockout AcMNPV (vAc78KO) constructed in this study had different phenotypes than those described in the previous studies. A few infectious BVs were detected using titer assays, immunoblot analyses and plaque assays, indicating that ac78 is not essential for BV formation. Electron microscopy confirmed that the ac78 deletion did not affect nucleocapsid assembly and ODV formation. However, the numbers of multiple nucleocapsid-enveloped ODVs and ODV-embedded occlusion bodies were significantly decreased. Subsequently, the highly conserved amino acid residues 2-25 and 64-88 of Ac78, which are homologous to an oxidoreductase and cytochrome c oxidase, respectively, were demonstrated to play a crucial role in the morphogenesis of multiple nucleocapsid-enveloped ODV. Immunoblot analysis found that Ac78 was an ODV envelope-associated protein. Consistently, amino acid residues 56-93 of Ac78 were identified as an inner nuclear membrane sorting motif, which may direct the localization of Ac78 to the ODV envelope. In vivo infectivity assays showed that the occlusion bodies of vAc78KO were unable to establish primary infection in the midgut of Trichoplusia ni larvae. Taken together, our results suggest that ac78 plays an important role in BV production and proper multiple nucleocapsid-enveloped ODV formation, as well as AcMNPV primary infection in vivo.
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Affiliation(s)
- Sai-Nan Li
- State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China; Department of Biology, Zhaoqing University, Zhaoqing 526061, China
| | - Jin-Yu Wang
- State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China
| | - Mei-Jin Yuan
- State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China
| | - Kai Yang
- State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China.
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Clem SA, Wu W, Passarelli AL. The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene. Virology 2014; 460-461:207-16. [PMID: 25010286 PMCID: PMC4101058 DOI: 10.1016/j.virol.2014.05.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2014] [Revised: 03/31/2014] [Accepted: 05/06/2014] [Indexed: 11/27/2022]
Abstract
The Autographa californica multiple nucleopolyhedrovirus ac92 is a conserved baculovirus gene with homology to flavin adenine dinucleotide-linked sulfhydryl oxidases. Its product, Ac92, is a functional sulfhydryl oxidase. Deletion of ac92 results in almost negligible levels of budded virus (BV) production, defects in occlusion-derived virus (ODV) co-envelopment and their inefficient incorporation into occlusion bodies. To determine the role of sulfhydryl oxidation in the production of BV, envelopment of nucleocapsids, and nucleocapsid incorporation into occlusion bodies, the Trichoplusia ni single nucleopolyhedrovirus ortholog, tn79, was substituted for ac92. Tn79 was found to be an active sulfhydryl oxidase that substituted for Ac92, resulting in the production of infectious BV, albeit about 10-fold less than an ac92-containing virus. Tn79 rescued defects in ODV morphogenesis caused by a lack of ac92. Active Tn79 sulfhydryl oxidase activity is required for efficient BV production, ODV envelopment, and their subsequent incorporation into occlusion bodies in the absence of ac92.
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Affiliation(s)
- Stian A Clem
- Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA
| | - Wenbi Wu
- Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA
| | - A Lorena Passarelli
- Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506-4901, USA.
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The virion of Cafeteria roenbergensis virus (CroV) contains a complex suite of proteins for transcription and DNA repair. Virology 2014; 466-467:82-94. [PMID: 24973308 DOI: 10.1016/j.virol.2014.05.029] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2014] [Revised: 05/25/2014] [Accepted: 05/27/2014] [Indexed: 11/20/2022]
Abstract
Cafeteria roenbergensis virus (CroV) is a giant virus of the Mimiviridae family that infects the marine phagotrophic flagellate C. roenbergensis. CroV possesses a DNA genome of ~730 kilobase pairs that is predicted to encode 544 proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified 141 virion-associated CroV proteins and 60 host proteins. Data are available via ProteomeXchange with identifier PXD000993. Predicted functions could be assigned to 36% of the virion proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. The overlapping virion proteome of CroV and Mimivirus reveals a set of conserved virion protein functions that were presumably present in the last common ancestor of the Mimiviridae.
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Yu HY, Zhu MH, Xiang DR, Li J, Sheng JF. High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma. Onco Targets Ther 2014; 7:887-93. [PMID: 24940072 PMCID: PMC4051792 DOI: 10.2147/ott.s61531] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis.
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Affiliation(s)
- Hai-Ying Yu
- State Key Laboratory of Infectious Disease and Department of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Man-Hua Zhu
- State Key Laboratory of Infectious Disease and Department of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Dai-Rong Xiang
- State Key Laboratory of Infectious Disease and Department of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Jun Li
- State Key Laboratory of Infectious Disease and Department of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People's Republic of China
| | - Ji-Fang Sheng
- State Key Laboratory of Infectious Disease and Department of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People's Republic of China
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Disulfide bond formation in prokaryotes: history, diversity and design. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2014; 1844:1402-14. [PMID: 24576574 DOI: 10.1016/j.bbapap.2014.02.014] [Citation(s) in RCA: 91] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Revised: 02/12/2014] [Accepted: 02/16/2014] [Indexed: 01/16/2023]
Abstract
The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.
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Maruri-Avidal L, Weisberg AS, Moss B. Association of the vaccinia virus A11 protein with the endoplasmic reticulum and crescent precursors of immature virions. J Virol 2013; 87:10195-206. [PMID: 23864611 PMCID: PMC3754016 DOI: 10.1128/jvi.01601-13] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2013] [Accepted: 07/03/2013] [Indexed: 12/17/2022] Open
Abstract
The apparent de novo formation of viral membranes within cytoplasmic factories is a mysterious, poorly understood first step in poxvirus morphogenesis. Genetic studies identified several viral proteins essential for membrane formation and the assembly of immature virus particles. Their repression results in abortive replication with the accumulation of dense masses of viroplasm. In the present study, we further characterized one of these proteins, A11, and investigated its association with cellular and viral membranes under normal and abortive replication conditions. We discovered that A11 colocalized in cytoplasmic factories with the endoplasmic reticulum (ER) and L2, another viral protein required for morphogenesis. Confocal microscopy and subcellular fractionation indicated that A11 was not membrane associated in uninfected cells, whereas L2 still colocalized with the ER. Cell-free transcription and translation experiments indicated that both A11 and L2 are tail-anchored proteins that associate posttranslationally with membranes and likely require specific cytoplasmic targeting chaperones. Transmission electron microscopy indicated that A11, like L2, associated with crescent membranes and immature virions during normal infection and with vesicles and tubules near masses of dense viroplasm during abortive infection in the absence of the A17 or A14 protein component of viral membranes. When the synthesis of A11 was repressed, "empty" immature-virion-like structures formed in addition to masses of viroplasm. The immature-virion-like structures were labeled with antibodies to A17 and to the D13 scaffold protein and were closely associated with calnexin-labeled ER. These studies revealed similarities and differences between A11 and L2, both of which may be involved in the recruitment of the ER for virus assembly.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
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