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1
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Münz C, Campbell GR, Esclatine A, Faure M, Labonte P, Lussignol M, Orvedahl A, Altan-Bonnet N, Bartenschlager R, Beale R, Cirone M, Espert L, Jung J, Leib D, Reggiori F, Sanyal S, Spector SA, Thiel V, Viret C, Wei Y, Wileman T, Wodrich H. Autophagy machinery as exploited by viruses. AUTOPHAGY REPORTS 2025; 4:27694127.2025.2464986. [PMID: 40201908 PMCID: PMC11921968 DOI: 10.1080/27694127.2025.2464986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Revised: 01/17/2025] [Accepted: 01/27/2025] [Indexed: 04/10/2025]
Abstract
Viruses adapt and modulate cellular pathways to allow their replication in host cells. The catabolic pathway of macroautophagy, for simplicity referred to as autophagy, is no exception. In this review, we discuss anti-viral functions of both autophagy and select components of the autophagy machinery, and how viruses have evaded them. Some viruses use the membrane remodeling ability of the autophagy machinery to build their replication compartments in the cytosol or efficiently egress from cells in a non-lytic fashion. Some of the autophagy machinery components and their remodeled membranes can even be found in viral particles as envelopes or single membranes around virus packages that protect them during spreading and transmission. Therefore, studies on autophagy regulation by viral infections can reveal functions of the autophagy machinery beyond lysosomal degradation of cytosolic constituents. Furthermore, they can also pinpoint molecular interactions with which the autophagy machinery can most efficiently be manipulated, and this may be relevant to develop effective disease treatments based on autophagy modulation.
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Affiliation(s)
- Christian Münz
- Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Zürich Switzerland
| | - Grant R Campbell
- Division of Basic Biomedical Sciences, Sanford School of Medicine, University of SD, Vermillion, SD, USA
| | - Audrey Esclatine
- Université Paris-Saclay, CEA, CNRS, 10 Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Mathias Faure
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Universite Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France
| | - Patrick Labonte
- eINRS-Centre Armand-Frappier Santé Biotechnologie, Laval, Canada
| | - Marion Lussignol
- Université Paris-Saclay, CEA, CNRS, 10 Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Anthony Orvedahl
- Department of Pediatrics, Washington University in St. Louis, St. Louis, MO, USA
- Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO, USA
| | - Nihal Altan-Bonnet
- Laboratory of Host-Pathogen Dynamics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
| | - Ralf Bartenschlager
- Heidelberg University, Medical Faculty Heidelberg, Department of Infectious Diseases, Molecular Virology, Heidelberg, Germany
- German Cancer Research Center (DKFZ), Division Virus-Associated Carcinogenesis, Heidelberg, Germany
- German Centre for Infection Research, Heidelberg partner site, Heidelberg, Germany
| | - Rupert Beale
- Cell Biology of Infection Laboratory, The Francis Crick Institute, London, UK
- Division of Medicine, University College London, London, UK
| | - Mara Cirone
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - Lucile Espert
- University of Montpellier, Montpellier, France
- CNRS, Institut de Recherche enInfectiologie deMontpellier (IRIM), Montpellier, France
| | - Jae Jung
- Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
| | - David Leib
- Guarini School of Graduate and Advanced Studies at Dartmouth, Hanover, NH, USA
| | - Fulvio Reggiori
- Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Aarhus C, Denmark
| | - Sumana Sanyal
- Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford, UK
- HKU-Pasteur Research Pole, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Stephen A. Spector
- Division of Infectious Diseases, Department of Pediatrics, University of California San Diego, La Jolla, CA, USA
- Rady Children’s Hospital, San Diego, CA, USA
| | - Volker Thiel
- Institute of Virology and Immunology, Bern and Mittelhäusern, Switzerland
- Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, and Multidisciplinary Center for Infectious Diseases, University of Bern, Bern, Switzerland
| | - Christophe Viret
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm, U1111, Universite Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France
| | - Yu Wei
- Institut Pasteur-Theravectys Joint Laboratory, Department of Virology, Institut Pasteur, Université Paris Cité, Paris, France
| | - Thomas Wileman
- Norwich Medical School, University of East Anglia
- Quadram Institute Bioscience, Norwich Research Park, Norfolk, UK
| | - Harald Wodrich
- sLaboratoire de Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR, Université de Bordeaux, Bordeaux, France
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2
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Druciarek T, Tzanetakis IE. Invisible vectors, visible impact: The role of eriophyoid mites in emaravirus disease dynamics. Virology 2025; 606:110478. [PMID: 40112629 DOI: 10.1016/j.virol.2025.110478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2024] [Revised: 02/25/2025] [Accepted: 02/27/2025] [Indexed: 03/22/2025]
Abstract
Emaraviruses are segmented, negative-sense RNA viruses that are transmitted by eriophyoid mites. Advances in virus detection and discovery have significantly improved our understanding of these viruses, yet several challenges persist. This review emphasizes the significant gaps in our knowledge of virus replication, transmission dynamics, and plant-virus-vector interactions and highlights the role of mite vectors in the epidemiology and control of emaraviruses. By bridging the knowledge gaps with advanced genomic tools such as high-throughput sequencing and bioinformatics and targeted acarological research we will achieve sustainable control strategies and reduce the impact of emaravirus-caused diseases.
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Affiliation(s)
- Tobiasz Druciarek
- Department of Entomology and Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, Arkansas, 72701, United States; Section of Applied Entomology, Department of Plant Protection, Institute of Horticultural Sciences, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland.
| | - Ioannis E Tzanetakis
- Department of Entomology and Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, Arkansas, 72701, United States.
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3
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Bakhache W, Shen A, Symonds-Orr W, Freeman MC, Dolan PT. Novel reporter constructs to accelerate antiviral and therapeutic discovery for Enterovirus-A71. Antiviral Res 2025; 235:106094. [PMID: 39900143 DOI: 10.1016/j.antiviral.2025.106094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2024] [Revised: 01/15/2025] [Accepted: 01/23/2025] [Indexed: 02/05/2025]
Abstract
Enterovirus A71 (EV-A71) is an important human pathogen and 'prototype pathogen' for studies of other Enteroviruses of pandemic potential. Understanding the biology of EV-A71 would inform generalizable strategies for antiviral drug, vaccine, and monoclonal antibody development. Such studies are accelerated by robust reagents to evaluate efficacy. Here, we describe and evaluate a suite of synthetic reporter constructs to accelerate EV-A71 research and therapeutic discovery. These constructs include replicons and infectious clones carrying luminescent and fluorescent reporter proteins. Among the reporters we tested were shorter luminescent and de novo-designed synthetic fluorescent proteins, which enhance genetic stability, reduce reporter gene loss and improve the utility of these reporters. This toolbox provides free access to robust and flexible assays for EV-A71 infection and replication through public repositories, promoting and accelerating open scientific discovery for this understudied emerging pathogen.
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Affiliation(s)
- William Bakhache
- Quantitative Virology and Evolution Unit, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA
| | - Ann Shen
- Quantitative Virology and Evolution Unit, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA
| | - Walker Symonds-Orr
- Quantitative Virology and Evolution Unit, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA
| | - Megan Culler Freeman
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, USA
| | - Patrick T Dolan
- Quantitative Virology and Evolution Unit, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA.
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4
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Heffner AL, Rouault TA. A Comparison of Conserved Features in the Human Coronavirus Family Shows That Studies of Viruses Less Pathogenic than SARS-CoV-2, Such as HCoV-OC43, Are Good Model Systems for Elucidating Basic Mechanisms of Infection and Replication in Standard Laboratories. Viruses 2025; 17:256. [PMID: 40007010 PMCID: PMC11860170 DOI: 10.3390/v17020256] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Revised: 02/05/2025] [Accepted: 02/06/2025] [Indexed: 02/27/2025] Open
Abstract
In 2021, at the height of the COVID-19 pandemic, coronavirus research spiked, with over 83,000 original research articles related to the word "coronavirus" added to the online resource PubMed. Just 2 years later, in 2023, only 30,900 original research articles related to the word "coronavirus" were added. While, irrefutably, the funding of coronavirus research drastically decreased, a possible explanation for the decrease in interest in coronavirus research is that projects on SARS-CoV-2, the causative agent of COVID-19, halted due to the challenge of establishing a good cellular or animal model system. Most laboratories do not have the capabilities to culture SARS-CoV-2 'in house' as this requires a Biosafety Level (BSL) 3 laboratory. Until recently, BSL 2 laboratory research on endemic coronaviruses was arduous due to the low cytopathic effect in isolated cell culture infection models and the lack of means to quantify viral loads. The purpose of this review article is to compare the human coronaviruses and provide an assessment of the latest techniques that use the endemic coronaviruses-HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1-as lower-biosafety-risk models for the more pathogenic coronaviruses-SARS-CoV-2, SARS-CoV, and MERS-CoV.
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Affiliation(s)
- Audrey L. Heffner
- Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
- Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
| | - Tracey A. Rouault
- Molecular Medicine Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
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5
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Liu Q, Long JE. Insight into the Life Cycle of Enterovirus-A71. Viruses 2025; 17:181. [PMID: 40006936 PMCID: PMC11861800 DOI: 10.3390/v17020181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2025] [Revised: 01/22/2025] [Accepted: 01/23/2025] [Indexed: 02/27/2025] Open
Abstract
Human enterovirus 71 (EV-A71), a member of the Picornaviridae family, is predominantly associated with hand, foot, and mouth disease in infants and young children. Additionally, EV-A71 can cause severe neurological complications, including aseptic meningitis, brainstem encephalitis, and fatalities. The molecular mechanisms underlying these symptoms are complex and involve the viral tissue tropism, evasion from the host immune responses, induction of the programmed cell death, and cytokine storms. This review article delves into the EV-A71 life cycle, with a particular emphasis on recent advancements in understanding the virion structure, tissue tropism, and the interplay between the virus and host regulatory networks during replication. The comprehensive review is expected to contribute to our understanding of EV-A71 pathogenesis and inform the development of antiviral therapies and vaccines.
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Affiliation(s)
- Qi Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China;
| | - Jian-Er Long
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China;
- Department of Pathogenic Biology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
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6
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Han S, Ye X, Yang J, Peng X, Jiang X, Li J, Zheng X, Zhang X, Zhang Y, Zhang L, Wang W, Li J, Xin W, Zhang X, Xiao G, Peng K, Zhang L, Du X, Zhou L, Liu W, Li H. Host specific sphingomyelin is critical for replication of diverse RNA viruses. Cell Chem Biol 2024; 31:2052-2068.e11. [PMID: 39566509 DOI: 10.1016/j.chembiol.2024.10.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Revised: 08/28/2024] [Accepted: 10/23/2024] [Indexed: 11/22/2024]
Abstract
Lipids and lipid metabolism play an important role in RNA virus replication, which typically occurs on host cell endomembrane structures in the cytoplasm through mechanisms that are not yet fully identified. We conducted genome-scale CRISPR screening and identified sphingomyelin synthase 1 (SMS1; encoded by SGMS1) as a critical host factor for infection by severe fever with thrombocytopenia syndrome virus (SFTSV). SGMS1 knockout reduced sphingomyelin (SM) (d18:1/16:1) levels, inhibiting SFTSV replication. A helix-turn-helix motif in SFTSV RNA-dependent RNA polymerase (RdRp) directly binds to SM(d18:1/16:1) in Golgi apparatus, which was also observed in SARS-CoV-2 and lymphocytic choriomeningitis virus (LCMV), both showing inhibited replication in SGMS1-KO cells. SM metabolic disturbance is associated with disease severity of viral infections. We designed a novel SMS1 inhibitor that protects mice against lethal SFTSV infection and reduce SARS-CoV-2 replication and pathogenesis. These findings highlight the critical role of SMS1 and SM(d18:1/16:1) in RNA virus replication, suggesting a broad-spectrum antiviral strategy.
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Affiliation(s)
- Shuo Han
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Xiaolei Ye
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Jintong Yang
- School of Pharmacy, Fudan University, Shanghai 201203, China
| | - Xuefang Peng
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Xiaming Jiang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Jin Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Xiaojie Zheng
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Xinchen Zhang
- School of Pharmacy, Fudan University, Shanghai 201203, China
| | - Yumin Zhang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Lingyu Zhang
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Wei Wang
- School of Pharmacy, Fudan University, Shanghai 201203, China
| | - Jiaxin Li
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Wenwen Xin
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Xiaoai Zhang
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China
| | - Gengfu Xiao
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Ke Peng
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Leike Zhang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
| | - Xuguang Du
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Lu Zhou
- School of Pharmacy, Fudan University, Shanghai 201203, China.
| | - Wei Liu
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China.
| | - Hao Li
- State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China.
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7
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Jassey A, Paudel B, Wagner MA, Pollack N, Cheng IT, Godoy-Ruiz R, Weber DJ, Jackson WT. Mitophagosomes induced during EV-D68 infection promote viral nonlytic release. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.05.627125. [PMID: 39677747 PMCID: PMC11643070 DOI: 10.1101/2024.12.05.627125] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Enterovirus-D68 (EV-D68) is a plus-strand RNA virus that primarily causes infant respiratory infections. In rare pediatric cases, infection with EV-D68 has been associated with acute flaccid myelitis, a polio-like paralytic disease. We have previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that EV-D68 induces mitophagy, the specific autophagic degradation of mitochondria. EV-D68 infection induces mitophagosome formation and several hallmarks of mitophagy, including mitochondrial fragmentation, mitochondrial membrane potential loss, and Parkin translocation to the mitochondria were observed in EV-D68 infected cells. The 3C protease of EV-D68 cleaves the mitochondrial fusion protein, mitofusin-2, near the C-terminal HR2 domain to induce mitochondrial fragmentation, and these fragmented mitochondria colocalized with double-stranded RNA (dsRNA), which labels viral RNA replication sites after peak viral RNA replication. Depleting components of mitophagy signaling specifically reduced EV-D68 release without impacting viral intracellular titers. Our results suggest that whereas the machinery of macroautophagy supports various stages of enterovirus replication, including viral genomic RNA replication and capsid maturation, mitophagy is the specific form of autophagy that regulates the nonlytic release of enteroviruses from cells.
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Affiliation(s)
- Alagie Jassey
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, 685 W. Baltimore Avenue, Baltimore, MD 21201, USA
| | - Bimal Paudel
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, 685 W. Baltimore Avenue, Baltimore, MD 21201, USA
| | - Michael A. Wagner
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, 685 W. Baltimore Avenue, Baltimore, MD 21201, USA
| | - Noah Pollack
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, 685 W. Baltimore Avenue, Baltimore, MD 21201, USA
| | - I-Ting Cheng
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore
- Institute for Bioscience and Biotechnology Research, Rockville
- The Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore
| | - Raquel Godoy-Ruiz
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore
- Institute for Bioscience and Biotechnology Research, Rockville
- The Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore
| | - David J. Weber
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore
- Institute for Bioscience and Biotechnology Research, Rockville
- The Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore
| | - William T. Jackson
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, 685 W. Baltimore Avenue, Baltimore, MD 21201, USA
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Wu B, Fan T, Chen X, He Y, Wang H. The class III phosphatidylinositol 3-kinase VPS34 supports EV71 replication by promoting viral replication organelle formation. J Virol 2024; 98:e0069524. [PMID: 39254312 PMCID: PMC11495007 DOI: 10.1128/jvi.00695-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Accepted: 08/20/2024] [Indexed: 09/11/2024] Open
Abstract
Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development.
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Affiliation(s)
- Bo Wu
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Tingting Fan
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Xinrui Chen
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Yingli He
- Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
| | - Hongliang Wang
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
- Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
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9
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Royet A, Ruedas R, Gargowitsch L, Gervais V, Habersetzer J, Pieri L, Ouldali M, Paternostre M, Hofmann I, Tubiana T, Fieulaine S, Bressanelli S. Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers. J Biol Chem 2024; 300:107724. [PMID: 39214299 PMCID: PMC11439542 DOI: 10.1016/j.jbc.2024.107724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 08/03/2024] [Accepted: 08/14/2024] [Indexed: 09/04/2024] Open
Abstract
Single-stranded, positive-sense RNA ((+)RNA) viruses replicate their genomes in virus-induced intracellular membrane compartments. (+)RNA viruses dedicate a significant part of their small genomes (a few thousands to a few tens of thousands of bases) to the generation of these compartments by encoding membrane-interacting proteins and/or protein domains. Noroviruses are a very diverse genus of (+)RNA viruses including human and animal pathogens. Human noroviruses are the major cause of acute gastroenteritis worldwide, with genogroup II genotype 4 (GII.4) noroviruses accounting for the vast majority of infections. Three viral proteins encoded in the N terminus of the viral replication polyprotein direct intracellular membrane rearrangements associated with norovirus replication. Of these three, nonstructural protein 4 (NS4) seems to be the most important, although its exact functions in replication organelle formation are unknown. Here, we produce, purify, and characterize GII.4 NS4. AlphaFold modeling combined with experimental data refines and corrects our previous crude structural model of NS4. Using simple artificial liposomes, we report an extensive characterization of the membrane properties of NS4. We find that NS4 self-assembles and thereby bridges liposomes together. Cryo-EM, NMR, and membrane flotation show formation of several distinct NS4 assemblies, at least two of them bridging pairs of membranes together in different fashions. Noroviruses belong to (+)RNA viruses whose replication compartment is extruded from the target endomembrane and generates double-membrane vesicles. Our data establish that the 21-kDa GII.4 human norovirus NS4 can, in the absence of any other factor, recapitulate in tubo several features, including membrane apposition, that occur in such processes.
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Affiliation(s)
- Adrien Royet
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Rémi Ruedas
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France; Sanofi, Integrated Drug Discovery, Vitry-sur-Seine, France
| | - Laetitia Gargowitsch
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France; Université Paris-Saclay, CNRS, Laboratoire de Physique des Solides, Orsay, France
| | - Virginie Gervais
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Johann Habersetzer
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Laura Pieri
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Malika Ouldali
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Maïté Paternostre
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Ilse Hofmann
- Core Facility Antibodies, German Cancer Research Center, Heidelberg, Germany
| | - Thibault Tubiana
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France
| | - Sonia Fieulaine
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
| | - Stéphane Bressanelli
- Université Paris-Saclay, CEA, CNRS - Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
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10
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Johnson P, Needham J, Lim N, Simon A. Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles. NAR Genom Bioinform 2024; 6:lqae104. [PMID: 39157584 PMCID: PMC11327873 DOI: 10.1093/nargab/lqae104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2024] [Revised: 07/23/2024] [Accepted: 08/01/2024] [Indexed: 08/20/2024] Open
Abstract
The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand reads and (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.
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Affiliation(s)
- Philip Z Johnson
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
| | - Jason M Needham
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
| | - Natalie K Lim
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
| | - Anne E Simon
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA
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11
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Wang X, Chen Y, Qi C, Li F, Zhang Y, Zhou J, Wu H, Zhang T, Qi A, Ouyang H, Xie Z, Pang D. Mechanism, structural and functional insights into nidovirus-induced double-membrane vesicles. Front Immunol 2024; 15:1340332. [PMID: 38919631 PMCID: PMC11196420 DOI: 10.3389/fimmu.2024.1340332] [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: 11/17/2023] [Accepted: 05/22/2024] [Indexed: 06/27/2024] Open
Abstract
During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.
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Affiliation(s)
- Xi Wang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Yiwu Chen
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Chunyun Qi
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Feng Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Yuanzhu Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Jian Zhou
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Heyong Wu
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Tianyi Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Aosi Qi
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
| | - Hongsheng Ouyang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
- Chongqing Research Institute, Jilin University, Chongqing, China
- Center for Animal Science and Technology Research, Chongqing Jitang Biotechnology Research Institute Co., Ltd, Chongqing, China
| | - Zicong Xie
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
- Chongqing Research Institute, Jilin University, Chongqing, China
| | - Daxin Pang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Sciences, Jilin University, Changchun, Jilin, China
- Chongqing Research Institute, Jilin University, Chongqing, China
- Center for Animal Science and Technology Research, Chongqing Jitang Biotechnology Research Institute Co., Ltd, Chongqing, China
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12
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Yang H, Fan T, Xun M, Wu B, Guo S, Li X, Zhao X, Yao H, Wang H. N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis. J Virol 2024; 98:e0174923. [PMID: 38189249 PMCID: PMC10878262 DOI: 10.1128/jvi.01749-23] [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: 11/06/2023] [Accepted: 12/07/2023] [Indexed: 01/09/2024] Open
Abstract
Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIβ (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIβ (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.
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Affiliation(s)
- Hang Yang
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Tingting Fan
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Meng Xun
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Bo Wu
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Shangrui Guo
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Xinyu Li
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Xiaohui Zhao
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
| | - Haoyan Yao
- Department of Gynecology and Obstetrics, The First Affiliated Hospital of Xi'an Jiaotong University, Xi’an, China
| | - Hongliang Wang
- Department of Pathogen Biology and Immunology, Xi’an Jiaotong University Health Science Center, Xi’an, China
- Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China
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13
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Grizer CS, Messacar K, Mattapallil JJ. Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children. FRONTIERS IN VIROLOGY (LAUSANNE, SWITZERLAND) 2024; 4:1328457. [PMID: 39246649 PMCID: PMC11378966 DOI: 10.3389/fviro.2024.1328457] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/10/2024]
Abstract
The past decade has seen the global reemergence and rapid spread of enterovirus D68 (EV-D68), a respiratory pathogen that causes severe respiratory illness and paralysis in children. EV-D68 was first isolated in 1962 from children with pneumonia. Sporadic cases and small outbreaks have been reported since then with a major respiratory disease outbreak in 2014 associated with an increased number of children diagnosed with polio-like paralysis. From 2014-2018, major outbreaks have been reported every other year in a biennial pattern with > 90% of the cases occurring in children under the age of 16. With the outbreak of SARS-CoV-2 and the subsequent COVID-19 pandemic, there was a significant decrease in the prevalence EV-D68 cases along with other respiratory diseases. However, since the relaxation of pandemic social distancing protocols and masking mandates the number of EV-D68 cases have begun to rise again - culminating in another outbreak in 2022. Here we review the virology, pathogenesis, and the immune response to EV-D68, and discuss the epidemiology of EV-D68 infections and the divergence of contemporary strains from historical strains. Finally, we highlight some of the key challenges in the field that remain to be addressed.
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Affiliation(s)
- Cassandra S Grizer
- Department of Microbiology & Immunology, The Henry M. Jackson Foundation for Military Medicine, Uniformed Services University, Bethesda, MD 20814, USA
| | - Kevin Messacar
- The Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, CO 80045, USA
| | - Joseph J Mattapallil
- Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD 20814, USA
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14
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Denker L, Dixon AM. The cell edit: Looking at and beyond non-structural proteins to understand membrane rearrangement in coronaviruses. Arch Biochem Biophys 2024; 752:109856. [PMID: 38104958 DOI: 10.1016/j.abb.2023.109856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2023] [Revised: 11/24/2023] [Accepted: 12/08/2023] [Indexed: 12/19/2023]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-stranded RNA virus that sits at the centre of the recent global pandemic. As a member of the coronaviridae family of viruses, it shares features such as a very large genome (>30 kb) that is replicated in a purpose-built replication organelle. Biogenesis of the replication organelle requires significant and concerted rearrangement of the endoplasmic reticulum membrane, a job that is carried out by a group of integral membrane non-structural proteins (NSP3, 4 and 6) expressed by the virus along with a host of viral replication enzymes and other factors that support transcription and replication. The primary sites for RNA replication within the replication organelle are double membrane vesicles (DMVs). The small size of DMVs requires generation of high membrane curvature, as well as stabilization of a double-membrane arrangement, but the mechanisms that underlie DMV formation remain elusive. In this review, we discuss recent breakthroughs in our understanding of the molecular basis for membrane rearrangements by coronaviruses. We incorporate established models of NSP3-4 protein-protein interactions to drive double membrane formation, and recent data highlighting the roles of lipid composition and host factor proteins (e.g. reticulons) that influence membrane curvature, to propose a revised model for DMV formation in SARS-CoV-2.
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Affiliation(s)
- Lea Denker
- Warwick Medical School, Biomedical Sciences, University of Warwick, Coventry, CV4 7AL, UK.
| | - Ann M Dixon
- Department of Chemistry, University of Warwick, Coventry, CV4 7SH, UK.
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15
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Lin J, Zhao J, Du L, Wang P, Sun B, Zhang C, Shi Y, Li H, Sun H. Activation of MAPK-mediated immunity by phosphatidic acid in response to positive-strand RNA viruses. PLANT COMMUNICATIONS 2024; 5:100659. [PMID: 37434356 PMCID: PMC10811337 DOI: 10.1016/j.xplc.2023.100659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Revised: 05/31/2023] [Accepted: 07/07/2023] [Indexed: 07/13/2023]
Abstract
Increasing evidence suggests that mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant defense against viruses. However, the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear. In this study, we discovered that phosphatidic acid (PA) represents a major class of lipids that respond to Potato virus Y (PVY) at an early stage of infection. We identified NbPLDα1 (Nicotiana benthamiana phospholipase Dα1) as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role. 6K2 of PVY interacts with NbPLDα1, leading to elevated PA levels. In addition, NbPLDα1 and PA are recruited by 6K2 to membrane-bound viral replication complexes. On the other hand, 6K2 also induces activation of the MAPK pathway, dependent on its interaction with NbPLDα1 and the derived PA. PA binds to WIPK/SIPK/NTF4, prompting their phosphorylation of WRKY8. Notably, spraying with exogenous PA is sufficient to activate the MAPK pathway. Knockdown of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA. 6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDα1 and induced the activation of MAPK-mediated immunity. Loss of function of NbPLDα1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation. Thus, activation of MAPK-mediated immunity by NbPLDα1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.
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Affiliation(s)
- Jiayu Lin
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Jinpeng Zhao
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Linlin Du
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Pengkun Wang
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Bingjian Sun
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Chao Zhang
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Yan Shi
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Honglian Li
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China
| | - Hangjun Sun
- The Engineering Research Center for Plant Health Protection Technology in Henan Province, College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan 450046, China.
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16
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Carella F, Prado P, De Vico G, Palić D, Villari G, García-March JR, Tena-Medialdea J, Cortés Melendreras E, Giménez-Casalduero F, Sigovini M, Aceto S. A widespread picornavirus affects the hemocytes of the noble pen shell ( Pinna nobilis), leading to its immunosuppression. Front Vet Sci 2023; 10:1273521. [PMID: 38164394 PMCID: PMC10758234 DOI: 10.3389/fvets.2023.1273521] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2023] [Accepted: 11/13/2023] [Indexed: 01/03/2024] Open
Abstract
Introduction The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. Methods We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. Results and discussion In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104-1.60 × 105 ml-1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus-a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.
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Affiliation(s)
- Francesca Carella
- Department of Biology, University of Naples Federico II, Naples, Italy
| | - Patricia Prado
- Institute of Agrifood Research and Technology (IRTA)-Sant Carles de la Ràpita, Tarragona, Spain
| | - Gionata De Vico
- Department of Biology, University of Naples Federico II, Naples, Italy
| | - Dušan Palić
- Chair for Fish Diseases and Fisheries Biology, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Munich, Germany
| | - Grazia Villari
- Department of Biology, University of Naples Federico II, Naples, Italy
| | - José Rafael García-March
- Instituto de Investigación en Medio Ambiente y Ciencia Marina, Universidad Católica de Valencia, Calpe, Spain
| | - José Tena-Medialdea
- Instituto de Investigación en Medio Ambiente y Ciencia Marina, Universidad Católica de Valencia, Calpe, Spain
| | | | - Francisca Giménez-Casalduero
- Department of Marine Science and Applied Biology, Research Marine Centre in Santa Pola (CIMAR), University of Alicante, Alicante, Spain
| | - Marco Sigovini
- Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, Venice, Italy
| | - Serena Aceto
- Department of Biology, University of Naples Federico II, Naples, Italy
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17
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Galitska G, Jassey A, Wagner MA, Pollack N, Miller K, Jackson WT. Enterovirus D68 capsid formation and stability requires acidic compartments. mBio 2023; 14:e0214123. [PMID: 37819109 PMCID: PMC10653823 DOI: 10.1128/mbio.02141-23] [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: 08/10/2023] [Accepted: 08/14/2023] [Indexed: 10/13/2023] Open
Abstract
IMPORTANCE The respiratory picornavirus enterovirus D68 is a causative agent of acute flaccid myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus that survives acidic environments when passing from host to host. Here, we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly, and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.
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Affiliation(s)
- Ganna Galitska
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Alagie Jassey
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Michael A. Wagner
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Noah Pollack
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Katelyn Miller
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - William T. Jackson
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
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18
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Viktorova EG, Gabaglio S, Moghimi S, Zimina A, Wynn BG, Sztul E, Belov GA. The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1. PLoS Pathog 2023; 19:e1011673. [PMID: 37721955 PMCID: PMC10538752 DOI: 10.1371/journal.ppat.1011673] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 09/28/2023] [Accepted: 09/08/2023] [Indexed: 09/20/2023] Open
Abstract
The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.
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Affiliation(s)
- Ekaterina G. Viktorova
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Samuel Gabaglio
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Seyedehmahsa Moghimi
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Anna Zimina
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Bridge G. Wynn
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - George A. Belov
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
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19
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Pham MT, Lee JY, Ritter C, Thielemann R, Meyer J, Haselmann U, Funaya C, Laketa V, Rohr K, Bartenschlager R. Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus. PLoS Pathog 2023; 19:e1011052. [PMID: 37506130 PMCID: PMC10411793 DOI: 10.1371/journal.ppat.1011052] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 08/09/2023] [Accepted: 07/07/2023] [Indexed: 07/30/2023] Open
Abstract
Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.
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Affiliation(s)
- Minh-Tu Pham
- Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Diseases Research, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
| | - Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Diseases Research, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
| | - Christian Ritter
- BioQuant Center, IPMB, Biomedical Computer Vision Group, Heidelberg University, Heidelberg, Germany
| | - Roman Thielemann
- BioQuant Center, IPMB, Biomedical Computer Vision Group, Heidelberg University, Heidelberg, Germany
| | - Janis Meyer
- BioQuant Center, IPMB, Biomedical Computer Vision Group, Heidelberg University, Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Diseases Research, Heidelberg University, Heidelberg, Germany
| | - Charlotta Funaya
- Electron Microscopy Core Facility (EMCF), Heidelberg University, Heidelberg, Germany
| | - Vibor Laketa
- German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
- Department of Infectious Diseases, Virology, Center for Integrative Infectious Diseases Research, Heidelberg University, Heidelberg, Germany
| | - Karl Rohr
- BioQuant Center, IPMB, Biomedical Computer Vision Group, Heidelberg University, Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Diseases Research, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
- Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
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20
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Galitska G, Jassey A, Wagner MA, Pollack N, Jackson WT. Enterovirus D68 capsid formation and stability requires acidic compartments. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.12.544695. [PMID: 37398138 PMCID: PMC10312662 DOI: 10.1101/2023.06.12.544695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/04/2023]
Abstract
Enterovirus D68 (EV-D68), a picornavirus traditionally associated with respiratory infections, has recently been linked to a polio-like paralytic condition known as acute flaccid myelitis (AFM). EV-D68 is understudied, and much of the field's understanding of this virus is based on studies of poliovirus. For poliovirus, we previously showed that low pH promotes virus capsid maturation, but here we show that, for EV-D68, inhibition of compartment acidification during a specific window of infection causes a defect in capsid formation and maintenance. These phenotypes are accompanied by radical changes in the infected cell, with viral replication organelles clustering in a tight juxtanuclear grouping. Organelle acidification is critical during a narrow window from 3-4hpi, which we have termed the "transition point," separating translation and peak RNA replication from capsid formation, maturation and egress. Our findings highlight that acidification is crucial only when vesicles convert from RNA factories to virion crucibles.
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Affiliation(s)
- Ganna Galitska
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA
| | - Alagie Jassey
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA
| | - Michael A Wagner
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA
| | - Noah Pollack
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA
| | - William T Jackson
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St, Baltimore, MD 21201, USA
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21
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Li Z, Zheng M, He Z, Qin Y, Chen M. Morphogenesis and functional organization of viral inclusion bodies. CELL INSIGHT 2023; 2:100103. [PMID: 37193093 PMCID: PMC10164783 DOI: 10.1016/j.cellin.2023.100103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 04/06/2023] [Accepted: 04/07/2023] [Indexed: 05/18/2023]
Abstract
Eukaryotic viruses are obligate intracellular parasites that rely on the host cell machinery to carry out their replication cycle. This complex process involves a series of steps, starting with virus entry, followed by genome replication, and ending with virion assembly and release. Negative strand RNA and some DNA viruses have evolved to alter the organization of the host cell interior to create a specialized environment for genome replication, known as IBs, which are precisely orchestrated to ensure efficient viral replication. The biogenesis of IBs requires the cooperation of both viral and host factors. These structures serve multiple functions during infection, including sequestering viral nucleic acids and proteins from innate immune responses, increasing the local concentration of viral and host factors, and spatially coordinating consecutive replication cycle steps. While ultrastructural and functional studies have improved our understanding of IBs, much remains to be learned about the precise mechanisms of IB formation and function. This review aims to summarize the current understanding of how IBs are formed, describe the morphology of these structures, and highlight the mechanism of their functions. Given that the formation of IBs involves complex interactions between the virus and the host cell, the role of both viral and cellular organelles in this process is also discussed.
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Affiliation(s)
- Zhifei Li
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, LuoJia Hill, Wuhan, 430072, China
| | - Miaomiao Zheng
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, LuoJia Hill, Wuhan, 430072, China
| | - Zhicheng He
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, LuoJia Hill, Wuhan, 430072, China
| | - Yali Qin
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, LuoJia Hill, Wuhan, 430072, China
| | - Mingzhou Chen
- State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, LuoJia Hill, Wuhan, 430072, China
- Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, China
- Hubei Jiangxia Laboratory, Wuhan, 430200, China
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22
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Frontini-López YR, Rivera L, Pocognoni CA, Roldán JS, Colombo MI, Uhart M, Delgui LR. Infectious Bursal Disease Virus Assembly Causes Endoplasmic Reticulum Stress and Lipid Droplet Accumulation. Viruses 2023; 15:1295. [PMID: 37376595 DOI: 10.3390/v15061295] [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: 03/26/2023] [Revised: 05/23/2023] [Accepted: 05/26/2023] [Indexed: 06/29/2023] Open
Abstract
Gumboro illness is caused by the highly contagious immunosuppressive infectious bursal disease virus (IBDV), which affects the poultry industry globally. We have previously shown that IBDV hijacks the endocytic pathway to construct viral replication complexes on endosomes linked to the Golgi complex (GC). Then, analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b, the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), and its substrate, the small GTPase ADP-ribosylation factor 1 (ARF1), for IBDV replication. In the current work, we focused on elucidating the IBDV assembly sites. We show that viral assembly occurs within single-membrane compartments closely associated with endoplasmic reticulum (ER) membranes, though we failed to elucidate the exact nature of the virus-wrapping membranes. Additionally, we show that IBDV infection promotes the stress of the ER, characterized by an accumulation of the chaperone binding protein (BiP) and lipid droplets (LDs) in the host cells. Overall, our results represent further original data showing the interplay between IBDV and the secretory pathway, making a substantial contribution to the field of birnaviruses-host cell interactions.
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Affiliation(s)
- Yesica R Frontini-López
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
| | - Lautaro Rivera
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
| | - Cristian A Pocognoni
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
- Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza 5500, Argentina
| | - Julieta S Roldán
- Instituto de Virología e Innovaciones Tecnológicas, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas, Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hurlingham 1686, Argentina
| | - María I Colombo
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
- Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza 5500, Argentina
| | - Marina Uhart
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
| | - Laura R Delgui
- Instituto de Histología y Embriología de Mendoza (IHEM), Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Mendoza 5500, Argentina
- Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza 5500, Argentina
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23
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Bryant JD, Lee JS, De Almeida A, Jacques J, Chang CH, Fassler W, Quéva C, Lerner L, Kennedy EM. Seneca Valley virus replicons are packaged in trans and have the capacity to overcome the limitations of viral transgene expression. Mol Ther Oncolytics 2023; 28:321-333. [PMID: 36938543 PMCID: PMC10018389 DOI: 10.1016/j.omto.2023.02.005] [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: 11/01/2022] [Accepted: 02/10/2023] [Indexed: 02/18/2023] Open
Abstract
Oncolytic viruses (OVs) promote the anti-tumor immune response as their replication, and the subsequent lysis of tumor cells, triggers the activation of immune-sensing pathways. Arming OVs by expressing transgenes with the potential to promote immune cell recruitment and activation is an attractive strategy to enhance OVs' therapeutic benefit. For picornaviruses, a family of OVs with clinical experience, the expression of a transgene is limited by multiple factors: genome physical packaging limits, high rates of recombination, and viral-mediated inhibition of transgene secretion. Here, we evaluated strategies for arming Seneca Valley virus (SVV) with relevant immunomodulatory transgenes. Specificially in the contex of arming SVV, we evaluated transgene maximum size and stabiltity, transgene secretion, and the impact of transgene inclusion on viral fitness. We find that SVV is not capable of expressing secreted payloads and has a transgene packaging capacity of ∼10% of viral genome size. To enable transgene expression, we developed SVV replicons with greater transgene size capacity and secretion capabilities. SVV replicons can be packaged in trans by virus in co-infected cells to express immunomodulatory transgenes in surrounding cells, thus providing a means to enhance the potential of this therapeutic to augment the anti-tumor immune response.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Edward M. Kennedy
- Oncorus, Inc., Andover, MA 01810, USA
- Corresponding author: Edward M. Kennedy, Oncorus, Inc., 4 Corporate Dr., Andover, MA 01810, USA.
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24
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Jassey A, Wagner MA, Galitska G, Paudel B, Miller K, Jackson WT. Starvation after infection restricts enterovirus D68 replication. Autophagy 2023; 19:112-125. [PMID: 35446171 PMCID: PMC9809931 DOI: 10.1080/15548627.2022.2062888] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Enterovirus D68 (EV-D68) is a respiratory pathogen associated with acute flaccid myelitis, a childhood paralysis disease. No approved vaccine or antiviral treatment exists against EV-D68. Infection with this virus induces the formation of autophagosomes to enhance its replication but blocks the downstream autophagosome- lysosome fusion steps. Here, we examined the impact of autophagy induction through starvation, either before (starvation before infection, SBI) or after (starvation after infection, SAI) EV-D68 infection. We showed that SAI, but not SBI, attenuated EV-D68 replication in multiple cell lines and abrogated the viral-mediated cleavage of host autophagic flux-related proteins. Furthermore, SAI induced autophagic flux during EV-D68 replication and prevented production of virus-induced membranes, which are required for picornavirus replication. Pharmacological inhibition of autophagic flux during SAI did not rescue EV-D68 titers. SAI had the same effect in multiple cell types, and restricted the replication of several medically relevant picornaviruses. Our results highlight the significance of autophagosomes for picornavirus replication and identify SAI as an attractive broad-spectrum anti-picornavirus strategy.Abbreviations: BAF: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; CVB3: coxsackievirus B3; EV-D68: enterovirus D68; hpi: hour post-infection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; NSP2B: nonstructural protein 2B; PV: poliovirus; RES: resveratrol; RV14: rhinovirus 14; SAI: starvation after infection; SBI: starvation before infection; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.
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Affiliation(s)
- Alagie Jassey
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Michael A. Wagner
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Ganna Galitska
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Bimal Paudel
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Katelyn Miller
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA
| | - William T. Jackson
- Department of Microbiology and Immunology and Center for Pathogen Research, University of Maryland School of Medicine, Baltimore, MD, USA,CONTACT William T. Jackson Department of Microbiology and Immunology and Center for Pathogen Research University of Maryland School of Medicine, Baltimore, USA
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25
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Miller K, Wagner MA, Jassey A, Jackson WT. SNAP23 is essential for germination of EV-D68 replication organelles. Virology 2023; 578:117-127. [PMID: 36527930 DOI: 10.1016/j.virol.2022.11.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2022] [Revised: 11/22/2022] [Accepted: 11/22/2022] [Indexed: 12/14/2022]
Abstract
Picornaviruses rearrange host cell membranes to facilitate their own replication. Here we investigate the Qbc SNARE, SNAP23, which is found at the plasma membrane and plays roles in exocytosis. We found that knockdown of SNAP23 expression inhibits virus replication but not release from cells. Knocking down SNAP23 inhibits viral RNA replication and synthesis of structural proteins. Normal cellular levels of SNAP23 are required for an early step in virus production, prior to or at the stage of virus RNA replication. We report that SNAP23 knockdown generates large, electron-light structures, and that infection of cells with these structures does not alter them, and those cells fail to generate viral RNA replication sites. We suggest that SNAP23 may play a role in maintaining membranes and lipids needed for generating virus replication organelles. Further investigation is needed to determine the precise role of this crucial SNARE protein in EV-D68 replication.
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Affiliation(s)
- Katelyn Miller
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W Baltimore St, Baltimore, MD, 21201, USA
| | - Michael A Wagner
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W Baltimore St, Baltimore, MD, 21201, USA
| | - Alagie Jassey
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W Baltimore St, Baltimore, MD, 21201, USA
| | - William T Jackson
- Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W Baltimore St, Baltimore, MD, 21201, USA.
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26
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Dahmane S, Kerviel A, Morado DR, Shankar K, Ahlman B, Lazarou M, Altan-Bonnet N, Carlson LA. Membrane-assisted assembly and selective secretory autophagy of enteroviruses. Nat Commun 2022; 13:5986. [PMID: 36216808 PMCID: PMC9550805 DOI: 10.1038/s41467-022-33483-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Accepted: 09/20/2022] [Indexed: 11/09/2022] Open
Abstract
Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.
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Affiliation(s)
- Selma Dahmane
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.,Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden.,The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden
| | - Adeline Kerviel
- Laboratory of Host-Pathogen Dynamics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
| | - Dustin R Morado
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden
| | - Kasturika Shankar
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.,Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden.,The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden
| | - Björn Ahlman
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.,Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden.,The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden
| | - Michael Lazarou
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia
| | - Nihal Altan-Bonnet
- Laboratory of Host-Pathogen Dynamics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
| | - Lars-Anders Carlson
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden. .,Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden. .,The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
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27
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Moghimi S, Viktorova EG, Gabaglio S, Zimina A, Budnik B, Wynn BG, Sztul E, Belov GA. A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication. PLoS Pathog 2022; 18:e1010906. [PMID: 36306280 PMCID: PMC9645661 DOI: 10.1371/journal.ppat.1010906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Revised: 11/09/2022] [Accepted: 09/30/2022] [Indexed: 11/07/2022] Open
Abstract
As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.
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Affiliation(s)
- Seyedehmahsa Moghimi
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Ekaterina G. Viktorova
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Samuel Gabaglio
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Anna Zimina
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Bogdan Budnik
- Mass Spectrometry and Proteomics Resource Laboratory (MSPRL), FAS Division of Science, Harvard University, Cambridge, Massachusetts, United States of America
| | - Bridge G. Wynn
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - George A. Belov
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
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28
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Fan YM, Zhang YL, Bahreyni A, Luo H, Mohamud Y. Coxsackievirus Protease 2A Targets Host Protease ATG4A to Impair Autophagy. Viruses 2022; 14:v14092026. [PMID: 36146840 PMCID: PMC9502984 DOI: 10.3390/v14092026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2022] [Revised: 09/04/2022] [Accepted: 09/09/2022] [Indexed: 01/18/2023] Open
Abstract
Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to usurp the cellular recycling process of autophagy for pro-viral functions, the precise manner by which this is accomplished remains to be elucidated. In the current manuscript, we sought to address the mechanism by which EVs subvert the autophagy pathway using Coxsackievirus B3 (CVB3) as a model. We showed that CVB3 infection selectively degrades the autophagy cysteine protease ATG4A but not other isoforms. Exogenous expression of an N-terminally Flag-labeled ATG4A demonstrated the emergence of a 43-kDa cleavage fragment following CVB3 infection. Furthermore, bioinformatics analysis coupled with site-directed mutagenesis and in vitro cleavage assays revealed that CVB3 protease 2A cleaves ATG4A before glycine 374. Using a combination of genetic silencing and overexpression studies, we demonstrated a novel pro-viral function for the autophagy protease ATG4A. Additionally, cleavage of ATG4A was associated with a loss of autophagy function of the truncated cleavage fragment. Collectively, our study identified ATG4A as a novel substrate of CVB3 protease, leading to disrupted host cellular function and sheds further light on viral mechanisms of autophagy dysregulation.
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Affiliation(s)
- Yiyun Michelle Fan
- Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
- Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC V6Z 1Y6, Canada
| | - Yizhuo Lyanne Zhang
- Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
- Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC V6Z 1Y6, Canada
| | - Amirhossein Bahreyni
- Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC V6Z 1Y6, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
| | - Honglin Luo
- Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC V6Z 1Y6, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
- Correspondence: (H.L.); (Y.M.)
| | - Yasir Mohamud
- Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, BC V6Z 1Y6, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
- Correspondence: (H.L.); (Y.M.)
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29
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Neufeldt CJ, Cortese M. Membrane architects: how positive-strand RNA viruses restructure the cell. J Gen Virol 2022; 103. [PMID: 35976091 DOI: 10.1099/jgv.0.001773] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Virus infection is a process that requires combined contributions from both virus and host factors. For this process to be efficient within the crowded host environment, viruses have evolved ways to manipulate and reorganize host structures to produce cellular microenvironments. Positive-strand RNA virus replication and assembly occurs in association with cytoplasmic membranes, causing a reorganization of these membranes to create microenvironments that support viral processes. Similarities between virus-induced membrane domains and cellular organelles have led to the description of these structures as virus replication organelles (vRO). Electron microscopy analysis of vROs in positive-strand RNA virus infected cells has revealed surprising morphological similarities between genetically diverse virus species. For all positive-strand RNA viruses, vROs can be categorized into two groups: those that make invaginations into the cellular membranes (In-vRO), and those that cause the production of protrusions from cellular membranes (Pr-vRO), most often in the form of double membrane vesicles (DMVs). In this review, we will discuss the current knowledge on the structure and biogenesis of these two different vRO classes as well as comparing morphology and function of vROs between various positive-strand RNA viruses. Finally, we will discuss recent studies describing pharmaceutical intervention in vRO formation as an avenue to control virus infection.
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Affiliation(s)
- Christopher John Neufeldt
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Mirko Cortese
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
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30
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Roingeard P, Eymieux S, Burlaud-Gaillard J, Hourioux C, Patient R, Blanchard E. The double-membrane vesicle (DMV): a virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses. Cell Mol Life Sci 2022; 79:425. [PMID: 35841484 PMCID: PMC9287701 DOI: 10.1007/s00018-022-04469-x] [Citation(s) in RCA: 52] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 05/16/2022] [Accepted: 06/30/2022] [Indexed: 12/18/2022]
Abstract
Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.
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Affiliation(s)
- Philippe Roingeard
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France. .,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France.
| | - Sébastien Eymieux
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France.,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France
| | - Julien Burlaud-Gaillard
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France.,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France
| | - Christophe Hourioux
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France.,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France
| | - Romuald Patient
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France.,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France
| | - Emmanuelle Blanchard
- INSERM U1259, Faculté de Médecine, Université François Rabelais de Tours and CHRU de Tours, 10 boulevard Tonnellé, 37032, Tours Cedex, France.,Plate-Forme IBiSA de Microscopie Electronique, Université de Tours and CHRU de Tours, Tours, France
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Chen D, Zhao YG, Zhang H. Endomembrane remodeling in SARS-CoV-2 infection. CELL INSIGHT 2022; 1:100031. [PMID: 37193051 PMCID: PMC9112566 DOI: 10.1016/j.cellin.2022.100031] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Revised: 05/09/2022] [Accepted: 05/09/2022] [Indexed: 12/18/2022]
Abstract
During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the viral proteins intimately interact with host factors to remodel the endomembrane system at various steps of the viral lifecycle. The entry of SARS-CoV-2 can be mediated by endocytosis-mediated internalization. Virus-containing endosomes then fuse with lysosomes, in which the viral S protein is cleaved to trigger membrane fusion. Double-membrane vesicles generated from the ER serve as platforms for viral replication and transcription. Virions are assembled at the ER-Golgi intermediate compartment and released through the secretory pathway and/or lysosome-mediated exocytosis. In this review, we will focus on how SARS-CoV-2 viral proteins collaborate with host factors to remodel the endomembrane system for viral entry, replication, assembly and egress. We will also describe how viral proteins hijack the host cell surveillance system-the autophagic degradation pathway-to evade destruction and benefit virus production. Finally, potential antiviral therapies targeting the host cell endomembrane system will be discussed.
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Affiliation(s)
- Di Chen
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Yan G. Zhao
- Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Hong Zhang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China
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32
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Gallo GL, López N, Loureiro ME. The Virus–Host Interplay in Junín Mammarenavirus Infection. Viruses 2022; 14:v14061134. [PMID: 35746604 PMCID: PMC9228484 DOI: 10.3390/v14061134] [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: 03/02/2022] [Revised: 04/26/2022] [Accepted: 04/28/2022] [Indexed: 02/06/2023] Open
Abstract
Junín virus (JUNV) belongs to the Arenaviridae family and is the causative agent of Argentine hemorrhagic fever (AHF), a severe human disease endemic to agricultural areas in Argentina. At this moment, there are no effective antiviral therapeutics to battle pathogenic arenaviruses. Cumulative reports from recent years have widely provided information on cellular factors playing key roles during JUNV infection. In this review, we summarize research on host molecular determinants that intervene in the different stages of the viral life cycle: viral entry, replication, assembly and budding. Alongside, we describe JUNV tight interplay with the innate immune system. We also review the development of different reverse genetics systems and their use as tools to study JUNV biology and its close teamwork with the host. Elucidating relevant interactions of the virus with the host cell machinery is highly necessary to better understand the mechanistic basis beyond virus multiplication, disease pathogenesis and viral subversion of the immune response. Altogether, this knowledge becomes essential for identifying potential targets for the rational design of novel antiviral treatments to combat JUNV as well as other pathogenic arenaviruses.
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33
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Nishikiori M, den Boon JA, Unchwaniwala N, Ahlquist P. Crowning Touches in Positive-Strand RNA Virus Genome Replication Complex Structure and Function. Annu Rev Virol 2022; 9:193-212. [PMID: 35610038 DOI: 10.1146/annurev-virology-092920-021307] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Masaki Nishikiori
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Johan A den Boon
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Nuruddin Unchwaniwala
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Current affiliation: Assembly Biosciences, Inc., South San Francisco, California, USA
| | - Paul Ahlquist
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
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34
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Lee HW, Jiang YF, Chang HW, Cheng IC. Foot-and-Mouth Disease Virus 3A Hijacks Sar1 and Sec12 for ER Remodeling in a COPII-Independent Manner. Viruses 2022; 14:v14040839. [PMID: 35458569 PMCID: PMC9028839 DOI: 10.3390/v14040839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 04/14/2022] [Accepted: 04/16/2022] [Indexed: 02/01/2023] Open
Abstract
Positive-stranded RNA viruses modify host organelles to form replication organelles (ROs) for their own replication. The enteroviral 3A protein has been demonstrated to be highly associated with the COPI pathway, in which factors operate on the ER-to-Golgi intermediate and the Golgi. However, Sar1, a COPII factor exerting coordinated action at endoplasmic reticulum (ER) exit sites rather than COPI factors, is required for the replication of foot-and-mouth disease virus (FMDV). Therefore, further understanding regarding FMDV 3A could be key to explaining the differences and to understanding FMDV’s RO formation. In this study, FMDV 3A was confirmed as a peripheral membrane protein capable of modifying the ER into vesicle-like structures, which were neither COPII vesicles nor autophagosomes. When the C-terminus of 3A was truncated, it was located at the ER without vesicular modification. This change was revealed using mGFP and APEX2 fusion constructs, and observed by fluorescence microscopy and electron tomography, respectively. For the other 3A truncation, the minimal region for modification was aa 42–92. Furthermore, we found that the remodeling was related to two COPII factors, Sar1 and Sec12; both interacted with 3A, but their binding domains on 3A were different. Finally, we hypothesized that the N-terminus of 3A would interact with Sar1, as its C-terminus simultaneously interacted with Sec12, which could possibly enhance Sar1 activation. On the ER membrane, active Sar1 interacted with regions of aa 42–59 and aa 76–92 from 3A for vesicle formation. This mechanism was distinct from the traditional COPII pathway and could be critical for FMDV RO formation.
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Affiliation(s)
- Heng-Wei Lee
- School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan; (H.-W.L.); (Y.-F.J.); (H.-W.C.)
| | - Yi-Fan Jiang
- School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan; (H.-W.L.); (Y.-F.J.); (H.-W.C.)
- Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan
| | - Hui-Wen Chang
- School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan; (H.-W.L.); (Y.-F.J.); (H.-W.C.)
- Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan
| | - Ivan-Chen Cheng
- School of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan; (H.-W.L.); (Y.-F.J.); (H.-W.C.)
- Correspondence:
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35
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Schneider WM, Hoffmann HH. Flavivirus-host interactions: an expanding network of proviral and antiviral factors. Curr Opin Virol 2022; 52:71-77. [PMID: 34896863 PMCID: PMC8655497 DOI: 10.1016/j.coviro.2021.11.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2021] [Accepted: 11/15/2021] [Indexed: 02/07/2023]
Abstract
Flaviviruses are zoonotic pathogens transmitted by the bite of infected mosquitos and ticks and represent a constant burden to human health. Here we review recent literature aimed at uncovering how flaviviruses interact with the cells that they infect. A better understanding of these interactions may ultimately lead to novel therapeutic targets. We highlight several studies that employed low-biased methods to discover new protein-protein, protein-RNA, and genetic interactions, and spotlight recent work characterizing the host protein, TMEM41B, which has been shown to be critical for infection by diverse flaviviruses and coronaviruses.
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Affiliation(s)
- William M Schneider
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.
| | - Hans-Heinrich Hoffmann
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.
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36
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Murer L, Volle R, Andriasyan V, Petkidis A, Gomez-Gonzalez A, Yang L, Meili N, Suomalainen M, Bauer M, Policarpo Sequeira D, Olszewski D, Georgi F, Kuttler F, Turcatti G, Greber UF. Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern. CURRENT RESEARCH IN VIROLOGICAL SCIENCE 2022; 3:100019. [PMID: 35072124 PMCID: PMC8760634 DOI: 10.1016/j.crviro.2022.100019] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 01/04/2022] [Accepted: 01/04/2022] [Indexed: 01/18/2023]
Abstract
Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.
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Affiliation(s)
- Luca Murer
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Romain Volle
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Vardan Andriasyan
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Anthony Petkidis
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Alfonso Gomez-Gonzalez
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Liliane Yang
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Nicole Meili
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Maarit Suomalainen
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Michael Bauer
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Daniela Policarpo Sequeira
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Dominik Olszewski
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Fanny Georgi
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Fabien Kuttler
- Biomolecular Screening Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 15, 1015, Lausanne, Switzerland
| | - Gerardo Turcatti
- Biomolecular Screening Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 15, 1015, Lausanne, Switzerland
| | - Urs F Greber
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
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37
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Gagliardi TB, Goldstein ME, Song D, Gray KM, Jung JW, Ignacio MA, Stroka KM, Duncan GA, Scull MA. Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium. PLoS Pathog 2022; 18:e1010159. [PMID: 34995322 PMCID: PMC8741012 DOI: 10.1371/journal.ppat.1010159] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Accepted: 11/29/2021] [Indexed: 12/21/2022] Open
Abstract
The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo. Rhinovirus C has a global distribution and significant clinical impact–especially in those with underlying lung disease. Although RV-C is genetically, structurally, and biologically distinct from RV-A and -B viruses, our understanding of the RV-C life cycle has been largely inferred from these and other related viruses. Here, we performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium. Our single-cell, microscopy-based approach revealed that–unlike other RVs–the endoplasmic reticulum is the primary site for RV-C15 replication. RV-C15 replication also stimulated STING expression, which was proviral, and triggered dramatic changes in cellular organization, including altered virus receptor distribution, fragmented Golgi stacks, and the induction of incomplete autophagy. Additionally, we observed a loss of epithelial barrier function and a decrease in mucociliary clearance, a major defense mechanism in the lung, during RV-C15 infection. Together, these data reveal novel insight into RV-C15 replication dynamics and resulting cytopathic effects in the primary target cells for infection, thereby furthering our understanding of the pathogenesis of RV-C. Our work highlights similar, as well as unique, aspects of RV-C15 replication compared to related pathogens, which will help guide future studies on the molecular mechanisms of RV-C infection.
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Affiliation(s)
- Talita B. Gagliardi
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America
| | - Monty E. Goldstein
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America
| | - Daniel Song
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States of America
| | - Kelsey M. Gray
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States of America
| | - Jae W. Jung
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States of America
| | - Maxinne A. Ignacio
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America
| | - Kimberly M. Stroka
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States of America
- Biophysics Program, University of Maryland, College Park, Maryland, United States of America
- Center for Stem Cell Biology and Regenerative Medicine, University of Maryland, Baltimore, Maryland, United States of America
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland, United States of America
| | - Gregg A. Duncan
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States of America
| | - Margaret A. Scull
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, United States of America
- * E-mail:
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38
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Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites. Viruses 2021; 13:v13122540. [PMID: 34960809 PMCID: PMC8708976 DOI: 10.3390/v13122540] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 12/01/2021] [Accepted: 12/15/2021] [Indexed: 12/22/2022] Open
Abstract
Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.
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39
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Twu WI, Lee JY, Kim H, Prasad V, Cerikan B, Haselmann U, Tabata K, Bartenschlager R. Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation. Cell Rep 2021; 37:110049. [PMID: 34788596 PMCID: PMC8577994 DOI: 10.1016/j.celrep.2021.110049] [Citation(s) in RCA: 78] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 09/02/2021] [Accepted: 11/02/2021] [Indexed: 02/09/2023] Open
Abstract
Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.
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Affiliation(s)
- Woan-Ing Twu
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Heeyoung Kim
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; Center for Infection Research (DZIF), Partner Site Heidelberg, 69120 Heidelberg, Germany
| | - Vibhu Prasad
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Berati Cerikan
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; Center for Infection Research (DZIF), Partner Site Heidelberg, 69120 Heidelberg, Germany
| | - Keisuke Tabata
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, 69120 Heidelberg, Germany; Center for Infection Research (DZIF), Partner Site Heidelberg, 69120 Heidelberg, Germany; Division Virus-Associated Carcinogenesis, German Cancer Research Center, 69120 Heidelberg, Germany.
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40
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High-Order Epistasis and Functional Coupling of Infection Steps Drive Virus Evolution toward Independence from a Host Pathway. Microbiol Spectr 2021; 9:e0080021. [PMID: 34468191 PMCID: PMC8557862 DOI: 10.1128/spectrum.00800-21] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The phosphatidylinositol-4 kinase IIIβ (PI4KB)/oxysterol-binding protein (OSBP) family I pathway serves as an essential host pathway for the formation of viral replication complex for viral plus-strand RNA synthesis; however, poliovirus (PV) could evolve toward substantial independence from this host pathway with four mutations. Recessive epistasis of the two mutations (3A-R54W and 2B-F17L) is essential for viral RNA replication. Quantitative analysis of effects of the other two mutations (2B-Q20H and 2C-M187V) on each step of infection reveals functional couplings between viral replication, growth, and spread conferred by the 2B-Q20H mutation, while no enhancing effect was conferred by the 2C-M187V mutation. The effects of the 2B-Q20H mutation occur only via another recessive epistasis between the 3A-R54W/2B-F17L mutations. These mutations confer enhanced replication in PI4KB/OSBP-independent infection concomitantly with an increased ratio of viral plus-strand RNA to the minus-strand RNA. This work reveals the essential roles of the functional coupling and high-order, multi-tiered recessive epistasis in viral evolution toward independence from an obligatory host pathway. IMPORTANCE Each virus has a different strategy for its replication, which requires different host factors. Enterovirus, a model RNA virus, requires host factors PI4KB and OSBP, which form an obligatory functional axis to support viral replication. In an experimental evolution system in vitro, virus mutants that do not depend on these host factors could arise only with four mutations. The two mutations (3A-R54W and 2B-F17L) are required for the replication but are not sufficient to support efficient infection. Another mutation (2B-Q20H) is essential for efficient spread of the virus. The order of introduction of the mutations in the viral genome is essential (known as “epistasis”), and functional couplings of infection steps (i.e., viral replication, growth, and spread) have substantial roles to show the effects of the 2B-Q20H mutation. These observations would provide novel insights into an evolutionary pathway of the virus to require host factors for infection.
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Abstract
Viruses have evolved precise mechanisms for using the cellular physiological pathways for their perpetuation. These virus-driven biochemical events must be separated in space and time from those of the host cell. In recent years, granular structures, known for over a century for rabies virus, were shown to host viral gene function and were named using terms such as viroplasms, replication sites, inclusion bodies, or viral factories (VFs). More recently, these VFs were shown to be liquid-like, sharing properties with membrane-less organelles driven by liquid–liquid phase separation (LLPS) in a process widely referred to as biomolecular condensation. Some of the best described examples of these structures come from negative stranded RNA viruses, where micrometer size VFs are formed toward the end of the infectious cycle. We here discuss some basic principles of LLPS in connection with several examples of VFs and propose a view, which integrates viral replication mechanisms with the biochemistry underlying liquid-like organelles. In this view, viral protein and RNA components gradually accumulate up to a critical point during infection where phase separation is triggered. This yields an increase in transcription that leads in turn to increased translation and a consequent growth of initially formed condensates. According to chemical principles behind phase separation, an increase in the concentration of components increases the size of the condensate. A positive feedback cycle would thus generate in which crucial components, in particular nucleoproteins and viral polymerases, reach their highest levels required for genome replication. Progress in understanding viral biomolecular condensation leads to exploration of novel therapeutics. Furthermore, it provides insights into the fundamentals of phase separation in the regulation of cellular gene function given that virus replication and transcription, in particular those requiring host polymerases, are governed by the same biochemical principles.
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Unchwaniwala N, Zhan H, den Boon JA, Ahlquist P. Cryo-electron microscopy of nodavirus RNA replication organelles illuminates positive-strand RNA virus genome replication. Curr Opin Virol 2021; 51:74-79. [PMID: 34601307 PMCID: PMC8504867 DOI: 10.1016/j.coviro.2021.09.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Revised: 09/10/2021] [Accepted: 09/13/2021] [Indexed: 11/18/2022]
Abstract
The nodavirus flock house virus recently provided a well-characterized model for the first cryo-electron microscope tomography of membrane-bound, positive-strand RNA ((+)RNA) virus genome replication complexes (RCs). The resulting first views of RC organization and complementary biochemical results showed that the viral RNA replication vesicle is tightly packed with the dsRNA genomic RNA replication intermediate, and that (+)ssRNA replication products are released through the vesicle neck to the cytosol through a 12-fold symmetric ring or crown of multi-functional viral RNA replication proteins, which likely also contribute to viral RNA synthesis. Subsequent studies identified similar crown-like RNA replication protein complexes in alphavirus and coronavirus RCs, indicating related mechanisms across highly divergent (+)RNA viruses. As outlined in this review, these results have significant implications for viral function, evolution and control.
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Affiliation(s)
- Nuruddin Unchwaniwala
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, WI, 53715, United States; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706, United States; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, 53705, United States
| | - Hong Zhan
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, WI, 53715, United States; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706, United States; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, 53705, United States
| | - Johan A den Boon
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, WI, 53715, United States; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706, United States; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, 53705, United States
| | - Paul Ahlquist
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, WI, 53715, United States; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706, United States; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, 53705, United States
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Hassan Z, Kumar ND, Reggiori F, Khan G. How Viruses Hijack and Modify the Secretory Transport Pathway. Cells 2021; 10:2535. [PMID: 34685515 PMCID: PMC8534161 DOI: 10.3390/cells10102535] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 08/28/2021] [Accepted: 09/06/2021] [Indexed: 12/23/2022] Open
Abstract
Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae,Poxviridae, Parvoviridae and Herpesviridae families.
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Affiliation(s)
- Zubaida Hassan
- Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain P.O. Box 17666, United Arab Emirates;
- Department of Microbiology, School of Life Sciences, Modibbo Adama University, Yola PMB 2076, Nigeria
| | - Nilima Dinesh Kumar
- Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands; (N.D.K.); (F.R.)
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands
| | - Fulvio Reggiori
- Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands; (N.D.K.); (F.R.)
| | - Gulfaraz Khan
- Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain P.O. Box 17666, United Arab Emirates;
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Nguyen-Dinh V, Herker E. Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses. Cells 2021; 10:cells10092407. [PMID: 34572055 PMCID: PMC8464962 DOI: 10.3390/cells10092407] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Revised: 08/12/2021] [Accepted: 09/02/2021] [Indexed: 11/25/2022] Open
Abstract
All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell’s surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.
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Elmasri Z, Nasal BL, Jose J. Alphavirus-Induced Membrane Rearrangements during Replication, Assembly, and Budding. Pathogens 2021; 10:984. [PMID: 34451448 PMCID: PMC8399458 DOI: 10.3390/pathogens10080984] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 07/30/2021] [Accepted: 08/02/2021] [Indexed: 01/01/2023] Open
Abstract
Alphaviruses are arthropod-borne viruses mainly transmitted by hematophagous insects that cause moderate to fatal disease in humans and other animals. Currently, there are no approved vaccines or antivirals to mitigate alphavirus infections. In this review, we summarize the current knowledge of alphavirus-induced structures and their functions in infected cells. Throughout their lifecycle, alphaviruses induce several structural modifications, including replication spherules, type I and type II cytopathic vacuoles, and filopodial extensions. Type I cytopathic vacuoles are replication-induced structures containing replication spherules that are sites of RNA replication on the endosomal and lysosomal limiting membrane. Type II cytopathic vacuoles are assembly induced structures that originate from the Golgi apparatus. Filopodial extensions are induced at the plasma membrane and are involved in budding and cell-to-cell transport of virions. This review provides an overview of the viral and host factors involved in the biogenesis and function of these virus-induced structures. Understanding virus-host interactions in infected cells will lead to the identification of new targets for antiviral discovery.
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Affiliation(s)
- Zeinab Elmasri
- Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA;
- Department of Biochemistry & Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802, USA;
| | - Benjamin L. Nasal
- Department of Biochemistry & Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802, USA;
| | - Joyce Jose
- Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA;
- Department of Biochemistry & Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802, USA;
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Kril V, Aïqui-Reboul-Paviet O, Briant L, Amara A. New Insights into Chikungunya Virus Infection and Pathogenesis. Annu Rev Virol 2021; 8:327-347. [PMID: 34255544 DOI: 10.1146/annurev-virology-091919-102021] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for major outbreaks of disease since 2004 in the Indian Ocean islands, South east Asia, and the Americas. CHIKV causes debilitating musculoskeletal disorders in humans that are characterized by fever, rash, polyarthralgia, and myalgia. The disease is often self-limiting and nonlethal; however, some patients experience atypical or severe clinical manifestations, as well as a chronic rheumatic syndrome. Unfortunately, no efficient antivirals against CHIKV infection are available so far, highlighting the importance of deepening our knowledge of CHIKV host cell interactions and viral replication strategies. In this review, we discuss recent breakthroughs in the molecular mechanisms that regulate CHIKV infection and lay down the foundations to understand viral pathogenesis. We describe the role of the recently identified host factors co-opted by the virus for infection and pathogenesis, and emphasize the importance of CHIKV nonstructural proteins in both replication complex assembly and host immune response evasion. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Vasiliya Kril
- Biology of Emerging Virus Team, INSERM U944, CNRS UMR 7212, Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, 75010 Paris, France;
| | - Olivier Aïqui-Reboul-Paviet
- RNA Viruses and Metabolism Team, CNRS UMR 9004, Institut de Recherche en Infectiologie de Montpellier, University of Montpellier, 34293 Montpellier, France;
| | - Laurence Briant
- RNA Viruses and Metabolism Team, CNRS UMR 9004, Institut de Recherche en Infectiologie de Montpellier, University of Montpellier, 34293 Montpellier, France;
| | - Ali Amara
- Biology of Emerging Virus Team, INSERM U944, CNRS UMR 7212, Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, 75010 Paris, France;
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Teo QW, van Leur SW, Sanyal S. Escaping the Lion's Den: redirecting autophagy for unconventional release and spread of viruses. FEBS J 2021; 288:3913-3927. [PMID: 33044763 DOI: 10.1111/febs.15590] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 10/01/2020] [Accepted: 10/08/2020] [Indexed: 12/30/2022]
Abstract
Autophagy is an evolutionarily conserved process, designed to maintain cellular homeostasis during a range of internal and external stimuli. Conventionally, autophagy is known for coordinated degradation and recycling of intracellular components and removal of cytosolic pathogens. More recently, several lines of evidence have indicated an unconventional, nondegradative role of autophagy for secretion of cargo that lacks a signal peptide. This process referred to as secretory autophagy has also been implicated in the infection cycle of several virus species. This review focuses on the current evidence available on the nondegradative features of autophagy, emphasizing its potential role and unresolved questions in the release and spread of (-) and (+) RNA viruses.
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Affiliation(s)
- Qi Wen Teo
- HKU-Pasteur Research Pole, School of Public Health, University of Hong Kong, Hong Kong
| | - Sophie Wilhelmina van Leur
- HKU-Pasteur Research Pole, School of Public Health, University of Hong Kong, Hong Kong.,Sir William Dunn School of Pathology, University of Oxford, UK
| | - Sumana Sanyal
- HKU-Pasteur Research Pole, School of Public Health, University of Hong Kong, Hong Kong.,Sir William Dunn School of Pathology, University of Oxford, UK
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Domanska A, Guryanov S, Butcher SJ. A comparative analysis of parechovirus protein structures with other picornaviruses. Open Biol 2021; 11:210008. [PMID: 34315275 PMCID: PMC8316810 DOI: 10.1098/rsob.210008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Accepted: 07/01/2021] [Indexed: 12/26/2022] Open
Abstract
Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.
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Affiliation(s)
- Aušra Domanska
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
| | - Sergey Guryanov
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
| | - Sarah J. Butcher
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
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Kellermann M, Scharte F, Hensel M. Manipulation of Host Cell Organelles by Intracellular Pathogens. Int J Mol Sci 2021; 22:ijms22126484. [PMID: 34204285 PMCID: PMC8235465 DOI: 10.3390/ijms22126484] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2021] [Revised: 06/03/2021] [Accepted: 06/04/2021] [Indexed: 12/13/2022] Open
Abstract
Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.
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Affiliation(s)
- Malte Kellermann
- Abt. Mikrobiologie, Fachbereich Biologie/Chemie, Barbarastr 11, Universität Osnabrück, 49076 Osnabrück, Germany; (M.K.); (F.S.)
| | - Felix Scharte
- Abt. Mikrobiologie, Fachbereich Biologie/Chemie, Barbarastr 11, Universität Osnabrück, 49076 Osnabrück, Germany; (M.K.); (F.S.)
| | - Michael Hensel
- Abt. Mikrobiologie, Fachbereich Biologie/Chemie, Barbarastr 11, Universität Osnabrück, 49076 Osnabrück, Germany; (M.K.); (F.S.)
- CellNanOs–Center of Cellular Nanoanalytics Osnabrück, Universität Osnabrück, Barbarastr 11, 49076 Osnabrück, Germany
- Correspondence: ; Tel.: +49-(0)-541-969-3940
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50
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Wong LH, Edgar JR, Martello A, Ferguson BJ, Eden ER. Exploiting Connections for Viral Replication. Front Cell Dev Biol 2021; 9:640456. [PMID: 33816489 PMCID: PMC8012536 DOI: 10.3389/fcell.2021.640456] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Accepted: 02/01/2021] [Indexed: 12/16/2022] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2-infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.
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Affiliation(s)
| | - James R. Edgar
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | | | - Brian J. Ferguson
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Emily R. Eden
- UCL Institute of Ophthalmology, London, United Kingdom
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