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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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Huntošová V, Benziane A, Zauška L, Ambro L, Olejárová S, Joniová J, Hlávková N, Wagnières G, Zelenková G, Diko P, Bednarčík J, Zákány F, Kovács T, Sedlák E, Vámosi G, Almáši M. The potential of metal-organic framework MIL-101(Al)-NH 2 in the forefront of antiviral protection of cells via interaction with SARS-CoV-2 spike RBD protein and their antibacterial action mediated with hypericin and photodynamic treatment. J Colloid Interface Sci 2025; 691:137454. [PMID: 40168900 DOI: 10.1016/j.jcis.2025.137454] [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: 01/27/2025] [Revised: 03/23/2025] [Accepted: 03/26/2025] [Indexed: 04/03/2025]
Abstract
The global pandemic of SARS-CoV-2 has highlighted the necessity for innovative therapeutic solutions. This research presents a new formulation utilising the metal-organic framework MIL-101(Al)-NH2, which is loaded with hypericin, aimed at addressing viral and bacterial challenges. Hypericin, recognised for its antiviral and antibacterial efficacy, was encapsulated to mitigate its hydrophobicity, improve bioavailability, and utilise its photodynamic characteristics. The MIL-101(Al)-NH2 Hyp complex was synthesised, characterised, and evaluated for its biological applications for the first time. The main objective of this study was to demonstrate the multimodal potential of such a construct, in particular the effect on SARS-CoV-2 protein levels and its interaction with cells. Both in vitro and in vivo experiments demonstrated the effective transport of hypericin to cells that express ACE2 receptors, thereby mimicking mechanisms of viral entry. In addition, hypericin found in the mitochondria showed selective phototoxicity when activated by light, leading to a decrease in the metabolic activity of glioblastoma cells. Importantly, the complex also showed antibacterial efficacy by selectively targeting Gram-positive Staphylococcus epidermidis compared to Gram-negative Escherichia coli under photodynamic therapy (PDT) conditions. To our knowledge, this study was the first to demonstrate the interaction between hypericin, MIL-101(Al)-NH2 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which inhibits cellular uptake and colocalises with ACE2-expressing cells. Therefore, the dual functionality of the complex - targeting the viral RBD and the antibacterial effect via PDT - emphasises its potential to mitigate complications of viral infections, such as secondary bacterial infections. In summary, these results suggest that MIL-101(Al)-NH2 Hyp is a promising multifunctional therapeutic agent for antiviral and antibacterial applications, potentially contributing to the improvement of COVID-19 treatment protocols and the treatment of co-infections.
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Affiliation(s)
- Veronika Huntošová
- Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic; Institute of Animal Biochemistry and Genetics, Centre of Biosciences, Slovak Academy of Sciences, Dúbravská cesta 9, SK-840 05 Bratislava, Slovak Republic.
| | - Anass Benziane
- Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Doctoral School of Molecular Medicine, Egyetem tér 1, H-4032 Debrecen, Hungary
| | - Luboš Zauška
- Department of Inorganic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, SK-041 54 Košice, Slovak Republic; BovaChem s.r.o, Laboratory-1, Kirejevská 22, SK-979 01 Rimavská Sobota, Slovak Republic
| | - Luboš Ambro
- Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic
| | - Soňa Olejárová
- Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic; Department of Biophysics, Faculty of Science, P. J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic
| | - Jaroslava Joniová
- Laboratory for Functional and Metabolic Imaging, Institute of Physics, Swiss Federal Institute of Technology in Lausanne (EPFL), Station 3, Building PH, CH-1015 Lausanne, Switzerland
| | - Nina Hlávková
- Department of Inorganic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, SK-041 54 Košice, Slovak Republic
| | - Georges Wagnières
- Laboratory for Functional and Metabolic Imaging, Institute of Physics, Swiss Federal Institute of Technology in Lausanne (EPFL), Station 3, Building PH, CH-1015 Lausanne, Switzerland
| | - Gabriela Zelenková
- Department of Chemistry, Faculty of Science, University of Ostrava, 30. Dubna 22, CZ-702 00 Ostrava, Czech Republic
| | - Pavel Diko
- Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, SK-040 01 Košice, Slovak Republic
| | - Jozef Bednarčík
- Depart of Condensed Matter Physics, Faculty of Science, P. J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic
| | - Florina Zákány
- Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Doctoral School of Molecular Medicine, Egyetem tér 1, H-4032 Debrecen, Hungary
| | - Tamás Kovács
- Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Doctoral School of Molecular Medicine, Egyetem tér 1, H-4032 Debrecen, Hungary
| | - Erik Sedlák
- Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University in Košice, Jesenná 5, SK-041 54 Košice, Slovak Republic; Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, SK-041 54 Košice, Slovak Republic
| | - György Vámosi
- Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Doctoral School of Molecular Medicine, Egyetem tér 1, H-4032 Debrecen, Hungary.
| | - Miroslav Almáši
- Department of Inorganic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Moyzesova 11, SK-041 54 Košice, Slovak Republic.
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Wang K, Wang HQ, Yang G, Wu S, Yan HY, Wu MY, Li YH, Jiang JD. Carrimycin exhibited broad spectrum inhibitory activities against coronaviruses replication through down-regulating host factor TMEM41B. Acta Pharmacol Sin 2025:10.1038/s41401-025-01577-9. [PMID: 40374896 DOI: 10.1038/s41401-025-01577-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/22/2024] [Accepted: 04/27/2025] [Indexed: 05/18/2025]
Abstract
We previously reported that carrimycin could inhibit pan-coronavirus including HCoV-229E, HCoV-OC43 and SARS-CoV-2. We found that carrimycin targeted the post-entry replicative events in coronavirus infection. Carrimycin could impede the viral protein translation switch from ORF1a to ORF1b by targeting programmed -1 ribosomal frameshifting (-1PRF). Carrimycin could also inhibit the newly synthesized (nascent) viral RNA. In this study we investigated whether carrimycin also inhibited the newly emerged SARS-CoV-2 variants. We showed that carrimycin (1.25-10 µM) dose-dependently inhibited both viral RNA and protein levels in Vero E6 cells. We further demonstrated that carrimycin disrupted the formation of SARS-CoV-2 double membrane vesicles (DMVs), and identified the host transmembrane protein B (TMEM41B) as the key factor involved in this process. Overexpression of TMEM41B increased viral protein levels and mRNA levels, whereas TMEM41B knockdown reduced viral replication including HCoV-229E, HCoV-OC43 and SARS-CoV-2. Moreover, overexpression of TMEM41B partially reversed the inhibitory effect of carrimycin, suggesting that carrimycin indeed exerted antiviral effects through regulation of TMEM41B. We revealed that carrimycin directly bound to TMEM41B and induced its K48 ubiquitination degradation, thereby inhibiting viral replication. These results expand the understanding of carrimycin's antiviral mechanisms, particularly its antiviral activity, and enrich our knowledge about the role of host factors in regulating viral replication.
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Affiliation(s)
- Kun Wang
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Hui-Qiang Wang
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Ge Yang
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Shuo Wu
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Hai-Yan Yan
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Meng-Yuan Wu
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
| | - Yu-Huan Li
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
| | - Jian-Dong Jiang
- CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
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Chen Y, Klute S, Sparrer KMJ, Serra-Moreno R. RAB5 is a host dependency factor for the generation of SARS-CoV-2 replication organelles. mBio 2025; 16:e0331424. [PMID: 40167317 PMCID: PMC12077180 DOI: 10.1128/mbio.03314-24] [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: 10/23/2024] [Accepted: 03/03/2025] [Indexed: 04/02/2025] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat due to the emergence of variants with increased transmissibility and enhanced escape from immune responses. Like other coronaviruses before, SARS-CoV-2 likely emerged after its transmission from bats. The successful propagation of SARS-CoV-2 in humans might have been facilitated by usurping evolutionarily conserved cellular factors to execute crucial steps in its life cycle, such as the generation of replication organelles-membrane structures where coronaviruses assemble their replication-transcription complex. In this study, we found that RAB5, which is highly conserved across mammals, is a critical host dependency factor for the replication of the SARS-CoV-2 genome. Our results also suggest that SARS-CoV-2 uses RAB5+ membranes to build replication organelles with the aid of COPB1, a component of the COP-I complex, and that the virus protein NSP6 participates in this process. Hence, targeting NSP6 represents a promising approach to interfere with SARS-CoV-2 RNA synthesis and halt its propagation.IMPORTANCEIn this study, we sought to identify the host dependency factors that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for the generation of replication organelles: cellular membranous structures that SARS-CoV-2 builds in order to support the replication and transcription of its genome. We uncovered that RAB5 is an important dependency factor for SARS-CoV-2 replication and the generation of replication organelles, and that the viral protein NSP6 participates in this process. Hence, NSP6 represents a promising target to halt SARS-CoV-2 replication.
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Affiliation(s)
- Yuexuan Chen
- Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA
| | - Susanne Klute
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Konstantin Maria Johannes Sparrer
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
- German Center for Neurodegenerative Diseases (DZNE), Ulm, Germany
| | - Ruth Serra-Moreno
- Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA
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Furusawa Y, Iwatsuki-Horimoto K, Yamayoshi S, Kawaoka Y. The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14. J Virol 2025:e0065625. [PMID: 40358207 DOI: 10.1128/jvi.00656-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2025] [Accepted: 04/21/2025] [Indexed: 05/15/2025] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants emerged at the end of 2021, and their subvariants are still circulating worldwide. While changes in the S protein of these variants have been extensively studied, the roles of amino acid substitutions in non-structural proteins have not been fully revealed. In this study, we found that SARS-CoV-2 bearing the NSP6-L260F substitution emerged repeatedly when we generated several SARS-CoV-2 variants by reverse genetics or when we passaged SARS-CoV-2 isolated from clinical samples and that it was selected under cell culture conditions. Although this substitution has been detected in BQ.1.1 and XBB.1.16 that circulated in nature, its effect on viral properties is unclear. Here, we generated SARS-CoV-2 with or without the NSP6-L260F by reverse genetics and found that NSP6-L260F promotes virus replication in vitro and in vivo by increasing viral polymerase activity and enhancing virus pathogenicity in hamsters. We also identified disadvantageous substitutions, NSP13-M233I and NSP14-D222Y, that reduced BQ.1.1 and XBB.1.16 replication, respectively. These adverse effects were compensated for by NSP6-L260F. Our findings suggest the importance of NSP6-L260F for virus replication and pathogenicity and reveal part of the evolutionary process of Omicron variants.IMPORTANCEAlthough the properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants continue to change through the acquisition of various amino acid substitutions, the roles of the amino acid substitutions in the non-structural proteins have not been fully explored. In this study, we found that the NSP6-L260F substitution enhances viral polymerase activity and is important for viral replication and pathogenicity. In addition, we found that the NSP13-M233I substitution in the BQ.1.1 lineage and the NSP14-D222Y substitution in the XBB.1.16 lineage reduce viral polymerase activity, and this adverse effect is compensated for by the NSP6-L260F substitution. Our results provide insight into the evolutionary process of SARS-CoV-2.
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Affiliation(s)
- Yuri Furusawa
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Shinjuku, Tokyo, Japan
- Division of Virology, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan
| | - Kiyoko Iwatsuki-Horimoto
- Division of Virology, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan
- The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center, Tokyo, Japan
| | - Seiya Yamayoshi
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Shinjuku, Tokyo, Japan
- Division of Virology, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan
- The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center, Tokyo, Japan
- International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan
| | - Yoshihiro Kawaoka
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Shinjuku, Tokyo, Japan
- Division of Virology, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan
- The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center, Tokyo, Japan
- Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
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6
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Tao X, Wang Y, Jin J, Yan H, Yang H, Wan X, Li P, Xiao Y, Yu Q, Liu L, Liu Y, Han T, Zhang W. NSP6 regulates calcium overload-induced autophagic cell death and is regulated by KLHL22-mediated ubiquitination. J Adv Res 2025:S2090-1232(25)00350-9. [PMID: 40373961 DOI: 10.1016/j.jare.2025.05.031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2025] [Revised: 05/07/2025] [Accepted: 05/12/2025] [Indexed: 05/17/2025] Open
Abstract
INTRODUCTION Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a substantial global threat. SARS-CoV-2 nonstructural proteins (NSPs) are essential for impeding the host replication mechanism while also assisting in the production and organization of new viral components. However, NSPs are not incorporated into viral particles, and their subsequent fate within host cells remains poorly understood. Additionally, their role in viral pathogenesis requires further investigation. OBJECTIVES This study aimed to discover the ultimate fate of NSP6 in host cells and to elucidate its role in viral pathogenesis. METHODS We investigated the effects of NSP6 on cell death and explored the underlying mechanism; moreover, we examined the degradation mechanism of NSP6 in human cells, along with analysing its correlation with coronavirus disease 2019 (COVID-19) severity in patient peripheral blood mononuclear cells (PBMCs). RESULTS NSP6 was demonstrated to induce cell death. Specifically, NSP6 interacted with EI24 autophagy-associated transmembrane protein (EI24) to increase intracellular Ca2+ levels, thereby enhancing the interactions between unc-51-like autophagy activating kinase 1 (ULK1) and RB1 inducible coiled-coil 1 (RB1CC1/FIP200), as well as beclin 1 (BECN1) and phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3). This cascade ultimately triggers autophagy, thus resulting in cell death. Additionally, we discovered that the homeostasis of the NSP6 protein was regulated by K48-linked ubiquitination. We identified kelch-like protein 22 (KLHL22) as the E3 ligase that was responsible for ubiquitinating and degrading NSP6, restoring intracellular calcium homeostasis and reversing NSP6-induced autophagic cell death. Moreover, NSP6 expression levels were observed to be positively associated with the severity of SARS-CoV-2-induced disease. CONCLUSION This study reveals that KLHL22-mediated ubiquitination controls NSP6 stability and that NSP6 induces autophagic cell death via calcium overload, highlighting its cytotoxic role and suggesting therapeutic strategies that target calcium signaling or promote NSP6 degradation as potential interventions against COVID-19.
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Affiliation(s)
- Xingyu Tao
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Yanan Wang
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Jiangbo Jin
- Department of Thoracic Surgery, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China
| | - Huilin Yan
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Hui Yang
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Xiaorui Wan
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Ping Li
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Yanghua Xiao
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Qi Yu
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Lingjiao Liu
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China
| | - Yang Liu
- China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China; Department of Clinical Microbiology, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China.
| | - Tianyu Han
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China.
| | - Wei Zhang
- Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City 330006 Jiangxi, China; Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City 330006 Jiangxi, China; China-Japan Friendship Jiangxi Hospital, National Regional Center for Respiratory Medicine, Nanchang City 330200 Jiangxi, China.
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7
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Hartmann S, Radochonski L, Ye C, Martinez-Sobrido L, Chen J. SARS-CoV-2 ORF3a drives dynamic dense body formation for optimal viral infectivity. Nat Commun 2025; 16:4393. [PMID: 40355429 PMCID: PMC12069715 DOI: 10.1038/s41467-025-59475-x] [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: 05/08/2024] [Accepted: 04/24/2025] [Indexed: 05/14/2025] Open
Abstract
SARS-CoV-2 hijacks multiple organelles for virion assembly, of which the mechanisms have not been fully understood. Here, we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs are unusual electron-dense and dynamic structures driven by the accessory protein ORF3a via remodeling a specific subset of the trans-Golgi network (TGN) and early endosomal membrane. 3DB formation is conserved in related bat and pangolin coronaviruses but was lost during the evolution to SARS-CoV. During SARS-CoV-2 infection, 3DB recruits the viral structural proteins spike (S) and membrane (M) and undergoes dynamic fusion/fission to maintain the optimal unprocessed-to-processed ratio of S on assembled virions. Disruption of 3DB formation resulted in virions assembled with an abnormal S processing rate, leading to a dramatic reduction in viral entry efficiency. Our study uncovers the crucial role of 3DB in maintaining maximal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.
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Affiliation(s)
- Stella Hartmann
- Department of Microbiology, University of Chicago, Chicago, IL, USA
- Howard Taylor Ricketts Laboratory, University of Chicago, Lemont, IL, USA
| | - Lisa Radochonski
- Department of Microbiology, University of Chicago, Chicago, IL, USA
- Howard Taylor Ricketts Laboratory, University of Chicago, Lemont, IL, USA
| | - Chengjin Ye
- Texas Biomedical Research Institute, San Antonio, TX, USA
| | | | - Jueqi Chen
- Department of Microbiology, University of Chicago, Chicago, IL, USA.
- Howard Taylor Ricketts Laboratory, University of Chicago, Lemont, IL, USA.
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8
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Miao X, Law MCY, Kumar J, Chng CP, Zeng Y, Tan YB, Wu J, Guo X, Huang L, Zhuang Y, Gao W, Huang C, Luo D, Zhao W. Saddle curvature association of nsP1 facilitates the replication complex assembly of Chikungunya virus in cells. Nat Commun 2025; 16:4282. [PMID: 40341088 PMCID: PMC12062417 DOI: 10.1038/s41467-025-59402-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Accepted: 04/22/2025] [Indexed: 05/10/2025] Open
Abstract
Positive-sense RNA viruses, including SARS-CoV-1 and -2, DENV, and CHIKV, replicate in curved membrane compartments within host cells. Non-structural proteins (nsPs) critically regulate these nanoscale membrane structures, yet their curvature-dependent assembly remains elusive due to the challenges of imaging nanoscale interaction on curved surfaces. Using vertically aligned nanostructures to generate pre-defined membrane curvatures, we here investigate the impact of curvature on nsPs assembly. Taking CHIKV as a model, we reveal that nsP1 preferentially binds and stabilizes on positively curved membranes, with stronger accumulation at radii ≤150 nm. This is driven by hydrophobic residues in the membrane association (MA) loops of individual nsP1. Molecular dynamics simulations further confirm the improved binding stability of nsP1 on curved membranes, particularly when it forms a dodecamer ring. Together, nsP1 supports a strong saddle curvature association, with flexible MA loops sensing a range of positive curvatures in the x-z plane while the rigid dodecamer stabilizing fixed negative curvature in the x-y plane - crucial for constraining the membrane spherule neck during replication progression. Moreover, CHIKV replication enriches on patterned nanoring structures, underscoring the curvature-guided assembly of the viral replication complex. Our findings highlight membrane curvature as a key regulator of viral nsPs organization, opening new avenues for studying membrane remodeling in viral replication.
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Affiliation(s)
- Xinwen Miao
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
| | - Michelle Cheok Yien Law
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
- NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
| | - Jatin Kumar
- School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore
| | - Choon-Peng Chng
- School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore
| | - Yongpeng Zeng
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
| | - Yaw Bia Tan
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
- NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
| | - Jiawei Wu
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
- State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China
| | - Xiangfu Guo
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
| | - Lizhen Huang
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
| | - Yinyin Zhuang
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
| | - Weibo Gao
- School of Electrical & Electronic Engineering, Nanyang Technological University, Singapore, Singapore
- School of Physics and Mathematical Science, Nanyang Technological University, Singapore, Singapore
| | - Changjin Huang
- School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, Singapore.
| | - Dahai Luo
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.
- NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore.
- National Centre for Infectious Diseases, Singapore, Singapore.
| | - Wenting Zhao
- School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore.
- Institute for Digital Molecular Analytics and Science, Nanyang Technological University, Singapore, Singapore.
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9
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Wang P, Tian B, Xiao K, Ji W, Li Z. The SARS-CoV-2 NSP4 T492I mutation promotes double-membrane vesicle formation to facilitate transmission. Virol Sin 2025; 40:225-235. [PMID: 40157604 DOI: 10.1016/j.virs.2025.03.010] [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: 12/24/2024] [Accepted: 03/24/2025] [Indexed: 04/01/2025] Open
Abstract
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in mutations not only in the spike protein, aiding immune evasion, but also in the NSP3/4/6 proteins, crucial for regulating double-membrane vesicle (DMV) formation. However, the functional consequences of these NSP3/4/6 mutations remain poorly understood. In this study, a systematic analysis was conducted to investigate the evolutionary patterns of NSP3/4/6 mutations and their impact on DMV formation. The findings revealed that the NSP4 T492I mutation, a prevalent mutation found in all Delta and Omicron sub-lineages, notably enhances DMV formation. Mechanistically, the NSP4 T492I mutation enhances its homodimerization, leading to an increase in the size of puncta induced by NSP3/4, and also augments endoplasmic reticulum (ER) membrane curvature, resulting in a higher DMV density per fluorescent puncta. This study underscores the significance of the NSP4 T492I mutation in modulating DMV formation, with potential implications for the transmission dynamics of SARS-CoV-2. It contributes valuable insights into how these mutations impact viral replication and pathogenesis.
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Affiliation(s)
- Pei Wang
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou National Laboratory Clinical Base, Guangzhou Medical University, Guangzhou, 510120, China; Guangzhou National Laboratory, Guangzhou, 510005, China
| | - Buyun Tian
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou National Laboratory Clinical Base, Guangzhou Medical University, Guangzhou, 510120, China; Guangzhou National Laboratory, Guangzhou, 510005, China
| | - Ke Xiao
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Wei Ji
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zonghong Li
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou National Laboratory Clinical Base, Guangzhou Medical University, Guangzhou, 510120, China; Guangzhou National Laboratory, Guangzhou, 510005, China.
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10
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Laporte M, Jochmans D, Bardiot D, Desmarets L, Debski-Antoniak OJ, Mizzon G, Abdelnabi R, Leyssen P, Chiu W, Zhang Z, Nomura N, Boland S, Ohto U, Stahl Y, Wuyts J, De Jonghe S, Stevaert A, van Hemert MJ, Bontes BW, Wanningen P, Groenewold GJM, Zegar A, Owczarek K, Joshi S, Koukni M, Arzel P, Klaassen H, Vanherck JC, Vandecaetsbeek I, Cremers N, Donckers K, Francken T, Van Buyten T, Rymenants J, Schepers J, Pyrc K, Hilgenfeld R, Dubuisson J, Bosch BJ, Van Kuppeveld F, Eydoux C, Decroly E, Canard B, Naesens L, Weynand B, Snijder EJ, Belouzard S, Shimizu T, Bartenschlager R, Hurdiss DL, Marchand A, Chaltin P, Neyts J. A coronavirus assembly inhibitor that targets the viral membrane protein. Nature 2025; 640:514-523. [PMID: 40140569 PMCID: PMC11981944 DOI: 10.1038/s41586-025-08773-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2024] [Accepted: 02/11/2025] [Indexed: 03/28/2025]
Abstract
The coronavirus membrane protein (M) is the main organizer of coronavirus assembly1-3. Here, we report on an M-targeting molecule, CIM-834, that blocks the assembly of SARS-CoV-2. CIM-834 was obtained through high-throughput phenotypic antiviral screening followed by medicinal-chemistry efforts and target elucidation. CIM-834 inhibits the replication of SARS-CoV-2 (including a broad panel of variants) and SARS-CoV. In SCID mice and Syrian hamsters intranasally infected with SARS-CoV-2, oral treatment reduced lung viral titres to nearly undetectable levels, even (as shown in mice) when treatment was delayed until 24 h before the end point. Treatment of infected hamsters prevented transmission to untreated sentinels. Transmission electron microscopy studies show that virion assembly is completely absent in cells treated with CIM-834. Single-particle cryo-electron microscopy reveals that CIM-834 binds and stabilizes the M protein in its short form, thereby preventing the conformational switch to the long form, which is required for successful particle assembly. In conclusion, we have discovered a new druggable target in the replication cycle of coronaviruses and a small molecule that potently inhibits it.
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Affiliation(s)
- Manon Laporte
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Dirk Jochmans
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | | | - Lowiese Desmarets
- CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Université de Lille, Lille, France
| | - Oliver J Debski-Antoniak
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | - Giulia Mizzon
- Department of Infectious Diseases, Molecular Virology, Medical Faculty Heidelberg, University of Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany
| | - Rana Abdelnabi
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
- Department of Microbiology, Immunology and Transplantation, VirusBank Platform, KU Leuven, Leuven, Belgium
| | - Pieter Leyssen
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Winston Chiu
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Zhikuan Zhang
- Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - Norimichi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | | | - Umeharu Ohto
- Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - Yannick Stahl
- Department of Infectious Diseases, Molecular Virology, Medical Faculty Heidelberg, University of Heidelberg, Heidelberg, Germany
| | | | - Steven De Jonghe
- Structural and Translational Virology Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Annelies Stevaert
- Structural and Translational Virology Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Martijn J van Hemert
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | - Brenda W Bontes
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | - Patrick Wanningen
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | - G J Mirjam Groenewold
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | - Aneta Zegar
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Katarzyna Owczarek
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Sanjata Joshi
- Institute of Molecular Medicine and German Center for Infection Research (DZIF), University of Lübeck, Lübeck, Germany
| | | | | | | | | | | | - Niels Cremers
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Kim Donckers
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Thibault Francken
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Tina Van Buyten
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Jasper Rymenants
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Joost Schepers
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Krzysztof Pyrc
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Rolf Hilgenfeld
- Institute of Molecular Medicine and German Center for Infection Research (DZIF), University of Lübeck, Lübeck, Germany
| | - Jean Dubuisson
- CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Université de Lille, Lille, France
| | - Berend-Jan Bosch
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | - Frank Van Kuppeveld
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | - Cecilia Eydoux
- Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Univ., CNRS, Faculté des Sciences Campus Luminy, Marseille, France
| | - Etienne Decroly
- Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Univ., CNRS, Faculté des Sciences Campus Luminy, Marseille, France
| | - Bruno Canard
- Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Univ., CNRS, Faculté des Sciences Campus Luminy, Marseille, France
| | - Lieve Naesens
- Structural and Translational Virology Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium
| | - Birgit Weynand
- Department of Imaging and Pathology, Division of Translational Cell and Tissue Research, KU Leuven, Leuven, Belgium
| | - Eric J Snijder
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | - Sandrine Belouzard
- CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017-CIIL-Center for Infection and Immunity of Lille, Université de Lille, Lille, France
| | - Toshiyuki Shimizu
- Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Medical Faculty Heidelberg, University of Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany
- German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Daniel L Hurdiss
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | | | - Patrick Chaltin
- CISTIM Leuven vzw, Leuven, Belgium
- Centre for Drug Design and Discovery (CD3), KU Leuven, Leuven, Belgium
| | - Johan Neyts
- Virology, Antiviral Drug & Vaccine Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium.
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11
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Kifer A, Pina F, Codallos N, Hermann A, Ziegler L, Niwa M. Orchestration of SARS-CoV-2 Nsp4 and host cell ESCRT proteins induces morphological changes of the endoplasmic reticulum. Mol Biol Cell 2025; 36:ar40. [PMID: 39937675 PMCID: PMC12005107 DOI: 10.1091/mbc.e24-12-0542] [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: 12/15/2024] [Revised: 01/17/2025] [Accepted: 02/04/2025] [Indexed: 02/14/2025] Open
Abstract
Upon entry into the host cell, the nonstructural proteins 3, 4, and 6 (Nsp3, Nsp 4, and Nsp6) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate the formation of double-membrane vesicles (DMVs) through extensive rearrangement of the host cell endoplasmic reticulum (ER) to replicate the viral genome and translate viral proteins. To dissect the functional roles of each Nsp and the molecular mechanisms underlying the ER changes, we exploited both yeast Saccharomyces cerevisiae and human cell experimental systems. Our results demonstrate that Nsp4 alone is sufficient to induce ER structural changes. Nsp4 expression led to robust activation of both the unfolded protein response (UPR) and the ER surveillance (ERSU) cell cycle checkpoint, resulting in cortical ER inheritance block and septin ring mislocalization. Interestingly, these ER morphological changes occurred independently of the canonical UPR and ERSU components but were mediated by the endosomal sorting complex for transport (ESCRT) proteins Vps4 and Vps24 in yeast. Similarly, ER structural changes occurred in human cells upon Nsp4 expression, providing a basis for a minimal experimental system for testing the involvement of human ESCRT proteins and ultimately advancing our understanding of DMV formation.
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Affiliation(s)
- Allison Kifer
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
| | - Franciso Pina
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
| | - Nicholas Codallos
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
| | - Anita Hermann
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
| | - Lauren Ziegler
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
| | - Maho Niwa
- School of Biological Sciences, Department of Molecular Biology, NSB, University of California, San Diego, San Diego, CA 92093-0377
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12
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Tanneti NS, Stillwell HA, Weiss SR. Human coronaviruses: activation and antagonism of innate immune responses. Microbiol Mol Biol Rev 2025; 89:e0001623. [PMID: 39699237 PMCID: PMC11948496 DOI: 10.1128/mmbr.00016-23] [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] [Indexed: 12/20/2024] Open
Abstract
SUMMARYHuman coronaviruses cause a range of respiratory diseases, from the common cold (HCoV-229E, HCoV-NL63, HCoV-OC43, and SARS-CoV-2) to lethal pneumonia (SARS-CoV, SARS-CoV-2, and MERS-CoV). Coronavirus interactions with host innate immune antiviral responses are an important determinant of disease outcome. This review compares the host's innate response to different human coronaviruses. Host antiviral defenses discussed in this review include frontline defenses against respiratory viruses in the nasal epithelium, early sensing of viral infection by innate immune effectors, double-stranded RNA and stress-induced antiviral pathways, and viral antagonism of innate immune responses conferred by conserved coronavirus nonstructural proteins and genus-specific accessory proteins. The common cold coronaviruses HCoV-229E and -NL63 induce robust interferon signaling and related innate immune pathways, SARS-CoV and SARS-CoV-2 induce intermediate levels of activation, and MERS-CoV shuts down these pathways almost completely.
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Affiliation(s)
- Nikhila S. Tanneti
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Helen A. Stillwell
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Susan R. Weiss
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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13
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Soultsioti M, de Jong AWM, Blomberg N, Tas A, Giera M, Snijder EJ, Bárcena M. Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles. J Virol 2025; 99:e0228224. [PMID: 39976449 PMCID: PMC11915874 DOI: 10.1128/jvi.02282-24] [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: 01/10/2025] [Accepted: 01/20/2025] [Indexed: 02/21/2025] Open
Abstract
Coronaviruses hijack host cell metabolic pathways and resources to support their replication. They induce extensive host endomembrane remodeling to generate viral replication organelles and exploit host membranes for assembly and budding of their enveloped progeny virions. Because of the overall significance of host membranes, we sought to gain insight into the role of host factors involved in lipid metabolism in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV). We employed a single-cycle infection approach in combination with pharmacological inhibitors, biochemical assays, lipidomics, and light and electron microscopy. Pharmacological inhibition of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), key host factors in de novo fatty acid biosynthesis, led to pronounced inhibition of MERS-CoV particle release. Inhibition of ACC led to a profound metabolic switch in Huh7 cells, altering their lipidomic profile and inducing lipolysis. However, despite the extensive changes induced by the ACC inhibitor, the biogenesis of viral replication organelles remained unaffected. Instead, ACC inhibition appeared to affect the trafficking and post-translational modifications of the MERS-CoV envelope proteins. Electron microscopy revealed an accumulation of nucleocapsids in early budding stages, indicating that MERS-CoV assembly is adversely impacted by ACC inhibition. Notably, inhibition of palmitoylation resulted in similar effects, while supplementation of exogenous palmitic acid reversed the compound's inhibitory effects, possibly reflecting a crucial need for palmitoylation of the MERS-CoV spike and envelope proteins for their role in virus particle assembly.IMPORTANCEMiddle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of a zoonotic respiratory disease of limited transmissibility between humans. However, MERS-CoV is still considered a high-priority pathogen and is closely monitored by WHO due to its high lethality rate of around 35% of laboratory-confirmed infections. Like other positive-strand RNA viruses, MERS-CoV relies on the host cell's endomembranes to support various stages of its replication cycle. However, in spite of this general reliance of MERS-CoV replication on host cell lipid metabolism, mechanistic insights are still very limited. In our study, we show that pharmacological inhibition of acetyl-CoA carboxylase (ACC), a key enzyme in the host cell's fatty acid biosynthesis pathway, significantly disrupts MERS-CoV particle assembly without exerting a negative effect on the biogenesis of viral replication organelles. Furthermore, our study highlights the potential of ACC as a target for the development of host-directed antiviral therapeutics against coronaviruses.
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Affiliation(s)
- M. Soultsioti
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center, Leiden, the Netherlands
| | - A. W. M. de Jong
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
| | - N. Blomberg
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands
| | - A. Tas
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center, Leiden, the Netherlands
| | - M. Giera
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands
| | - E. J. Snijder
- Molecular Virology Laboratory, Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center, Leiden, the Netherlands
| | - M. Bárcena
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
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14
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Khatun O, Kaur S, Tripathi S. Anti-interferon armamentarium of human coronaviruses. Cell Mol Life Sci 2025; 82:116. [PMID: 40074984 PMCID: PMC11904029 DOI: 10.1007/s00018-025-05605-z] [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: 07/01/2024] [Revised: 12/15/2024] [Accepted: 01/23/2025] [Indexed: 03/14/2025]
Abstract
Cellular innate immune pathways are formidable barriers against viral invasion, creating an environment unfavorable for virus replication. Interferons (IFNs) play a crucial role in driving and regulating these cell-intrinsic innate antiviral mechanisms through the action of interferon-stimulated genes (ISGs). The host IFN response obstructs viral replication at every stage, prompting viruses to evolve various strategies to counteract or evade this response. Understanding the interplay between viral proteins and cell-intrinsic IFN-mediated immune mechanisms is essential for developing antiviral and anti-inflammatory strategies. Human coronaviruses (HCoVs), including SARS-CoV-2, MERS-CoV, SARS-CoV, and seasonal coronaviruses, encode a range of proteins that, through shared and distinct mechanisms, inhibit IFN-mediated innate immune responses. Compounding the issue, a dysregulated early IFN response can lead to a hyper-inflammatory immune reaction later in the infection, resulting in severe disease. This review provides a brief overview of HCoV replication and a detailed account of its interaction with host cellular innate immune pathways regulated by IFN.
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Affiliation(s)
- Oyahida Khatun
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India
| | - Sumandeep Kaur
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India
| | - Shashank Tripathi
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India.
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India.
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15
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Evdokimova M, Feng S, Caobi A, Moreira FR, Jones D, Alysandratos KD, Tully ES, Kotton DN, Boyd DF, Banach BS, Kirchdoerfer RN, Saeed M, Baker SC. Coronavirus endoribonuclease antagonizes ZBP1-mediated necroptosis and delays multiple cell death pathways. Proc Natl Acad Sci U S A 2025; 122:e2419620122. [PMID: 40035769 PMCID: PMC11912388 DOI: 10.1073/pnas.2419620122] [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: 09/30/2024] [Accepted: 01/13/2025] [Indexed: 03/06/2025] Open
Abstract
Identifying conserved mechanisms used by viruses to delay host innate responses can reveal potential targets for antiviral therapeutics. Here, we investigated coronavirus nonstructural protein 15 (nsp15), which encodes a highly conserved endoribonuclease (EndoU). EndoU functions as an immune antagonist by limiting the accumulation of viral replication intermediates that would otherwise be sensed by the host. Despite being a promising antiviral target, it has been difficult to develop small-molecule inhibitors that target the EndoU active site. We generated nsp15 mutants of the coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mouse hepatitis virus (MHV)-A59 and identified conserved residues within the amino-terminal domain that are required for EndoU activity. Loss of EndoU activity caused the activation of host sensors, which limited viral replication in interferon-responsive cells and attenuated disease in MHV-infected mice. Using transcriptional profiling, we found that MHV EndoU mutant viruses upregulate multiple host sensors, including Z-form nucleic acid-binding protein 1 (ZBP1). We found that nsp15 mutants induced early, robust ZBP1-mediated necroptosis. EndoU mutant viruses also induced ZBP1-independent apoptosis and pyroptosis pathways, causing early, robust cell death that limits virus replication and pathogenesis. Overall, we document the importance of the amino-terminal domain for EndoU function. We also highlight the importance of nsp15/EndoU activity for evading host sensors, delaying cell death, and promoting pathogenesis.
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Affiliation(s)
- Monika Evdokimova
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL60153
| | - Shuchen Feng
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL60153
| | - Allen Caobi
- Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, Boston University, Boston, MA02118
- National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA02118
| | - Fernando R. Moreira
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL60153
| | - Dakota Jones
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA02118
- The Pulmonary Center and Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA02118
| | - Konstantinos-Dionysios Alysandratos
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA02118
- The Pulmonary Center and Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA02118
| | - Ena S. Tully
- Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI53706
| | - Darrell N. Kotton
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA02118
- The Pulmonary Center and Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA02118
| | - David F. Boyd
- Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA95064
| | - Bridget S. Banach
- Department of Pathology, Delnor Hospital-Northwestern Medicine, Geneva, IL60134
| | - Robert N. Kirchdoerfer
- Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI53706
| | - Mohsan Saeed
- Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, Boston University, Boston, MA02118
- National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA02118
| | - Susan C. Baker
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL60153
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16
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Baazim H, Koyuncu E, Tuncman G, Burak MF, Merkel L, Bahour N, Karabulut ES, Lee GY, Hanifehnezhad A, Karagoz ZF, Földes K, Engin I, Erman AG, Oztop S, Filazi N, Gul B, Ceylan A, Cinar OO, Can F, Kim H, Al-Hakeem A, Li H, Semerci F, Lin X, Yilmaz E, Ergonul O, Ozkul A, Hotamisligil GS. FABP4 as a therapeutic host target controlling SARS-CoV-2 infection. EMBO Mol Med 2025; 17:414-440. [PMID: 39843629 PMCID: PMC11904229 DOI: 10.1038/s44321-024-00188-x] [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: 05/09/2024] [Revised: 12/16/2024] [Accepted: 12/17/2024] [Indexed: 01/24/2025] Open
Abstract
Host metabolic fitness is a critical determinant of infectious disease outcomes. Obesity, aging, and other related metabolic disorders are recognized as high-risk disease modifiers for respiratory infections, including coronavirus infections, though the underlying mechanisms remain unknown. Our study highlights fatty acid-binding protein 4 (FABP4), a key regulator of metabolic dysfunction and inflammation, as a modulator of SARS-CoV-2 pathogenesis, correlating strongly with disease severity in COVID-19 patients. We demonstrate that loss of FABP4 function, by genetic or pharmacological means, reduces SARS-CoV-2 replication and disrupts the formation of viral replication organelles in adipocytes and airway epithelial cells. Importantly, FABP4 inhibitor treatment of infected hamsters diminished lung viral titers, alleviated lung damage and reduced collagen deposition. These findings highlight the therapeutic potential of targeting host metabolism in limiting coronavirus replication and mitigating the pathogenesis of infection.
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Affiliation(s)
- Hatoon Baazim
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | | | - Gürol Tuncman
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - M Furkan Burak
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
- Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
| | - Lea Merkel
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Nadine Bahour
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Ezgi Simay Karabulut
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Grace Yankun Lee
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Alireza Hanifehnezhad
- Ankara University, Faculty of Veterinary Medicine, Department of Virology, Ankara, Türkiye
| | | | | | - Ilayda Engin
- Ankara University, Biotechnology Institute, Ankara, Türkiye
| | | | - Sidika Oztop
- Ankara Medipol University, School of Medicine, Department of Medical Biology, Ankara, Türkiye
| | - Nazlican Filazi
- Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Virology, Hatay, Türkiye
| | - Buket Gul
- Ankara University, Faculty of Veterinary Medicine, Department of Virology, Ankara, Türkiye
| | - Ahmet Ceylan
- Ankara University, Faculty of Veterinary Medicine, Department of Histology and Embryology, Ankara, Türkiye
| | - Ozge Ozgenc Cinar
- Ankara University, Faculty of Veterinary Medicine, Department of Histology and Embryology, Ankara, Türkiye
| | - Fusun Can
- Koç University, School of Medicine, Department of Infectious Diseases, Istanbul, Türkiye
| | - Hahn Kim
- Crescenta Biosciences Inc, Irvine, CA, USA
- Princeton University Small Molecule Screening Center, Princeton University, Princeton, NJ, USA
- Department of Chemistry, Princeton University, Princeton, NJ, USA
| | | | - Hui Li
- Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | | | - Xihong Lin
- Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Erkan Yilmaz
- Ankara University, Biotechnology Institute, Ankara, Türkiye
| | - Onder Ergonul
- Koç University, School of Medicine, Department of Infectious Diseases, Istanbul, Türkiye
| | - Aykut Ozkul
- Ankara University, Faculty of Veterinary Medicine, Department of Virology, Ankara, Türkiye.
| | - Gökhan S Hotamisligil
- Sabri Ülker Center for Metabolic Research, Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
- Harvard-MIT Broad Institute, Cambridge, MA, USA.
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17
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Gong X, Feng S, Wang J, Gao B, Xue W, Chu H, Fang S, Yuan Y, Cheng Y, Liao M, Sun Y, Tan L, Song C, Qiu X, Ding C, Tijhaar E, Forlenza M, Liao Y. Coronavirus endoribonuclease nsp15 suppresses host protein synthesis and evades PKR-eIF2α-mediated translation shutoff to ensure viral protein synthesis. PLoS Pathog 2025; 21:e1012987. [PMID: 40096172 PMCID: PMC11975131 DOI: 10.1371/journal.ppat.1012987] [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: 08/21/2024] [Revised: 04/07/2025] [Accepted: 02/16/2025] [Indexed: 03/19/2025] Open
Abstract
The endoribonuclease (EndoU) nsp15 of coronaviruses plays a crucial role in evading host innate immune responses by reducing the abundance of viral double-stranded RNA (dsRNA). However, our understanding of its interactions with host cellular targets remains limited. In this study, we demonstrate that overexpression of nsp15 from four coronavirus genera inhibits cellular protein synthesis and causes nuclear retention of PABPC1. Mutation analysis confirms the essential role of EndoU activity in these processes. Fluorescence in situ hybridization (FISH) analysis shows that cellular mRNA co-localizes with nsp15 in certain cells. Real time RT-PCR indicates that the mRNA levels of several antiviral genes decrease in cells expressing nsp15, and this reduction depends on the EndoU activity of nsp15. Using infectious bronchitis virus (IBV) as a model, we investigate the inhibitory effect of nsp15 on protein translation during infection. We find that infection with IBV with functional nsp15 suppresses protein synthesis in a PKR-eIF2α independent manner, with PABPC1 mainly located in the cytoplasm. However, infection with EndoU activity-deficiency mutant virus rIBV-nsp15-H238A results in the accumulation of viral dsRNA, triggering a PKR-eIF2α-dependent shutdown of protein synthesis and leading to the nuclear relocation of PABPC1. In the absence of the PKR-eIF2α pathway, IBV is still able to suppress host protein synthesis, while the inhibitory effect of rIBV-nsp15-H238A on protein synthesis was significantly reduced. Although nsp15 locates to replication-transcription complex (RTC) during infection, RNA immunoprecipitation (RIP)-Seq analysis confirms that IBV nsp15 binds to six viral RNAs and 237 cellular RNAs. The proteins encoded by the nsp15-associated cellular RNAs predominantly involved in translation. Additionally, proteomic analysis of the nsp15 interactome identifies 809 cellular proteins, which are significantly enriched in pathways related to ribosome biogenesis, RNA processing, and translation. Therefore, nsp15 helps virus circumvent the detrimental PKR-eIF2α pathway by reducing viral dsRNA accumulation and suppresses host protein synthesis by targeting host RNAs and proteins. This study reveals unique yet conserved mechanisms of protein synthesis shutdown by catalytically active nsp15 EndoU, shedding light on how coronaviruses regulate host protein expression.
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Affiliation(s)
- Xiaoqian Gong
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
- Cell Biology and Immunology Group, Wageningen University and Research, Department of Animal Sciences, Wageningen, the Netherlands,
| | - Shanhuan Feng
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Jiehuang Wang
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Bo Gao
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Wenxiang Xue
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Hongyan Chu
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Shouguo Fang
- College of Agriculture, College of Animal Sciences, Yangtze University, Jingzhou, China,
| | - Yanmei Yuan
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Yuqiang Cheng
- Shanghai Key Laboratory of Veterinary Biotechnology, Key Laboratory of Urban Agriculture (South), School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China,
| | - Min Liao
- Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, China,
| | - Yingjie Sun
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Lei Tan
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Cuiping Song
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Xusheng Qiu
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
| | - Chan Ding
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
- Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China,
| | - Edwin Tijhaar
- Cell Biology and Immunology Group, Wageningen University and Research, Department of Animal Sciences, Wageningen, the Netherlands,
| | - Maria Forlenza
- Cell Biology and Immunology Group, Wageningen University and Research, Department of Animal Sciences, Wageningen, the Netherlands,
- Host-Microbe Interactomics Group, Wageningen University and Research, Department of Animal Sciences, Wageningen, the Netherlands
| | - Ying Liao
- Department of Avian Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China,
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18
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de
Lima IL, Cataldi TR, Brites C, Labate MT, Vaz SN, Deminco F, da Cunha GS, Labate CA, Eberlin MN. 4D-DIA Proteomics Uncovers New Insights into Host Salivary Response Following SARS-CoV-2 Omicron Infection. J Proteome Res 2025; 24:499-514. [PMID: 39803891 PMCID: PMC11812090 DOI: 10.1021/acs.jproteome.4c00630] [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: 07/23/2024] [Revised: 12/04/2024] [Accepted: 12/30/2024] [Indexed: 02/08/2025]
Abstract
Since late 2021, Omicron variants have dominated the epidemiological scenario as the most successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineages, driving new and breakthrough infections globally over the past two years. In this study, we investigated for the first time the host salivary response of COVID-19 patients infected with Omicron variants (BA.1, BA.2, and BA.4/5) by using an untargeted four-dimensional data-independent acquisition (4D-DIA)-based proteomics approach. We identified 137 proteins whose abundance levels differed between the COVID-19 positive and negative groups. Salivary signatures were mainly enriched in ribosomal proteins, linked to mRNAviral translation, protein synthesis and processing, immune innate, and antiapoptotic signaling. The higher abundance of 14-3-3 proteins (YWHAG, YWHAQ, YWHAE, and SFN) in saliva, first reported here, may be associated with increased infectivity and improved viral replicative fitness. We also identified seven proteins (ACTN1, H2AC2, GSN, NDKA, CD109, GGH, and PCYOX) that yielded comprehension into Omicron infection and performed outstandingly in screening patients with COVID-19 in a hospital setting. This panel also presented an enhanced anti-COVID-19 and anti-inflammatory signature, providing insights into disease severity, supported by comparisons with other proteome data sets. The salivary signature provided valuable insights into the host's response to SARS-CoV-2 Omicron infection, shedding light on the pathophysiology of COVID-19, particularly in cases associated with mild disease. It also underscores the potential clinical applications of saliva for disease screening in hospital settings. Data are available via ProteomeXchange with the identifier PXD054133.
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Affiliation(s)
- Iasmim Lopes de
Lima
- PPGEMN,
School of Engineering, Mackenzie Presbyterian University & MackGraphe
- Mackenzie Institute for Research in Graphene and Nanotechnologies, Mackenzie Presbyterian Institute, São Paulo, São
Paulo 01302-907, Brazil
| | - Thais Regiani Cataldi
- Department
of Genetics, “Luiz de Queiroz”
College of Agriculture, University of São Paulo/ESALQ, Piracicaba, São Paulo 13418-900, Brazil
| | - Carlos Brites
- LAPI
- Laboratory of Research in Infectology, University Hospital Professor
Edgard Santos (HUPES), Federal University
of Bahia (UFBA), Salvador, Bahia 40110-060, Brazil
| | - Mônica Teresa
Veneziano Labate
- Department
of Genetics, “Luiz de Queiroz”
College of Agriculture, University of São Paulo/ESALQ, Piracicaba, São Paulo 13418-900, Brazil
| | - Sara Nunes Vaz
- LAPI
- Laboratory of Research in Infectology, University Hospital Professor
Edgard Santos (HUPES), Federal University
of Bahia (UFBA), Salvador, Bahia 40110-060, Brazil
| | - Felice Deminco
- LAPI
- Laboratory of Research in Infectology, University Hospital Professor
Edgard Santos (HUPES), Federal University
of Bahia (UFBA), Salvador, Bahia 40110-060, Brazil
| | - Gustavo Santana da Cunha
- PPGEMN,
School of Engineering, Mackenzie Presbyterian University & MackGraphe
- Mackenzie Institute for Research in Graphene and Nanotechnologies, Mackenzie Presbyterian Institute, São Paulo, São
Paulo 01302-907, Brazil
| | - Carlos Alberto Labate
- Department
of Genetics, “Luiz de Queiroz”
College of Agriculture, University of São Paulo/ESALQ, Piracicaba, São Paulo 13418-900, Brazil
| | - Marcos Nogueira Eberlin
- PPGEMN,
School of Engineering, Mackenzie Presbyterian University & MackGraphe
- Mackenzie Institute for Research in Graphene and Nanotechnologies, Mackenzie Presbyterian Institute, São Paulo, São
Paulo 01302-907, Brazil
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19
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Renner DM, Parenti NA, Bracci N, Weiss SR. Betacoronaviruses Differentially Activate the Integrated Stress Response to Optimize Viral Replication in Lung-Derived Cell Lines. Viruses 2025; 17:120. [PMID: 39861909 PMCID: PMC11769277 DOI: 10.3390/v17010120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Revised: 01/13/2025] [Accepted: 01/14/2025] [Indexed: 01/27/2025] Open
Abstract
The betacoronavirus genus contains five of the seven human coronaviruses, making it a particularly critical area of research to prepare for future viral emergence. We utilized three human betacoronaviruses, one from each subgenus-HCoV-OC43 (embecovirus), SARS-CoV-2 (sarbecovirus), and MERS-CoV (merbecovirus)-, to study betacoronavirus interactions with the PKR-like ER kinase (PERK) pathway of the integrated stress response (ISR)/unfolded protein response (UPR). The PERK pathway becomes activated by an abundance of unfolded proteins within the endoplasmic reticulum (ER), leading to phosphorylation of eIF2α and translational attenuation. We demonstrate that MERS-CoV, HCoV-OC43, and SARS-CoV-2 all activate PERK and induce responses downstream of p-eIF2α, while only SARS-CoV-2 induces detectable p-eIF2α during infection. Using a small molecule inhibitor of eIF2α dephosphorylation, we provide evidence that MERS-CoV and HCoV-OC43 maximize viral replication through p-eIF2α dephosphorylation. Interestingly, genetic ablation of growth arrest and DNA damage-inducible protein (GADD34) expression, an inducible protein phosphatase 1 (PP1)-interacting partner targeting eIF2α for dephosphorylation, did not significantly alter HCoV-OC43 or SARS-CoV-2 replication, while siRNA knockdown of the constitutive PP1 partner, constitutive repressor of eIF2α phosphorylation (CReP), dramatically reduced HCoV-OC43 replication. Combining GADD34 knockout with CReP knockdown had the maximum impact on HCoV-OC43 replication, while SARS-CoV-2 replication was unaffected. Overall, we conclude that eIF2α dephosphorylation is critical for efficient protein production and replication during MERS-CoV and HCoV-OC43 infection. SARS-CoV-2, however, appears to be insensitive to p-eIF2α and, during infection, may even downregulate dephosphorylation to limit host translation.
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Affiliation(s)
- David M. Renner
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (D.M.R.); (N.A.P.); (N.B.)
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Nicholas A. Parenti
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (D.M.R.); (N.A.P.); (N.B.)
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Nicole Bracci
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (D.M.R.); (N.A.P.); (N.B.)
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Susan R. Weiss
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (D.M.R.); (N.A.P.); (N.B.)
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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20
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Sekine R, Takeda K, Suenaga T, Tsuno S, Kaiya T, Kiso M, Yamayoshi S, Takaku Y, Ohno S, Yamaguchi Y, Nishizawa S, Sumitomo K, Ikuta K, Kanda T, Kawaoka Y, Nishimura H, Kuge S. G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication. Commun Biol 2025; 8:27. [PMID: 39815031 PMCID: PMC11735773 DOI: 10.1038/s42003-024-07351-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Accepted: 12/03/2024] [Indexed: 01/18/2025] Open
Abstract
Future pandemic threats may be caused by novel coronaviruses and influenza A viruses. Here we show that when directly added to a cell culture, 12mer guanine RNA (G12) and its phosphorothioate-linked derivatives (G12(S)), rapidly entered cytoplasm and suppressed the propagation of human coronaviruses and influenza A viruses to between 1/100 and nearly 1/1000 of normal virus infectivity without cellular toxicity and induction of innate immunity. Moreover, G12(S) alleviated the weight loss caused by coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with left-handed parallel-stranded G-quadruplex, and inhibit the replication process by impeding interaction between viral nucleoproteins and viral RNA in the cytoplasm. Unlike previous antiviral strategies that target the G-quadruplexes of the viral genome, we now show that excess exogenous G-quadruplex-forming small RNA displaces genomic RNA from ribonucleoprotein, effectively inhibiting viral replication. The approach has the potential to facilitate the creation of versatile middle-molecule antivirals featuring lipid nanoparticle-free delivery.
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Affiliation(s)
- Ryoya Sekine
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Kouki Takeda
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Tsukasa Suenaga
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Satsuki Tsuno
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Takumi Kaiya
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Maki Kiso
- Division of Virology, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo, 108-8639, Japan
- The University of Tokyo, Pandemic Preparedness, Infection, and Advanced Research Center, Tokyo, Japan
| | - Seiya Yamayoshi
- Division of Virology, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo, 108-8639, Japan
- The University of Tokyo, Pandemic Preparedness, Infection, and Advanced Research Center, Tokyo, Japan
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan
| | - Yoshihide Takaku
- Department of Chemistry, Graduate School of Science, Tohoku University, 6-3 Azaaoba, Aoba-ku, Sendai, Miyagi, 980-8578, Japan
| | - Shiho Ohno
- Division of Structural Glycobiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Yoshiki Yamaguchi
- Division of Structural Glycobiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan
| | - Seiichi Nishizawa
- Department of Chemistry, Graduate School of Science, Tohoku University, 6-3 Azaaoba, Aoba-ku, Sendai, Miyagi, 980-8578, Japan
| | - Kazuhiro Sumitomo
- Division of Geriatric and Community Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 1-15-1, Fukumuro, Miyagino-ku, Sendai, Miyagi, 983-8536, Japan
| | - Kazufumi Ikuta
- Division of Microbiology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 1-15-1, Fukumuro, Miyagino-ku, Sendai, Miyagi, 983-8536, Japan
| | - Teru Kanda
- Division of Microbiology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 1-15-1, Fukumuro, Miyagino-ku, Sendai, Miyagi, 983-8536, Japan
| | - Yoshihiro Kawaoka
- Division of Virology, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo, 108-8639, Japan
- The University of Tokyo, Pandemic Preparedness, Infection, and Advanced Research Center, Tokyo, Japan
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan
| | - Hidekazu Nishimura
- Virus Research Center, Clinical Research Division, National Hospital Organization Sendai Medical Center, 2-1-12, Miyagino, Miyagino-ku, Sendai, Miyagi, 983-8520, Japan
| | - Shusuke Kuge
- Division of Microbiology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsuhima, Aoba-ku, Sendai, Miyagi, 981-8558, Japan.
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21
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Sotoudeheian M, Mirahmadi SMS, Pirhayati M, Farahmandian N, Azarbad R, Toroudi HP. Targeting SIRT1 by Scopoletin to Inhibit XBB.1.5 COVID-19 Life Cycle. Curr Rev Clin Exp Pharmacol 2025; 20:4-13. [PMID: 38441021 DOI: 10.2174/0127724328281178240225082456] [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: 09/07/2023] [Revised: 12/21/2023] [Accepted: 01/05/2024] [Indexed: 03/06/2024]
Abstract
Natural products have historically driven pharmaceutical discovery, but their reliance has diminished with synthetic drugs. Approximately 35% of medicines originate from natural products. Scopoletin, a natural coumarin compound found in herbs, exhibits antioxidant, hepatoprotective, antiviral, and antimicrobial properties through diverse intracellular signaling mechanisms. Furthermore, it also enhances the activity of antioxidants. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes viral pneumonia through cytokine storms and systemic inflammation. Cellular autophagy pathways play a role in coronavirus replication and inflammation. The Silent Information Regulator 1 (SIRT1) pathway, linked to autophagy, protects cells via FOXO3, inhibits apoptosis, and modulates SIRT1 in type-II epithelial cells. SIRT1 activation by adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) enhances the autophagy cascade. This pathway holds therapeutic potential for alveolar and pulmonary diseases and is crucial in lung inflammation. Angiotensin-converting enzyme 2 (ACE-2) activation, inhibited by reduced expression, prevents COVID-19 virus entry into type-II epithelial cells. The coronavirus disease 2019 (COVID-19) virus binds ACE-2 to enter into the host cells, and XBB.1.5 COVID-19 displays high ACE-2-binding affinity. ACE-2 expression in pneumocytes is regulated by signal transducers and activators of transcription-3 (STAT3), which can increase COVID-19 virus replication. SIRT1 regulates STAT3, and the SIRT1/STAT3 pathway is involved in lung diseases. Therapeutic regulation of SIRT1 protects the lungs from inflammation caused by viral-mediated oxidative stress. Scopoletin, as a modulator of the SIRT1 cascade, can regulate autophagy and inhibit the entry and life cycle of XBB.1.5 COVID-19 in host cells.
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Affiliation(s)
| | | | | | - Navid Farahmandian
- Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Reza Azarbad
- Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
| | - Hamidreza Pazoki Toroudi
- Physiology Research Center and Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
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22
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Grelewska‐Nowotko K, Elhag AE, Turowski TW. Transcription Kinetics in the Coronavirus Life Cycle. WILEY INTERDISCIPLINARY REVIEWS. RNA 2025; 16:e70000. [PMID: 39757745 PMCID: PMC11701415 DOI: 10.1002/wrna.70000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Revised: 12/04/2024] [Accepted: 12/05/2024] [Indexed: 01/07/2025]
Abstract
Coronaviruses utilize a positive-sense single-strand RNA, functioning simultaneously as mRNA and the genome. An RNA-dependent RNA polymerase (RdRP) plays a dual role in transcribing genes and replicating the genome, making RdRP a critical target in therapies against coronaviruses. This review explores recent advancements in understanding the coronavirus transcription machinery, discusses it within virus infection context, and incorporates kinetic considerations on RdRP activity. We also address steric limitations in coronavirus replication, particularly during early infection phases, and outline hypothesis regarding translation-transcription conflicts, postulating the existence of mechanisms that resolve these issues. In cells infected by coronaviruses, abundant structural proteins are synthesized from subgenomic RNA fragments (sgRNAs) produced via discontinuous transcription. During elongation, RdRP can skip large sections of the viral genome, resulting in the creation of shorter sgRNAs that reflects the stoichiometry of viral structural proteins. Although the precise mechanism of discontinuous transcription remains unknown, we discuss recent hypotheses involving long-distance RNA-RNA interactions, helicase-mediated RdRP backtracking, dissociation and reassociation of RdRP, and RdRP dimerization.
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Affiliation(s)
| | - Ahmed Eisa Elhag
- Institute of Biochemistry and BiophysicsPolish Academy of SciencesWarsawPoland
- Department of Preventive Medicine and Clinical Studies, Faculty of Veterinary SciencesUniversity of GadarifAl QadarifSudan
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23
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Zhou Q, Lok SM. Visualizing the virus world inside the cell by cryo-electron tomography. J Virol 2024; 98:e0108523. [PMID: 39494908 PMCID: PMC11650999 DOI: 10.1128/jvi.01085-23] [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] [Indexed: 11/05/2024] Open
Abstract
Structural studies on purified virus have revealed intricate architectures, but there is little structural information on how viruses interact with host cells in situ. Cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have emerged as revolutionary tools in structural biology to visualize the dynamic conformational of viral particles and their interactions with host factors within infected cells. Here, we review the state-of-the-art cryo-ET technique for in situ viral structure studies and highlight exemplary studies that showcase the remarkable capabilities of cryo-ET in capturing the dynamic virus-host interaction, advancing our understanding of viral infection and pathogenesis.
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Affiliation(s)
- Qunfei Zhou
- Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore
| | - Shee-Mei Lok
- Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore, Singapore
- Department of Biological Sciences, Centre for BioImaging Sciences, National University of Singapore, Singapore, Singapore
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24
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Kirk NM, Liang Y, Ly H. Pathogenesis and virulence of coronavirus disease: Comparative pathology of animal models for COVID-19. Virulence 2024; 15:2316438. [PMID: 38362881 PMCID: PMC10878030 DOI: 10.1080/21505594.2024.2316438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Accepted: 02/04/2024] [Indexed: 02/17/2024] Open
Abstract
Animal models that can replicate clinical and pathologic features of severe human coronavirus infections have been instrumental in the development of novel vaccines and therapeutics. The goal of this review is to summarize our current understanding of the pathogenesis of coronavirus disease 2019 (COVID-19) and the pathologic features that can be observed in several currently available animal models. Knowledge gained from studying these animal models of SARS-CoV-2 infection can help inform appropriate model selection for disease modelling as well as for vaccine and therapeutic developments.
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Affiliation(s)
- Natalie M. Kirk
- Department of Veterinary & Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, MN, USA
| | - Yuying Liang
- Department of Veterinary & Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, MN, USA
| | - Hinh Ly
- Department of Veterinary & Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Twin Cities, MN, USA
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25
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Bezerra PR, Vasconcelos AA, Almeida VS, Neves-Martins TC, Mebus-Antunes NC, Almeida FCL. 1H, 15N, and 13C resonance assignments of the N-terminal domain and ser-arg-rich intrinsically disordered region of the nucleocapsid protein of the SARS-CoV-2. BIOMOLECULAR NMR ASSIGNMENTS 2024; 18:219-225. [PMID: 39174826 DOI: 10.1007/s12104-024-10191-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Accepted: 08/17/2024] [Indexed: 08/24/2024]
Abstract
The nucleocapsid (N) protein of SARS-CoV-2 is a multifunctional protein involved in nucleocapsid assembly and various regulatory functions. It is the most abundant protein during viral infection. Its functionality is closely related to its structure, which comprises two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by intrinsically disordered regions. The linker between the NTD and CTD includes a Serine-Arginine rich (SR) region, which is crucial for the regulation of the N protein's function. Here, we report the near-complete assignment of the construct containing the NTD followed by the SR region (NTD-SR). Additionally, we describe the dynamic nature of the SR region and compare it with all other available chemical shift assignments reported for the SR region.
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Affiliation(s)
- Peter R Bezerra
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
- National Center of Nuclear Magnetic Resonance (CNRMN), National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Ariana A Vasconcelos
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
- National Center of Nuclear Magnetic Resonance (CNRMN), National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Vitor S Almeida
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
- National Center of Nuclear Magnetic Resonance (CNRMN), National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Thais C Neves-Martins
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Nathane C Mebus-Antunes
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Fabio C L Almeida
- National Center of Nuclear Magnetic Resonance, Institute of Medical Biochemistry (IBqM), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
- National Center of Nuclear Magnetic Resonance (CNRMN), National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
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26
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Torres J, Pervushin K, Surya W. Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations. Comput Struct Biotechnol J 2024; 23:3730-3740. [PMID: 39525089 PMCID: PMC11543627 DOI: 10.1016/j.csbj.2024.10.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2024] [Revised: 10/01/2024] [Accepted: 10/15/2024] [Indexed: 11/16/2024] Open
Abstract
The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, 'polar-in', and one where this residue is in an interhelical orientation 'polar-inter'. For the SARS models, the comparison with the two experimental models 'closed' (PDB: 7K3G) and 'open' (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.
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Affiliation(s)
- Jaume Torres
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Konstantin Pervushin
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Wahyu Surya
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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27
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Soares VC, Dias SSG, Santos JC, Bozza PT. Unlocking secrets: lipid metabolism and lipid droplet crucial roles in SARS-CoV-2 infection and the immune response. J Leukoc Biol 2024; 116:1254-1268. [PMID: 39087951 DOI: 10.1093/jleuko/qiae170] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 06/11/2024] [Accepted: 07/31/2024] [Indexed: 08/02/2024] Open
Abstract
Lipid droplets (LDs) are crucial for maintaining lipid and energy homeostasis within cells. LDs are highly dynamic organelles that present a phospholipid monolayer rich in neutral lipids. Additionally, LDs are associated with structural and nonstructural proteins, rapidly mobilizing lipids for various biological processes. Lipids play a pivotal role during viral infection, participating during viral membrane fusion, viral replication, and assembly, endocytosis, and exocytosis. SARS-CoV-2 infection often induces LD accumulation, which is used as a source of energy for the replicative process. These findings suggest that LDs are a hallmark of viral infection, including SARS-CoV-2 infection. Moreover, LDs participate in the inflammatory process and cell signaling, activating pathways related to innate immunity and cell death. Accumulating evidence demonstrates that LD induction by SARS-CoV-2 is a highly coordinated process, aiding replication and evading the immune system, and may contribute to the different cell death process observed in various studies. Nevertheless, recent research in the field of LDs suggests these organelles according to the pathogen and infection conditions may also play roles in immune and inflammatory responses, protecting the host against viral infection. Understanding how SARS-CoV-2 influences LD biogenesis is crucial for developing novel drugs or repurposing existing ones. By targeting host lipid metabolic pathways exploited by the virus, it is possible to impact viral replication and inflammatory responses. This review seeks to discuss and analyze the role of LDs during SARS-CoV-2 infection, specifically emphasizing their involvement in viral replication and the inflammatory response.
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Affiliation(s)
- Vinicius Cardoso Soares
- Laboratory of Immunopharmacology, Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
- Center for Research, Innovation and Surveillance in COVID-19 and Heath Emergencies, Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
- Program of Immunology and Inflammation, Federal University of Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, 21941-902, Brazil
| | - Suelen Silva Gomes Dias
- Laboratory of Immunopharmacology, Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
- Center for Research, Innovation and Surveillance in COVID-19 and Heath Emergencies, Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
| | - Julia Cunha Santos
- Laboratory of Immunopharmacology, Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
- Center for Research, Innovation and Surveillance in COVID-19 and Heath Emergencies, Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
| | - Patrícia T Bozza
- Laboratory of Immunopharmacology, Oswaldo Cruz Institute (IOC), Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
- Center for Research, Innovation and Surveillance in COVID-19 and Heath Emergencies, Oswaldo Cruz Foundation, Fiocruz, Brasil Ave, Rio de Janeiro, RJ, 21040-361, Brazil
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28
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Yu J, Ge S, Li J, Zhang Y, Xu J, Wang Y, Liu S, Yu X, Wang Z. Interaction between coronaviruses and the autophagic response. Front Cell Infect Microbiol 2024; 14:1457617. [PMID: 39650836 PMCID: PMC11621220 DOI: 10.3389/fcimb.2024.1457617] [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: 07/01/2024] [Accepted: 10/18/2024] [Indexed: 12/11/2024] Open
Abstract
In recent years, the emergence and widespread dissemination of the coronavirus SARS-CoV-2 has posed a significant threat to global public health and social development. In order to safely and effectively prevent and control the spread of coronavirus diseases, a profound understanding of virus-host interactions is paramount. Cellular autophagy, a process that safeguards cells by maintaining cellular homeostasis under diverse stress conditions. Xenophagy, specifically, can selectively degrade intracellular pathogens, such as bacteria, fungi, viruses, and parasites, thus establishing a robust defense mechanism against such intruders. Coronaviruses have the ability to induce autophagy, and they manipulate this pathway to ensure their efficient replication. While progress has been made in elucidating the intricate relationship between coronaviruses and autophagy, a comprehensive summary of how autophagy either benefits or hinders viral replication remains elusive. In this review, we delve into the mechanisms that govern how different coronaviruses regulate autophagy. We also provide an in-depth analysis of virus-host interactions, particularly focusing on the latest data pertaining to SARS-CoV-2. Our aim is to lay a theoretical foundation for the development of novel coronavirus vaccines and the screening of potential drug targets.
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Affiliation(s)
- Jiarong Yu
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Shengqiang Ge
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Jinming Li
- China Animal Health and Epidemiology Center, Qingdao, China
| | | | - Jiao Xu
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Yingli Wang
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Shan Liu
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Xiaojing Yu
- China Animal Health and Epidemiology Center, Qingdao, China
| | - Zhiliang Wang
- China Animal Health and Epidemiology Center, Qingdao, China
- College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China
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29
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Chen A, Lupan AM, Quek RT, Stanciu SG, Asaftei M, Stanciu GA, Hardy KS, de Almeida Magalhães T, Silver PA, Mitchison TJ, Salic A. A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles. SCIENCE ADVANCES 2024; 10:eadq9580. [PMID: 39514670 PMCID: PMC11546809 DOI: 10.1126/sciadv.adq9580] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/07/2024] [Accepted: 10/04/2024] [Indexed: 11/16/2024]
Abstract
Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, resulting in broad-spectrum anticoronaviral activity.
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Affiliation(s)
- Anan Chen
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Ana-Mihaela Lupan
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Rui Tong Quek
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Stefan G. Stanciu
- Center for Microscopy-Microanalysis and Information Processing, National University of Science and Technology Politehnica Bucharest, 313 Splaiul Independenței, 060042 Bucharest, Romania
| | - Mihaela Asaftei
- Center for Microscopy-Microanalysis and Information Processing, National University of Science and Technology Politehnica Bucharest, 313 Splaiul Independenței, 060042 Bucharest, Romania
- Department of Microbiology, University of Bucharest, Aleea Portocalelor nr. 1-3, 060101 Bucharest, Romania
| | - George A. Stanciu
- Center for Microscopy-Microanalysis and Information Processing, National University of Science and Technology Politehnica Bucharest, 313 Splaiul Independenței, 060042 Bucharest, Romania
| | - Kierra S. Hardy
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
| | | | - Pamela A. Silver
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
| | | | - Adrian Salic
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
- Faculty of Chemistry, University of Bucharest, Șoseaua Panduri nr. 90, 050663 Bucharest, Romania
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30
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Song K, Hasan A, Hao W, Wu Y, Sun Y, Li W, Wang L, Li S. Stimulator of interferon genes (STING) inhibits coronavirus infection by disrupting viral replication organelles. J Med Virol 2024; 96:e70020. [PMID: 39470032 PMCID: PMC11534302 DOI: 10.1002/jmv.70020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2024] [Revised: 09/18/2024] [Accepted: 10/08/2024] [Indexed: 10/30/2024]
Abstract
Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) protein that plays a crucial role in cytosolic DNA-mediated innate immunity. Both STING agonists and antagonists have demonstrated their ability to enhance mouse survival against coronavirus, however, the physiological role of endogenous STING in coronavirus infection remains unclear. Our research unveils that STING inhibits coronavirus replication by impeding the formation of the ER-derived double-membrane vesicles (DMVs), the organelles in which coronavirus replicates. We found that STING was still capable of inhibiting coronavirus OC43 infection in cells, regardless of the knockout of cGAS or MAVS, or blocking type I interferon receptor. Moreover, STING disrupted the interaction between two crucial proteins, NSP4 and NSP6, involved in DMV formation, leading to the disruption of DMV formation. Taken together, our study sheds light on a novel antiviral role of STING in coronavirus infection, elucidating how it disrupts the formation of viral replication organelles, thereby impeding the replication process.
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Affiliation(s)
- Kun Song
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Abdul Hasan
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Wenzhuo Hao
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Yakun Wu
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Yiwen Sun
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Wenjun Li
- Department of Craniofacial Biomedicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Lingyan Wang
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
| | - Shitao Li
- Department of Microbiology and Immunology, Tulane University, New Orleans, LA 70112, USA
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31
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Wu H, Fujioka Y, Sakaguchi S, Suzuki Y, Nakano T. Electron Tomography as a Tool to Study SARS-CoV-2 Morphology. Int J Mol Sci 2024; 25:11762. [PMID: 39519314 PMCID: PMC11547116 DOI: 10.3390/ijms252111762] [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: 09/30/2024] [Revised: 10/29/2024] [Accepted: 10/30/2024] [Indexed: 11/16/2024] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel betacoronavirus, is the causative agent of COVID-19, which has caused economic and social disruption worldwide. To date, many drugs and vaccines have been developed for the treatment and prevention of COVID-19 and have effectively controlled the global epidemic of SARS-CoV-2. However, SARS-CoV-2 is highly mutable, leading to the emergence of new variants that may counteract current therapeutic measures. Electron microscopy (EM) is a valuable technique for obtaining ultrastructural information about the intracellular process of virus replication. In particular, EM allows us to visualize the morphological and subcellular changes during virion formation, which would provide a promising avenue for the development of antiviral agents effective against new SARS-CoV-2 variants. In this review, we present our recent findings using transmission electron microscopy (TEM) combined with electron tomography (ET) to reveal the morphologically distinct types of SARS-CoV-2 particles, demonstrating that TEM and ET are valuable tools for visually understanding the maturation status of SARS-CoV-2 in infected cells. This review also discusses the application of EM analysis to the evaluation of genetically engineered RNA viruses.
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Affiliation(s)
- Hong Wu
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka 565-0871, Japan; (Y.F.); (S.S.); (T.N.)
| | | | | | - Youichi Suzuki
- Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka 565-0871, Japan; (Y.F.); (S.S.); (T.N.)
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32
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Lin CH, Lin HY, Yang CC, Hsu HW, Hsieh FC, Yang CY, Wu HY. Preferential cleavage of the coronavirus defective viral genome by cellular endoribonuclease with characteristics of RNase L. Virol J 2024; 21:273. [PMID: 39487538 PMCID: PMC11529150 DOI: 10.1186/s12985-024-02549-x] [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: 08/16/2024] [Accepted: 10/18/2024] [Indexed: 11/04/2024] Open
Abstract
In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses.
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Affiliation(s)
- Ching-Hung Lin
- Department of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, 91201, Pingtung, Taiwan
| | - Hsuan-Yung Lin
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Chun-Chun Yang
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Hsuan-Wei Hsu
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Feng-Cheng Hsieh
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Cheng-Yao Yang
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Hung-Yi Wu
- Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan.
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Bezerra PR, Almeida FCL. Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization. J Biol Chem 2024; 300:107834. [PMID: 39343000 PMCID: PMC11541846 DOI: 10.1016/j.jbc.2024.107834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2024] [Revised: 09/17/2024] [Accepted: 09/18/2024] [Indexed: 10/01/2024] Open
Abstract
The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membrane-less compartments through liquid-liquid phase separation, thereby facilitating the congregation of the replication and transcription complex. Considering the information available regarding the catalytic activities and binding signatures of the N protein-RNA interaction, this review focuses on the regulatory role of the SARS-CoV-2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities.
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Affiliation(s)
- Peter R Bezerra
- Program of Structural Biology, Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; National Center of Nuclear Magnetic Resonance (CNRMN), CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Fabio C L Almeida
- Program of Structural Biology, Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; National Center of Nuclear Magnetic Resonance (CNRMN), CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
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Renner DM, Parenti NA, Weiss SR. BETACORONAVIRUSES DIFFERENTIALLY ACTIVATE THE INTEGRATED STRESS RESPONSE TO OPTIMIZE VIRAL REPLICATION IN LUNG DERIVED CELL LINES. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.25.614975. [PMID: 39386680 PMCID: PMC11463420 DOI: 10.1101/2024.09.25.614975] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 10/12/2024]
Abstract
The betacoronavirus genus contains five of the seven human viruses, making it a particularly critical area of research to prepare for future viral emergence. We utilized three human betacoronaviruses, one from each subgenus- HCoV-OC43 (embecovirus), SARS-CoV-2 (sarbecovirus) and MERS-CoV (merbecovirus)- to study betacoronavirus interaction with the PKR-like ER kinase (PERK) pathway of the integrated stress response (ISR)/unfolded protein response (UPR). The PERK pathway becomes activated by an abundance of unfolded proteins within the endoplasmic reticulum (ER), leading to phosphorylation of eIF2α and translational attenuation in lung derived cell lines. We demonstrate that MERS-CoV, HCoV-OC43, and SARS-CoV-2 all activate PERK and induce responses downstream of p-eIF2α, while only SARS-CoV-2 induces detectable p-eIF2α during infection. Using a small molecule inhibitor of eIF2α dephosphorylation, we provide evidence that MERS-CoV and HCoV-OC43 maximize replication through p-eIF2α dephosphorylation. Interestingly, genetic ablation of GADD34 expression, an inducible phosphatase 1 (PP1)-interacting partner targeting eIF2α for dephosphorylation, did not significantly alter HCoV-OC43 or SARS-CoV-2 replication, while siRNA knockdown of the constitutive PP1 partner, CReP, dramatically reduced HCoV-OC43 replication. Combining growth arrest and DNA damage-inducible protein (GADD34) knockout with peripheral ER membrane-targeted protein (CReP) knockdown had the maximum impact on HCoV-OC43 replication, while SARS-CoV-2 replication was unaffected. Overall, we conclude that eIF2α dephosphorylation is critical for efficient protein production and replication during MERS-CoV and HCoV-OC43 infection. SARS-CoV-2, however, appears to be insensitive to p-eIF2α and, during infection, may even downregulate dephosphorylation to limit host translation. IMPORTANCE Lethal human betacoronaviruses have emerged three times in the last two decades, causing two epidemics and a pandemic. Here, we demonstrate differences in how these viruses interact with cellular translational control mechanisms. Utilizing inhibitory compounds and genetic ablation, we demonstrate that MERS-CoV and HCoV-OC43 benefit from keeping p-eIF2α levels low to maintain high rates of virus translation while SARS-CoV-2 tolerates high levels of p-eIF2α. We utilized a PP1:GADD34/CReP inhibitor, GADD34 KO cells, and CReP-targeting siRNA to investigate the therapeutic potential of these pathways. While ineffective for SARS-CoV-2, we found that HCoV-OC43 seems to primarily utilize CReP to limit p-eIF2a accumulation. This work highlights the need to consider differences amongst these viruses, which may inform the development of host-directed pan-coronavirus therapeutics.
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Affiliation(s)
- David M. Renner
- Departments of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
| | - Nicholas A. Parenti
- Departments of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
| | - Susan R. Weiss
- Departments of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
- Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 19104-6076
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Attiq A, Afzal S, Wahab HA, Ahmad W, Kandeel M, Almofti YA, Alameen AO, Wu YS. Cytokine Storm-Induced Thyroid Dysfunction in COVID-19: Insights into Pathogenesis and Therapeutic Approaches. Drug Des Devel Ther 2024; 18:4215-4240. [PMID: 39319193 PMCID: PMC11421457 DOI: 10.2147/dddt.s475005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2024] [Accepted: 08/26/2024] [Indexed: 09/26/2024] Open
Abstract
Angiotensin-converting enzyme 2 receptors (ACE2R) are requisite to enter the host cells for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). ACE2R is constitutive and functions as a type I transmembrane metallo-carboxypeptidase in the renin-angiotensin system (RAS). On thyroid follicular cells, ACE2R allows SARS-CoV-2 to invade the thyroid gland, impose cytopathic effects and produce endocrine abnormalities, including stiff back, neck pain, muscle ache, lethargy, and enlarged, inflamed thyroid gland in COVID-19 patients. Further damage is perpetuated by the sudden bursts of pro-inflammatory cytokines, which is suggestive of a life-threatening syndrome known as a "cytokine storm". IL-1β, IL-6, IFN-γ, and TNF-α are identified as the key orchestrators of the cytokine storm. These inflammatory mediators upregulate transcriptional turnover of nuclear factor-kappa B (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and mitogen-activated protein kinase (MAPK), paving the pathway for cytokine storm-induced thyroid dysfunctions including euthyroid sick syndrome, autoimmune thyroid diseases, and thyrotoxicosis in COVID-19 patients. Targeted therapies with corticosteroids (dexamethasone), JAK inhibitor (baricitinib), nucleotide analogue (remdesivir) and N-acetyl-cysteine have demonstrated effectiveness in terms of attenuating the severity and frequency of cytokine storm-induced thyroid dysfunctions, morbidity and mortality in severe COVID-19 patients. Here, we review the pathogenesis of cytokine storms and the mechanisms and pathways that establish the connection between thyroid disorder and COVID-19. Moreover, cross-talk interactions of signalling pathways and therapeutic strategies to address COVID-19-associated thyroid diseases are also discussed herein.
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Affiliation(s)
- Ali Attiq
- School of Pharmaceutical Sciences, Universiti Sains Malaysia, Gelugor, Penang, 11800, Malaysia
| | - Sheryar Afzal
- Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, Al Ahsa, 31982, Saudi Arabia
| | - Habibah A Wahab
- School of Pharmaceutical Sciences, Universiti Sains Malaysia, Gelugor, Penang, 11800, Malaysia
| | - Waqas Ahmad
- School of Pharmaceutical Sciences, Universiti Sains Malaysia, Gelugor, Penang, 11800, Malaysia
| | - Mahmoud Kandeel
- Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, Al Ahsa, 31982, Saudi Arabia
- Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrel Sheikh, 6860404, Egypt
| | - Yassir A Almofti
- Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, Al Ahsa, 31982, Saudi Arabia
- Department of Biochemistry, Molecular Biology and Bioinformatics, College of Veterinary Medicine, University of Bahri, Khartoum, 12217, Sudan
| | - Ahmed O Alameen
- Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, Al Ahsa, 31982, Saudi Arabia
- Department of Physiology, Faculty of Veterinary Medicine, University of Khartoum, Shambat, 13314, Sudan
| | - Yuan Seng Wu
- Sunway Microbiome Centre, School of Medical and Life Sciences, Sunway University, Subang Jaya, Selangor, 47500, Malaysia
- Department of Biological Sciences, School of Medical and Life Sciences, Sunway University, Subang Jaya, Selangor, 47500, Malaysia
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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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37
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Quinteros JA, Browning GF, Mardani K, Noormohammadi AH, Stevenson MA, Coppo MJC, Loncoman CA, Ficorilli N, Diaz-Méndez A. Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells. J Virol Methods 2024; 329:114989. [PMID: 38917942 DOI: 10.1016/j.jviromet.2024.114989] [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: 02/15/2024] [Revised: 06/04/2024] [Accepted: 06/16/2024] [Indexed: 06/27/2024]
Abstract
Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.
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Affiliation(s)
- José A Quinteros
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia.
| | - Glenn F Browning
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Karim Mardani
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Amir H Noormohammadi
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Werribee, VIC 3030, Australia
| | - Mark A Stevenson
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Mauricio J C Coppo
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Carlos A Loncoman
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Nino Ficorilli
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Andrés Diaz-Méndez
- Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
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38
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Sergio MC, Ricciardi S, Guarino AM, Giaquinto L, De Matteis MA. Membrane remodeling and trafficking piloted by SARS-CoV-2. Trends Cell Biol 2024; 34:785-800. [PMID: 38262893 DOI: 10.1016/j.tcb.2023.12.006] [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: 10/20/2023] [Revised: 12/14/2023] [Accepted: 12/21/2023] [Indexed: 01/25/2024]
Abstract
The molecular mechanisms underlying SARS-CoV-2 host cell invasion and life cycle have been studied extensively in recent years, with a primary focus on viral entry and internalization with the aim of identifying antiviral therapies. By contrast, our understanding of the molecular mechanisms involved in the later steps of the coronavirus life cycle is relatively limited. In this review, we describe what is known about the host factors and viral proteins involved in the replication, assembly, and egress phases of SARS-CoV-2, which induce significant host membrane rearrangements. We also discuss the limits of the current approaches and the knowledge gaps still to be addressed.
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Affiliation(s)
- Maria Concetta Sergio
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; University of Naples Federico II, Naples, Italy
| | | | - Andrea M Guarino
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; University of Naples Federico II, Naples, Italy
| | - Laura Giaquinto
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; University of Naples Federico II, Naples, Italy
| | - Maria Antonietta De Matteis
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; University of Naples Federico II, Naples, Italy.
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39
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Stancheva VG, Sanyal S. Positive-strand RNA virus replication organelles at a glance. J Cell Sci 2024; 137:jcs262164. [PMID: 39254430 PMCID: PMC11423815 DOI: 10.1242/jcs.262164] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/11/2024] Open
Abstract
Membrane-bound replication organelles (ROs) are a unifying feature among diverse positive-strand RNA viruses. These compartments, formed as alterations of various host organelles, provide a protective niche for viral genome replication. Some ROs are characterised by a membrane-spanning pore formed by viral proteins. The RO membrane separates the interior from immune sensors in the cytoplasm. Recent advances in imaging techniques have revealed striking diversity in RO morphology and origin across virus families. Nevertheless, ROs share core features such as interactions with host proteins for their biogenesis and for lipid and energy transfer. The restructuring of host membranes for RO biogenesis and maintenance requires coordinated action of viral and host factors, including membrane-bending proteins, lipid-modifying enzymes and tethers for interorganellar contacts. In this Cell Science at a Glance article and the accompanying poster, we highlight ROs as a universal feature of positive-strand RNA viruses reliant on virus-host interplay, and we discuss ROs in the context of extensive research focusing on their potential as promising targets for antiviral therapies and their role as models for understanding fundamental principles of cell biology.
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Affiliation(s)
- Viktoriya G. Stancheva
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
| | - Sumana Sanyal
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
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40
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Chen XN, Cai ST, Liang YF, Weng ZJ, Song TQ, Li X, Sun YS, Peng YZ, Huang Z, Gao Q, Tang SQ, Zhang GH, Gong L. Subcellular localization of viral proteins after porcine epidemic diarrhea virus infection and their roles in the viral life cycle. Int J Biol Macromol 2024; 274:133401. [PMID: 38925184 DOI: 10.1016/j.ijbiomac.2024.133401] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Revised: 06/20/2024] [Accepted: 06/22/2024] [Indexed: 06/28/2024]
Abstract
Porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases affecting the pig industry globally. Due to the emergence of novel strains, no effective vaccines are available for prevention and control. Investigating the pathogenic mechanisms of PEDV may provide insights for creating clinical interventions. This study constructed and expressed eukaryotic expression vectors containing PEDV proteins (except NSP11) with a 3' HA tag in Vero cells. The subcellular localization of PEDV proteins was examined using endogenous protein antibodies to investigate their involvement in the viral life cycle, including endocytosis, intracellular trafficking, genome replication, energy metabolism, budding, and release. We systematically analyzed the potential roles of all PEDV viral proteins in the virus life cycle. We found that the endosome sorting complex required for transport (ESCRT) machinery may be involved in the replication and budding processes of PEDV. Our study provides insight into the molecular mechanisms underlying PEDV infection. IMPORTANCE: The global swine industry has suffered immense losses due to the spread of PEDV. Currently, there are no effective vaccines available for clinical protection. Exploring the pathogenic mechanisms of PEDV may provide valuable insights for clinical interventions. This study investigated the involvement of viral proteins in various stages of the PEDV lifecycle in the state of viral infection and identified several previously unreported interactions between viral and host proteins. These findings contribute to a better understanding of the pathogenic mechanisms underlying PEDV infection and may serve as a basis for further research and development of therapeutic strategies.
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Affiliation(s)
- Xiong-Nan Chen
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Shaoguan University, Shaoguan, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Shao-Tong Cai
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China
| | - Yi-Fan Liang
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Zhi-Jun Weng
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Tian-Qi Song
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Key Laboratory of Animal Vaccine Development, Ministry of Agriculture and Rural Affairs, People's Republic of China
| | - Xi Li
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Ying-Shuo Sun
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Yun-Zhao Peng
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Key Laboratory of Animal Vaccine Development, Ministry of Agriculture and Rural Affairs, People's Republic of China
| | - Zhao Huang
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Shaoguan University, Shaoguan, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Qi Gao
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China
| | - Sheng-Qiu Tang
- Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Shaoguan University, Shaoguan, People's Republic of China
| | - Gui-Hong Zhang
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China; Key Laboratory of Animal Vaccine Development, Ministry of Agriculture and Rural Affairs, People's Republic of China; National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou, People's Republic of China.
| | - Lang Gong
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China; Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong, People's Republic of China; Key Laboratory of Animal Vaccine Development, Ministry of Agriculture and Rural Affairs, People's Republic of China; National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou, People's Republic of China.
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41
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den Boon JA, Nishikiori M, Zhan H, Ahlquist P. Positive-strand RNA virus genome replication organelles: structure, assembly, control. Trends Genet 2024; 40:681-693. [PMID: 38724328 DOI: 10.1016/j.tig.2024.04.003] [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: 02/27/2024] [Revised: 04/09/2024] [Accepted: 04/10/2024] [Indexed: 08/09/2024]
Abstract
Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.
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Affiliation(s)
- Johan A den Boon
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Masaki Nishikiori
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Hong Zhan
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
| | - Paul Ahlquist
- Rowe Center for Virology, Morgridge Institute for Research, Madison, WI, USA; Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI; McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI.
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42
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Melo RCN, Silva TP. Eosinophil activation during immune responses: an ultrastructural view with an emphasis on viral diseases. J Leukoc Biol 2024; 116:321-334. [PMID: 38466831 DOI: 10.1093/jleuko/qiae058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 02/17/2024] [Accepted: 02/21/2024] [Indexed: 03/13/2024] Open
Abstract
Eosinophils are cells of the innate immune system that orchestrate complex inflammatory responses. The study of the cell biology of eosinophils, particularly associated with cell activation, is of great interest to understand their immune responses. From a morphological perspective, activated eosinophils show ultrastructural signatures that have provided critical insights into the comprehension of their functional capabilities. Application of conventional transmission electron microscopy in combination with quantitative assessments (quantitative transmission electron microscopy), molecular imaging (immunoEM), and 3-dimensional electron tomography have generated important insights into mechanisms of eosinophil activation. This review explores a multitude of ultrastructural events taking place in eosinophils activated in vitro and in vivo as key players in allergic and inflammatory diseases, with an emphasis on viral infections. Recent progress in our understanding of biological processes underlying eosinophil activation, including in vivo mitochondrial remodeling, is discussed, and it can bring new thinking to the field.
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Affiliation(s)
- Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Rua José Lourenço Kelmer, campus, Juiz de Fora, MG, 36036-900, Brazil
| | - Thiago P Silva
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Rua José Lourenço Kelmer, campus, Juiz de Fora, MG, 36036-900, Brazil
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43
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Bilotti K, Keep S, Sikkema AP, Pryor JM, Kirk J, Foldes K, Doyle N, Wu G, Freimanis G, Dowgier G, Adeyemi O, Tabatabaei SK, Lohman GJS, Bickerton E. One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system. PLoS One 2024; 19:e0307655. [PMID: 39052682 PMCID: PMC11271894 DOI: 10.1371/journal.pone.0307655] [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: 03/04/2024] [Accepted: 07/09/2024] [Indexed: 07/27/2024] Open
Abstract
Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.
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Affiliation(s)
- Katharina Bilotti
- New England Biolabs, Ipswich, Massachusetts, United States of America
| | - Sarah Keep
- The Pirbright Institute, Woking, United Kingdom
| | - Andrew P. Sikkema
- New England Biolabs, Ipswich, Massachusetts, United States of America
| | - John M. Pryor
- New England Biolabs, Ipswich, Massachusetts, United States of America
| | - James Kirk
- The Pirbright Institute, Woking, United Kingdom
| | | | | | - Ge Wu
- The Pirbright Institute, Woking, United Kingdom
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Caohuy H, Eidelman O, Chen T, Mungunsukh O, Yang Q, Walton NI, Pollard BS, Khanal S, Hentschel S, Florez C, Herbert AS, Pollard HB. Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 spike protein. Sci Rep 2024; 14:16895. [PMID: 39043712 PMCID: PMC11266487 DOI: 10.1038/s41598-024-66473-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Accepted: 07/01/2024] [Indexed: 07/25/2024] Open
Abstract
SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air-liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.
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Affiliation(s)
- Hung Caohuy
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Collaborative Health Initiative Research Program (CHIRP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Consortium for Health and Military Performance (CHAMP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | - Ofer Eidelman
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Collaborative Health Initiative Research Program (CHIRP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | - Tinghua Chen
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Collaborative Health Initiative Research Program (CHIRP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Consortium for Health and Military Performance (CHAMP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | - Ognoon Mungunsukh
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Consortium for Health and Military Performance (CHAMP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Center for Military Precision Health, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | - Qingfeng Yang
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Center for the Study of Traumatic Stress (CSTS), and Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | - Nathan I Walton
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Collaborative Health Initiative Research Program (CHIRP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
- Consortium for Health and Military Performance (CHAMP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA
| | | | - Sara Khanal
- Virology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD, 21702, USA
- The Geneva Foundation, Tacoma, WA, 98402, USA
| | - Shannon Hentschel
- Virology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD, 21702, USA
- Cherokee Nation Assurance, Catoosa, OK, 74015, USA
| | - Catalina Florez
- Virology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD, 21702, USA
- The Geneva Foundation, Tacoma, WA, 98402, USA
| | - Andrew S Herbert
- Virology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD, 21702, USA
| | - Harvey B Pollard
- Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.
- Collaborative Health Initiative Research Program (CHIRP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.
- Consortium for Health and Military Performance (CHAMP), Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.
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Girard J, Le Bihan O, Lai-Kee-Him J, Girleanu M, Bernard E, Castellarin C, Chee M, Neyret A, Spehner D, Holy X, Favier AL, Briant L, Bron P. In situ fate of Chikungunya virus replication organelles. J Virol 2024; 98:e0036824. [PMID: 38940586 PMCID: PMC11265437 DOI: 10.1128/jvi.00368-24] [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: 02/26/2024] [Accepted: 06/02/2024] [Indexed: 06/29/2024] Open
Abstract
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.
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Affiliation(s)
- Justine Girard
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
- Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS, Montpellier, France
| | - Olivier Le Bihan
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Joséphine Lai-Kee-Him
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
| | - Maria Girleanu
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Eric Bernard
- Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS, Montpellier, France
| | - Cedric Castellarin
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Matthew Chee
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
| | - Aymeric Neyret
- Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS, Montpellier, France
| | - Danièle Spehner
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Xavier Holy
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Anne-Laure Favier
- Institut de Recherche Biomédicale des Armées (IRBA), Ministère des armées, Brétigny-sur-Orge, France
| | - Laurence Briant
- Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS, Montpellier, France
| | - Patrick Bron
- Centre de Biologie Structurale (CBS), Université de Montpellier, CNRS, INSERM, Montpellier, France
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Abstract
Coronavirus Disease-19 (COVID-19) pandemic is caused by SARS-CoV-2 that has infected more than 600 million people and killed more than 6 million people worldwide. This infection affects mainly certain groups of people that have high susceptibility to present severe COVID-19 due to comorbidities. Moreover, the long-COVID-19 comprises a series of symptoms that may remain in some patients for months after infection that further compromises their health. Thus, since this pandemic is profoundly affecting health, economy, and social life of societies, a deeper understanding of viral replication cycle could help to envisage novel therapeutic alternatives that limit or stop COVID-19. Several findings have unexpectedly discovered that mitochondria play a critical role in SARS-CoV-2 cell infection. Indeed, it has been suggested that this organelle could be the origin of its replication niches, the double membrane vesicles (DMV). In this regard, mitochondria derived vesicles (MDV), involved in mitochondria quality control, discovered almost 15 years ago, comprise a subpopulation characterized by a double membrane. MDV shedding is induced by mitochondrial stress, and it has a fast assembly dynamic, reason that perhaps has precluded their identification in electron microscopy or tomography studies. These and other features of MDV together with recent SARS-CoV-2 protein interactome and other findings link SARS-CoV-2 to mitochondria and support that these vesicles are the precursors of SARS-CoV-2 induced DMV. In this work, the morphological, biochemical, molecular, and cellular evidence that supports this hypothesis is reviewed and integrated into the current model of SARS-CoV-2 cell infection. In this scheme, some relevant questions are raised as pending topics for research that would help in the near future to test this hypothesis. The intention of this work is to provide a novel framework that could open new possibilities to tackle SARS-CoV-2 pandemic through mitochondria and DMV targeted therapies.
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Affiliation(s)
- Pavel Montes de Oca-B
- Neurociencia Cognitiva, Instituto de Fisiologia-UNAM, CDMX, CDMX, 04510, Mexico
- Unidad de Neurobiologia Dinamica, Instituto Nacional de Neurologia y Neurocirugia, CDMX, CDMX, 14269, Mexico
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47
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Park H, Higgs PG. Evolution of RNA Viruses: Reasons for the Existence of Separate Plus, Minus, and Double-Strand Replication Strategies. Viruses 2024; 16:1081. [PMID: 39066243 PMCID: PMC11281585 DOI: 10.3390/v16071081] [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: 06/07/2024] [Revised: 06/28/2024] [Accepted: 07/02/2024] [Indexed: 07/28/2024] Open
Abstract
Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.
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Affiliation(s)
| | - Paul G. Higgs
- Department of Physics and Astronomy, McMaster University, Hamilton, ON L8S 4L8, Canada;
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48
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Miranda GASC, Corrêa IA, Amorim ÉA, Caldas LA, Carneiro FÁ, da Costa LJ, Granjeiro JM, Tanuri A, de Souza W, Baptista LS. Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening. Artif Organs 2024; 48:723-733. [PMID: 38385713 DOI: 10.1111/aor.14729] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 01/15/2024] [Accepted: 02/07/2024] [Indexed: 02/23/2024]
Abstract
BACKGROUND The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.
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Affiliation(s)
| | - Isadora Alonso Corrêa
- Laboratório de Genética e Imunologia das Infecções Virais, Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Érica Almeida Amorim
- Gcell 3D, Rio de Janeiro, Brazil
- Laboratório de Ultraestrutura celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Lucio Ayres Caldas
- Laboratório de Ultraestrutura celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Núcleo Multidisciplinar de Pesquisa (Numpex-bio), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Fabiana Ávila Carneiro
- Laboratório de Ultraestrutura celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Núcleo Multidisciplinar de Pesquisa (Numpex-bio), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Luciana Jesus da Costa
- Laboratório de Genética e Imunologia das Infecções Virais, Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - José Mauro Granjeiro
- Laboratório de Biologia de Células Eucarióticas, Duque de Caxias, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil
- Laboratório de Pesquisa Clínica em Odontologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil
| | - Amilcar Tanuri
- Laboratório de Genética e Imunologia das Infecções Virais, Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Wanderley de Souza
- Laboratório de Ultraestrutura celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Centro de Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Leandra Santos Baptista
- Núcleo Multidisciplinar de Pesquisa (Numpex-bio), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Laboratório de Biologia de Células Eucarióticas, Duque de Caxias, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil
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Prasanth MI, Wannigama DL, Reiersen AM, Thitilertdecha P, Prasansuklab A, Tencomnao T, Brimson S, Brimson JM. A systematic review and meta-analysis, investigating dose and time of fluvoxamine treatment efficacy for COVID-19 clinical deterioration, death, and Long-COVID complications. Sci Rep 2024; 14:13462. [PMID: 38862591 PMCID: PMC11166997 DOI: 10.1038/s41598-024-64260-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Accepted: 06/06/2024] [Indexed: 06/13/2024] Open
Abstract
There have been 774,075,242 cases of COVID-19 and 7,012,986 deaths worldwide as of January 2024. In the early stages of the pandemic, there was an urgent need to reduce the severity of the disease and prevent the need for hospitalization to avoid stress on healthcare systems worldwide. The repurposing of drugs to prevent clinical deterioration of COVID-19 patients was trialed in many studies using many different drugs. Fluvoxamine (an SSRI and sigma-1 receptor agonist) was initially identified to potentially provide beneficial effects in COVID-19-infected patients, preventing clinical deterioration and the need for hospitalization. Fourteen clinical studies have been carried out to date, with seven of those being randomized placebo-controlled studies. This systematic review and meta-analysis covers the literature from the outbreak of SARS-CoV-2 in late 2019 until January 2024. Search terms related to fluvoxamine, such as its trade names and chemical names, along with words related to COVID-19, such as SARS-CoV-2 and coronavirus, were used in literature databases including PubMed, Google Scholar, Scopus, and the ClinicalTrials.gov database from NIH, to identify the trials used in the subsequent analysis. Clinical deterioration and death data were extracted from these studies where available and used in the meta-analysis. A total of 7153 patients were studied across 14 studies (both open-label and double-blind placebo-controlled). 681 out of 3553 (19.17%) in the standard care group and 255 out of 3600 (7.08%) in the fluvoxamine-treated group experienced clinical deterioration. The estimated average log odds ratio was 1.087 (95% CI 0.200 to 1.973), which differed significantly from zero (z = 2.402, p = 0.016). The seven placebo-controlled studies resulted in a log odds ratio of 0.359 (95% CI 0.1111 to 0.5294), which differed significantly from zero (z = 3.103, p = 0.002). The results of this study identified fluvoxamine as effective in preventing clinical deterioration, and subgrouping analysis suggests that earlier treatment with a dose of 200 mg or above provides the best outcomes. We hope the outcomes of this study can help design future studies into respiratory viral infections and potentially improve clinical outcomes.
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Affiliation(s)
- Mani Iyer Prasanth
- Natural Products for Neuroprotection and Anti-Ageing (Neur-Age Natura) Research Unit, Chulalongkorn University, Bangkok, 10330, Thailand
- Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Dhammika Leshan Wannigama
- Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata, Japan
- Department of Microbiology, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Chulalongkorn University, Thai Red Cross Society, Bangkok, Thailand
- Yamagata Prefectural University of Health Sciences, Kamiyanagi, Yamagata, 990-2212, Japan
- Pathogen Hunter's Research Collaborative Team, Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata, Japan
| | - Angela Michelle Reiersen
- Department of Psychiatry, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA
| | - Premrutai Thitilertdecha
- Siriraj Research Group in Immunobiology and Therapeutic Sciences, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Anchalee Prasansuklab
- Natural Products for Neuroprotection and Anti-Ageing (Neur-Age Natura) Research Unit, Chulalongkorn University, Bangkok, 10330, Thailand
- College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Tewin Tencomnao
- Natural Products for Neuroprotection and Anti-Ageing (Neur-Age Natura) Research Unit, Chulalongkorn University, Bangkok, 10330, Thailand
- Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - Sirikalaya Brimson
- Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand
| | - James Michael Brimson
- Natural Products for Neuroprotection and Anti-Ageing (Neur-Age Natura) Research Unit, Chulalongkorn University, Bangkok, 10330, Thailand.
- Research, Innovation and International Affairs, Faculty of Allied Health Sciences, Chulalongkorn University, 154 Rama 1 Road, Pathumwan, Wang Mai, Bangkok, 10330, Thailand.
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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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