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Zhang DW, Xu XS, Xie L, Xu L, Fu Z, Li Y, Xu X. Natural product sennoside B disrupts liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein by inhibiting its RNA-binding activity. J Enzyme Inhib Med Chem 2025; 40:2501743. [PMID: 40371698 PMCID: PMC12082725 DOI: 10.1080/14756366.2025.2501743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2025] [Revised: 04/02/2025] [Accepted: 04/29/2025] [Indexed: 05/16/2025] Open
Abstract
The nucleocapsid protein (NP) of SARS-CoV-2, an RNA-binding protein, is capable of undergoing liquid-liquid phase separation (LLPS) during viral infection, which plays a crucial role in virus assembly, replication, and immune regulation. In this study, we developed a homogeneous time-resolved fluorescence (HTRF) method for identifying inhibitors of the SARS-CoV-2 NP-RNA interaction. Using this HTRF-based approach, we identified two natural products, sennoside A and sennoside B, as effective blockers of this interaction. Bio-layer interferometry assays confirmed that both sennosides directly bind to the NP, with binding sites located within the C-terminal domain. Additionally, fluorescence recovery after photobleaching (FRAP) experiments revealed that sennoside B significantly inhibited RNA-induced LLPS of the NP, while sennoside A displayed comparatively weaker activity. Thus, the developed HTRF-based assay is a valuable tool for identifying novel compounds that disrupt the RNA-binding activity and LLPS of the SARS-CoV-2 NP. Our findings may facilitate the development of antiviral drugs targeting SARS-CoV-2 NP.
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Affiliation(s)
- Da-Wei Zhang
- Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
| | - Xiao-Shuang Xu
- Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
| | - Liangxu Xie
- Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
| | - Lei Xu
- Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
| | - Zhiguo Fu
- Department of Orthopedics, Changzhou Hospital of Traditional Chinese Medicine, Changzhou, China
| | - Yimin Li
- College of Pharmacy and Key Laboratory for Research and Development of "Qin Medicine" of Shaanxi Administration of Chinese Medicine, Shaanxi University of Chinese Medicine, Xixian New District, China
| | - Xiaojun Xu
- Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
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2
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Inamoto S, Yoshida A, Yamagishi A, Kaneko N, Otsuka Y. Cryo-transfer energy dispersive X-ray spectroscopic tomography for morphological and elemental analysis of moisture-containing sunscreen creams. Micron 2025; 195:103824. [PMID: 40233543 DOI: 10.1016/j.micron.2025.103824] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2025] [Revised: 04/08/2025] [Accepted: 04/09/2025] [Indexed: 04/17/2025]
Abstract
Electron tomography based on a tilt-series of energy dispersive X-ray spectroscopy (EDX) maps is effective for analyzing the three-dimensional elemental distribution of materials containing multiple elements. However, EDX mapping requires a high electron dose, making it challenging to apply EDX tomography to electron-beam-sensitive materials. In this study, we demonstrated cryo-transfer EDX tomography of water-containing sunscreen cream, which easily dissolves under electron beam irradiation. Under optimal experimental conditions, the three-dimensional dispersion state of titanium oxide particles, zinc oxide particles, silicone oil, and emulsified water particles in the sunscreen cream was successfully visualized. This study provides valuable insights into the dispersion state of sunscreen components, which is crucial for improving product design and performance.
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Affiliation(s)
- Shin Inamoto
- Toray Research Center, Inc., 2-11, Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan.
| | - Akiyo Yoshida
- Toray Research Center, Inc., 2-11, Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan
| | - Ayaka Yamagishi
- Toray Research Center, Inc., 2-11, Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan
| | - Naoto Kaneko
- Toray Research Center, Inc., 2-11, Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan
| | - Yuji Otsuka
- Toray Research Center, Inc., 2-11, Sonoyama 3-chome, Otsu, Shiga 520-8567, Japan
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3
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Loonen S, van Steenis L, Bauer M, Šoštarić N. Phosphorylation Changes SARS-CoV-2 Nucleocapsid Protein's Structural Dynamics and Its Interaction With RNA. Proteins 2025. [PMID: 40375582 DOI: 10.1002/prot.26842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2024] [Revised: 05/01/2025] [Accepted: 05/07/2025] [Indexed: 05/18/2025]
Abstract
The SARS-CoV-2 nucleocapsid protein, or N-protein, is a structural protein that plays an important role in the SARS-CoV-2 life cycle. The N-protein takes part in the regulation of viral RNA replication and drives highly specific packaging of full-length genomic RNA prior to virion formation. One regulatory mechanism that is proposed to drive the switch between these two operating modes is the phosphorylation state of the N-protein. Here, we assess the dynamic behavior of non-phosphorylated and phosphorylated versions of the N-protein homodimer through atomistic molecular dynamics simulations. We show that the introduction of phosphorylation yields a more dynamic protein structure and decreases the binding affinity between the N-protein and RNA. Furthermore, we find that secondary structure is essential for the preferential binding of particular RNA elements from the 5' UTR of the viral genome to the N-terminal domain of the N-protein. Altogether, we provide detailed molecular insights into N-protein dynamics, N-protein:RNA interactions, and phosphorylation. Our results corroborate the hypothesis that phosphorylation of the N-protein serves as a regulatory mechanism that determines N-protein function.
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Affiliation(s)
- Stefan Loonen
- Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, HZ, the Netherlands
| | - Lina van Steenis
- Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, HZ, the Netherlands
| | - Marianne Bauer
- Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, HZ, the Netherlands
| | - Nikolina Šoštarić
- Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, HZ, the Netherlands
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4
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Aliyari SR, Xie G, Xia X, Wang L, Zhou ZH, Cheng G. Infectivity and structure of SARS-CoV-2 after hydrogen peroxide treatment. mBio 2025; 16:e0399424. [PMID: 40257280 PMCID: PMC12077155 DOI: 10.1128/mbio.03994-24] [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: 01/15/2025] [Accepted: 02/18/2025] [Indexed: 04/22/2025] Open
Abstract
Hydrogen peroxide (H2O2) exhibits broad-spectrum antiviral activity and is commonly used as an over-the-counter disinfecting agent. However, its potential activities against SARS-CoV-2 have not been systematically evaluated, and mechanisms of action are not well understood. In this study, we investigate H2O2's antiviral activity against SARS-CoV-2 infection and its impact on the virion's structural integrity as compared to the commonly used fixative agent paraformaldehyde (PFA). We show that H2O2 rapidly and directly inactivates SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 0.0015%. Cryogenic electron tomography (cryo-ET) with subtomogram averaging reveals that treatment with PFA induced the viral trimeric spike protein (S) to adopt a post-fusion conformation, and treatment of viral particles with H2O2 locked S in its pre-fusion conformation. Therefore, H2O2 treatment likely has induced modifications, such as oxidation of cysteine residues within the S subunits of the spike trimer that locked them in their pre-fusion conformation. Locking of the meta-stable pre-fusion trimer prevents its transition to the post-fusion conformation, a process essential for viral fusion with host cells and entry into host cells. Together, our cellular, biochemical, and structural studies established that hydrogen peroxide can inactivate SARS-CoV-2 in tissue culture and uncovered its underlying molecular mechanism.IMPORTANCEHydrogen peroxide (H2O2) is the commonly used, over-the-counter antiseptic solution available in pharmacies, but its effect against the SARS-CoV-2 virus has not been evaluated systematically. In this study, we show that H2O2 inactivates the SARS-CoV-2 infectivity and establish the effective concentration of this activity. Cryogenic electron tomography and sub-tomogram averaging reveal a detailed structural understanding of how H2O2 affects the SARS-CoV-2 spike in comparison with that of the commonly used fixative PFA under identical conditions. We found that PFA promoted a post-fusion conformation of the viral spike protein, while H2O2 could potentially lock the spike in its pre-fusion state. Our findings not only substantiate the disinfectant efficacy of H2O2 as a potent agent against SARS-CoV-2 but also lay the groundwork for future investigations into targeted antiviral therapies that may leverage the virus' structural susceptibilities. In addition, this study may have significant implications for developing new antiviral strategies and improving existing disinfection protocols.
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Affiliation(s)
- Saba R. Aliyari
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
| | - Guodong Xie
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
- California NanoSystems Institute, UCLA, Los Angeles, California, USA
| | - Xian Xia
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
- California NanoSystems Institute, UCLA, Los Angeles, California, USA
| | - Lulan Wang
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
| | - Z. Hong Zhou
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
- California NanoSystems Institute, UCLA, Los Angeles, California, USA
| | - Genhong Cheng
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California, USA
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5
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Augusto I, Lemos M, Girard-Dias W, Oliveira Filho JDA, Pascutti PG, de Souza W, Miranda K. New dimensions in acidocalcisome research: the potential of cryo-EM to uncover novel aspects of protozoan parasite physiology. mBio 2025; 16:e0166224. [PMID: 40197013 PMCID: PMC12077218 DOI: 10.1128/mbio.01662-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/09/2025] Open
Abstract
Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling high-resolution, near-native visualization of macromolecular structures and entire cells. Its application to etiologic agents of diseases is an expanding field, particularly for those caused by viruses or unicellular eukaryotes, such as protozoan parasites and fungi. This review focuses on acidocalcisomes-ion-rich, multifunctional organelles essential for cell physiology and survival in several pathogens. The structure and function of these organelles are examined through a range of electron microscopy techniques, using Trypanosoma cruzi as a model. The advantages and limitations of the methods employed to study acidocalcisome morphofunctional organization-such as chemical fixation, plunge and high-pressure freezing, cryo-electron microscopy of vitrified sections (CEMOVIS), freeze-drying, freeze substitution, tomography, and microanalysis using X rays and inelastic scattered electrons-are discussed, alongside their contributions to our current understanding of acidocalcisome structure and function. Recent advances in cryo-EM and its potential to address longstanding questions and fill existing gaps in our understanding of parasite ion mobilization mechanisms and physiology are also discussed.
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Affiliation(s)
- Ingrid Augusto
- Laboratório de Ultraestrutura Celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem—Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Moara Lemos
- Laboratório de Ultraestrutura Celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Institut Pasteur, Paris, France
| | - Wendell Girard-Dias
- Laboratório de Ultraestrutura Celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Plataforma de Microscopia Eletrônica Rudolf Barth, Instituto Oswaldo Cruz–Fiocruz, Rio de Janeiro, Brazil
| | - José de Anchieta Oliveira Filho
- Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Pedro G. Pascutti
- Laboratório de Modelagem e Dinâmica Molecular, Instituto de Biofísica Carlos Chagas Filho, 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 and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem—Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Centro Multiusuário para Análise de Fenômenos Biomédicos, Universidade do Estado do Amazonas, Amazonas, Brazil
| | - Kildare Miranda
- Laboratório de Ultraestrutura Celular Hertha Meyer, Centro de Pesquisa em Medicina de Precisão, Instituto de Biofísica Carlos Chagas Filho and Centro Nacional de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem—Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- Centro Multiusuário para Análise de Fenômenos Biomédicos, Universidade do Estado do Amazonas, Amazonas, Brazil
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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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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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Manwatkar S, Vaidyanathan R, Chaudhary N, Kumar A, Priyadarshini P, Bagaria D, Gupta A, Sagar S, Kumar S, Mishra B. Impact of SARS-CoV-2 Infection on the Outcomes of Trauma Patients at a Level I Trauma Center: An Ambispective Observational Study. Cureus 2025; 17:e82162. [PMID: 40370923 PMCID: PMC12076266 DOI: 10.7759/cureus.82162] [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] [Accepted: 04/12/2025] [Indexed: 05/16/2025] Open
Abstract
INTRODUCTION Trauma remained a leading cause of hospital admissions even during the COVID-19 pandemic. Trauma and surgical interventions are known to impair the patient's immune function. Clinically, some asymptomatic COVID-19 patients experienced rapid deterioration following surgery. Surgeons and anesthesiologists need to be aware that acute lung injury caused by COVID-19 could be present preoperatively or may worsen postoperatively. Hence, an ambispective observational study was planned to assess the impact of severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection on trauma patient outcomes. AIMS AND OBJECTIVES This study aims to evaluate the impact of SARS-CoV-2 infection on the outcomes of trauma patients at a level I trauma center. MATERIALS AND METHODS This ambispective observational study was conducted at a level 1 trauma center and included patients admitted under the trauma surgery service in the COVID-19 facility. Their outcomes were compared with those of patients admitted to the non-COVID-19 facility from March 2020 to March 2022. RESULTS A total of 2,017 patients were admitted under the Division of Trauma Surgery and Critical Care from March 2020 to March 2022. The mean duration of intercostal drainage (ICD) was significantly longer in SARS-CoV-2-positive trauma patients (7.03 ± 3.69 days) compared to SARS-CoV-2-negative trauma patients (5.28 ± 2.75). Acute respiratory distress syndrome (ARDS) was also more common among SARS-CoV-2-positive trauma patients. Additionally, these patients had a longer hospital stay. Notably, SARS-CoV-2-positive trauma patients who died had a significantly lower average injury severity score (ISS) compared to SARS-CoV-2-negative counterparts. DISCUSSION Although the average ISS was lower and the average trauma and injury severity score (TRISS) was higher in SARS-CoV-2-positive trauma patients who died compared to SARS-CoV-2-negative trauma patients, overall mortality rates were comparable between the two groups. CONCLUSION Trauma patients with concomitant SARS-CoV-2 infection had a longer duration of ICD, along with an increased incidence of chest infections and ARDS. A greater proportion of SARS-CoV-2-positive trauma patients required ventilatory support. The mortality observed in SARS-CoV-2-positive trauma patients is likely attributed to the concomitant SARS-CoV-2 infection.
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Affiliation(s)
- Shrikant Manwatkar
- Trauma Surgery and Critical Care, Command Hospital Air Force, Bengaluru, IND
| | - Ramesh Vaidyanathan
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Narendra Chaudhary
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Abhinav Kumar
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | | | - Dinesh Bagaria
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Amit Gupta
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Sushma Sagar
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Subodh Kumar
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
| | - Biplab Mishra
- Trauma Surgery and Critical Care, All India Institute of Medical Sciences, New Delhi, IND
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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 PMCID: PMC12131028 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] [Download PDF] [Figures] [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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Kubinski HC, Despres HW, Johnson BA, Schmidt MM, Jaffrani SA, Turner AH, Fanuele CD, Mills MG, Lokugamage KG, Dumas CM, Shirley DJ, Estes LK, Pekosz A, Crothers JW, Roychoudhury P, Greninger AL, Jerome KR, Di Genova BM, Walker DH, Ballif BA, Ladinsky MS, Bjorkman PJ, Menachery VD, Bruce EA. Variant mutation G215C in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation. PLoS Biol 2025; 23:e3003115. [PMID: 40299982 PMCID: PMC12040272 DOI: 10.1371/journal.pbio.3003115] [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: 02/19/2025] [Accepted: 03/12/2025] [Indexed: 05/01/2025] Open
Abstract
The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of spike are not well studied, the entire viral genome is undergoing evolutionary selection, with several variants containing mutations in the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a more stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Mechanistically, we show that the N:G215C mutant has more encapsidation as measured by increased RNA binding to N, N incorporation into virions, and electron microscopy showing that individual virions are larger, with elongated morphologies.
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Affiliation(s)
- Hannah C. Kubinski
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Hannah W. Despres
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Bryan A. Johnson
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America
- Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, Texas, United States of America
- Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Madaline M. Schmidt
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Sara A. Jaffrani
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Allyson H. Turner
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Conor D. Fanuele
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Margaret G. Mills
- Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America
| | - Kumari G. Lokugamage
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Caroline M. Dumas
- Department of Biology, University of Vermont, Burlington, Vermont, United States of America
| | - David J. Shirley
- Faraday, Inc. Data Science Department, Burlington, Vermont, United States of America
| | - Leah K. Estes
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Andrew Pekosz
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Jessica W. Crothers
- Department of Pathology and Laboratory Medicine, Robert Larner, MD College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - Pavitra Roychoudhury
- Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America
| | - Alexander L. Greninger
- Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle Washington, United States of America
| | - Keith R. Jerome
- Virology Division, Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle Washington, United States of America
| | - Bruno Martorelli Di Genova
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
| | - David H. Walker
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America
- Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston, Texas, United States of America
- Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America
- Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Bryan A. Ballif
- Department of Biology, University of Vermont, Burlington, Vermont, United States of America
| | - Mark S. Ladinsky
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America
| | - Pamela J. Bjorkman
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America
| | - Vineet D. Menachery
- Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America
- World Reference Center of Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, Texas, United States of America
- Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America
- Department of Pediatrics and Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America
| | - Emily A. Bruce
- Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, Vermont, United States of America
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11
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Streif S, Baeumner AJ. Advances in Surrogate Neutralization Tests for High-Throughput Screening and the Point-of-Care. Anal Chem 2025; 97:5407-5423. [PMID: 40035475 PMCID: PMC11923957 DOI: 10.1021/acs.analchem.5c00666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/05/2025]
Affiliation(s)
- Simon Streif
- Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany
| | - Antje J Baeumner
- Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany
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12
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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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13
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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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14
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Baruah N, Midya J, Gompper G, Dasanna AK, Auth T. Adhesion-driven vesicle translocation through membrane-covered pores. Biophys J 2025; 124:740-752. [PMID: 39863923 PMCID: PMC11897550 DOI: 10.1016/j.bpj.2025.01.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 11/24/2024] [Accepted: 01/21/2025] [Indexed: 01/27/2025] Open
Abstract
Translocation across barriers and through constrictions is a mechanism that is often used in vivo for transporting material between compartments. A specific example is apicomplexan parasites invading host cells through the tight junction that acts as a pore, and a similar barrier crossing is involved in drug delivery using lipid vesicles penetrating intact skin. Here, we use triangulated membranes and energy minimization to study the translocation of vesicles through pores with fixed radii. The vesicles bind to a lipid bilayer spanning the pore, the adhesion-energy gain drives the translocation, and the vesicle deformation induces an energy barrier. In addition, the deformation-energy cost for deforming the pore-spanning membrane hinders the translocation. Increasing the bending rigidity of the pore-spanning membrane and decreasing the pore size both increase the barrier height and shift the maximum to smaller fractions of translocated vesicle membrane. We compare the translocation of initially spherical vesicles with fixed membrane area and freely adjustable volume to that of initially prolate vesicles with fixed membrane area and volume. In the latter case, translocation can be entirely suppressed. Our predictions may help rationalize the invasion of apicomplexan parasites into host cells and design measures to combat the diseases they transmit.
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Affiliation(s)
- Nishant Baruah
- Theoretical Physics of Living Matter, Institute of Biological Information Processing and Institute for Advanced Simulation, Forschungszentrum Jülich, Jülich, Germany.
| | - Jiarul Midya
- Theoretical Physics of Living Matter, Institute of Biological Information Processing and Institute for Advanced Simulation, Forschungszentrum Jülich, Jülich, Germany; School of Basic Sciences, Indian Institute of Technology, Bhubaneswar, India.
| | - Gerhard Gompper
- Theoretical Physics of Living Matter, Institute of Biological Information Processing and Institute for Advanced Simulation, Forschungszentrum Jülich, Jülich, Germany.
| | - Anil Kumar Dasanna
- Department of Theoretical Physics and Center for Biophysics, Saarland University, Saarbrücken, Germany; INM-Leibniz Institute for New Materials, Saarbrücken, Germany; Department of Physical Sciences, Indian Institute of Science Education and Research Mohali, Knowledge City, Manauli, India.
| | - Thorsten Auth
- Theoretical Physics of Living Matter, Institute of Biological Information Processing and Institute for Advanced Simulation, Forschungszentrum Jülich, Jülich, Germany.
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15
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Ruma YN, Nannenga BL, Gonen T. Unraveling atomic complexity from frozen samples. STRUCTURAL DYNAMICS (MELVILLE, N.Y.) 2025; 12:020901. [PMID: 40255534 PMCID: PMC12009148 DOI: 10.1063/4.0000303] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/24/2025] [Accepted: 03/26/2025] [Indexed: 04/22/2025]
Abstract
Cryo-electron microscopy (cryo-EM) is a significant driver of recent advances in structural biology. Cryo-EM is comprised of several distinct and complementary methods, which include single particle analysis, cryo-electron tomography, and microcrystal electron diffraction. In this Perspective, we will briefly discuss the different branches of cryo-EM in structural biology and the current challenges in these areas.
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Affiliation(s)
| | | | - Tamir Gonen
- Author to whom correspondence should be addressed:
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16
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Rosas‐Lemus M, Minasov G, Brunzelle JS, Taha TY, Lemak S, Yin S, Shuvalova L, Rosecrans J, Khanna K, Seifert HS, Savchenko A, Stogios PJ, Ott M, Satchell KJF. Torsional twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains. Protein Sci 2025; 34:e70050. [PMID: 39969084 PMCID: PMC11837046 DOI: 10.1002/pro.70050] [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: 08/13/2024] [Revised: 01/03/2025] [Accepted: 01/19/2025] [Indexed: 02/20/2025]
Abstract
Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.
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Affiliation(s)
- Monica Rosas‐Lemus
- Department of Microbiology‐Immunology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
- Center for Structural Biology of Infectious Diseases, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
- Present address:
Department of Molecular Genetics and Microbiology, School of Medicine, Health Sciences CenterUniversity of New MexicoAlbuquerqueNew MexicoUSA
| | - George Minasov
- Department of Microbiology‐Immunology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
- Center for Structural Biology of Infectious Diseases, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
| | - Joseph S. Brunzelle
- Center for Structural Biology of Infectious Diseases, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
- Northwestern Synchrotron Research Center, Life Sciences Collaborative Access TeamNorthwestern UniversityArgonneIllinoisUSA
| | - Taha Y. Taha
- Gladstone Institute of VirologyGladstone InstitutesSan FranciscoCaliforniaUSA
| | - Sofia Lemak
- BioZone, Department of Chemical Engineering and Applied ChemistryUniversity of TorontoTorontoOntarioCanada
| | - Shaohui Yin
- Department of Microbiology‐Immunology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
| | - Ludmilla Shuvalova
- Department of Pharmacology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
| | - Julia Rosecrans
- Gladstone Institute of VirologyGladstone InstitutesSan FranciscoCaliforniaUSA
| | - Kanika Khanna
- Gladstone Institute of VirologyGladstone InstitutesSan FranciscoCaliforniaUSA
| | - H. Steven Seifert
- Department of Microbiology‐Immunology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
| | - Alexei Savchenko
- Department of Microbiology, Immunology and Infectious DiseasesUniversity of CalgaryCalgaryAlbertaCanada
| | - Peter J. Stogios
- Department of Microbiology, Immunology and Infectious DiseasesUniversity of CalgaryCalgaryAlbertaCanada
| | - Melanie Ott
- Gladstone Institute of VirologyGladstone InstitutesSan FranciscoCaliforniaUSA
- Department of MedicineUniversity of California at San FranciscoSan FranciscoCaliforniaUSA
| | - Karla J. F. Satchell
- Department of Microbiology‐Immunology, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
- Center for Structural Biology of Infectious Diseases, Feinberg School of MedicineNorthwestern UniversityChicagoIllinoisUSA
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17
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Dewangan N, Jana ID, Yadav S, Sardar A, Mallick AI, Mondal A, Tarafdar PK. Design of Flavonoid-Based Lipid Domains as Fusion Inhibitors to Efficiently Block Coronavirus and Other Enveloped Virus Infection. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2025; 21:e2410727. [PMID: 39828665 DOI: 10.1002/smll.202410727] [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: 11/11/2024] [Revised: 12/27/2024] [Indexed: 01/22/2025]
Abstract
Developing a broad-spectrum antiviral is imperative in light of the recent emergence of recurring viral infections. The critical role of host-virus attachment and membrane fusion during enveloped virus entry is a suitable target for developing broad-spectrum antivirals. A new class of flavonoid-based fusion inhibitors are designed to alter the membrane's physical properties. These flavonoid-based molecules (MFDA; myristoyl flavonoid di-aspartic acid) are self-assembled in the membrane, creating distinct nanodomains and effectively inhibiting membrane fusion by modulating the membrane's interfacial properties. The broad-spectrum antiviral efficacy of these compounds are established in effectively blocking the entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Type A Influenza, Human coronavirus OC43 (HCoV-OC43), and Vesicular stomatitis virus (VSV). A slightly more effectivity of MFDA in coronavirus infection than other enveloped viruses may be attributed to its secondary interaction with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. A membrane nanodomain formation strategy is highlighted with natural-product-based fusion inhibitors, effectively thwarting the infection of several enveloped viruses, entailing their broad-spectrum antiviral functionality.
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Affiliation(s)
- Nikesh Dewangan
- Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India
| | - Indrani Das Jana
- Department of Bioscience & Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Sandeep Yadav
- Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India
| | - Avijit Sardar
- Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India
| | - Amirul I Mallick
- Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India
| | - Arindam Mondal
- Department of Bioscience & Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Pradip K Tarafdar
- Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India
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18
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Farci D, Graça AT, Hall M, Haniewicz P, Kereïche S, Faull P, Kirkpatrick J, Tramontano E, Schröder WP, Piano D. Characterization of SARS-CoV-2 nucleocapsid protein oligomers. J Struct Biol 2025; 217:108162. [PMID: 39675446 DOI: 10.1016/j.jsb.2024.108162] [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: 08/25/2024] [Revised: 11/05/2024] [Accepted: 12/12/2024] [Indexed: 12/17/2024]
Abstract
Oligomers of the SARS-CoV-2 nucleocapsid (N) protein are characterized by pronounced instability resulting in fast degradation. This property likely relates to two contrasting behaviors of the N protein: genome stabilization through a compact nucleocapsid during cell evasion and genome release by nucleocapsid disassembling during infection. In vivo, the N protein forms rounded complexes of high molecular mass from its interaction with the viral genome. To study the N protein and understand its instability, we analyzed degradation profiles under different conditions by size-exclusion chromatography and characterized samples by mass spectrometry and cryo-electron microscopy. We identified self-cleavage properties of the N protein based on specific Proprotein convertases activities, with Cl- playing a key role in modulating stability and degradation. These findings allowed isolation of a stable oligomeric complex of N, for which we report the 3D structure at ∼6.8 Å resolution. Findings are discussed considering available knowledge about the coronaviruses' infection cycle.
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Affiliation(s)
- Domenica Farci
- Department of Plant Physiology, Institute of Biology, Warsaw University of Life Sciences - SGGW, Warsaw, Poland; Department of Chemistry, Umeå University, Umeå, Sweden; Laboratory of Plant Physiology and Photobiology, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy; ReGenFix Laboratories, R&D Department, Sardara, Italy.
| | - André T Graça
- Department of Chemistry, Umeå University, Umeå, Sweden
| | - Michael Hall
- Department of Chemistry, Umeå University, Umeå, Sweden
| | - Patrycja Haniewicz
- Department of Plant Physiology, Institute of Biology, Warsaw University of Life Sciences - SGGW, Warsaw, Poland
| | - Sami Kereïche
- Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Peter Faull
- The Francis Crick Institute, London, United Kingdom; Proteomics Facility, University of Texas at Austin, Austin, USA
| | | | - Enzo Tramontano
- Laboratory of Molecular Virology, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
| | | | - Dario Piano
- Department of Plant Physiology, Institute of Biology, Warsaw University of Life Sciences - SGGW, Warsaw, Poland; Laboratory of Plant Physiology and Photobiology, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy; ReGenFix Laboratories, R&D Department, Sardara, Italy.
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19
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Gao X, Chen X, Yu L, Zhao S, Jiu Y. Host cytoskeleton and membrane network remodeling in the regulation of viral replication. BIOPHYSICS REPORTS 2025; 11:34-45. [PMID: 40070659 PMCID: PMC11891074 DOI: 10.52601/bpr.2024.240040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2024] [Accepted: 10/15/2024] [Indexed: 03/14/2025] Open
Abstract
Viral epidemics pose major threats to global health and economies. A hallmark of viral infection is the reshaping of host cell membranes and cytoskeletons to form organelle-like structures, known as viral factories, which support viral genome replication. Viral infection in many cases induces the cytoskeletal network to form cage-like structures around viral factories, including actin rings, microtubule cages, and intermediate filament cages. Viruses hijack various organelles to create these replication factories, such as viroplasms, spherules, double-membrane vesicles, tubes, and nuclear viral factories. This review specifically examines the roles of cytoskeletal elements and the endomembrane system in material transport, structural support, and biochemical regulation during viral factory formation. Furthermore, we discuss the broader implications of these interactions for viral replication and highlight potential future research directions.
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Affiliation(s)
- Xuedi Gao
- Unit of Cell Biology and Imaging Study of Pathogen Host Interaction, The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xinming Chen
- Unit of Cell Biology and Imaging Study of Pathogen Host Interaction, The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Letian Yu
- Unit of Cell Biology and Imaging Study of Pathogen Host Interaction, The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuangshuang Zhao
- Unit of Cell Biology and Imaging Study of Pathogen Host Interaction, The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yaming Jiu
- Unit of Cell Biology and Imaging Study of Pathogen Host Interaction, The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai 200031, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Key Laboratory of Virology and Biosafety, Chinese Academy of Sciences, Wuhan 430071, China
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20
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Burkova EE, Bakhno IA. Sequences in the Cytoplasmic Tail Contribute to the Intracellular Trafficking and the Cell Surface Localization of SARS-CoV-2 Spike Protein. Biomolecules 2025; 15:280. [PMID: 40001583 PMCID: PMC11853650 DOI: 10.3390/biom15020280] [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: 12/04/2024] [Revised: 01/09/2025] [Accepted: 01/22/2025] [Indexed: 02/27/2025] Open
Abstract
Spike protein is a surface glycoprotein of the SARS-CoV-2 coronavirus, providing interaction of the coronavirus with angiotensin-converting enzyme 2 (ACE2) on the host cell. The cytoplasmic tail of the S protein plays an important role in an intracellular transport and translocation of the glycoprotein to the plasma membrane. The cytoplasmic domain of the S protein contains binding sites for COPI, COPII, and SNX27, which are required for the intracellular trafficking of this glycoprotein. In addition, the cytoplasmic domain of the S protein contains S-palmitoylation sites. S-palmitoylation increases the hydrophobicity of the S protein by regulating its transport to the plasma membrane. The cytoplasmic tail of the S protein has a signaling sequence that provides interaction with the ERM family proteins, which may mediate communication between the cell membrane and the actin cytoskeleton. This review examines the role of the cytoplasmic tail of the SARS-CoV-2 S protein in its intracellular transport and translocation to the plasma membrane. Understanding these processes is necessary not only for the development of vaccines based on mRNA or adenovirus vectors encoding the full-length spike (S) protein, but also for the therapy of the new coronavirus infection (COVID-19).
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Affiliation(s)
- Evgeniya E. Burkova
- SB RAS Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk, Russia;
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21
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Albalawi W, Thomas J, Mughal F, Kotsiri A, Roper KJ, Alshehri A, Kelbrick M, Pollakis G, Paxton WA. SARS-CoV-2 S, M, and E Structural Glycoproteins Differentially Modulate Endoplasmic Reticulum Stress Responses. Int J Mol Sci 2025; 26:1047. [PMID: 39940816 PMCID: PMC11816748 DOI: 10.3390/ijms26031047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 01/15/2025] [Accepted: 01/16/2025] [Indexed: 02/16/2025] Open
Abstract
We have previously shown that the hepatitis C virus (HCV) E1E2 envelope glycoprotein can regulate HIV-1 long-terminal repeat (LTR) activity through disruption to NF-κB activation. This response is associated with upregulation of the endoplasmic reticulum (ER) stress response pathway. Here, we demonstrate that the SARS-CoV-2 S, M, and E but not the N structural protein can perform similar downmodulation of HIV-1 LTR activation, and in a dose-dependent manner, in both HEK293 and lung BEAS-2B cell lines. This effect is highest with the SARS-CoV-2 Wuhan S strain and decreases over time for the subsequent emerging variants of concern (VOC), with Omicron providing the weakest effect. We developed pseudo-typed viral particle (PVP) viral tools that allowed for the generation of cell lines constitutively expressing the four SARS-CoV-2 structural proteins and utilising the VSV-g envelope protein to deliver the integrated gene construct. Differential gene expression analysis (DGEA) was performed on cells expressing S, E, M, or N to determine cell activation status. Gene expression differences were found in a number of interferon-stimulated genes (ISGs), including IF16, IFIT1, IFIT2, and ISG15, as well as for a number of heat shock protein (HSP) genes, including HSPH1, HSPA6, and HSPBP1, with all four SARS-CoV-2 structural proteins. There were also differences observed in expression patterns of transcription factors, with both SP1 and MAVS upregulated in the presence of S, M, and E but not the N protein. Collectively, the results indicate that gene expression patterns associated with ER stress pathways can be activated by SARS-CoV-2 envelope glycoprotein expression. The results suggest the SARS-CoV-2 infection can modulate an array of cell pathways, resulting in disruption to NF-κB signalling, hence providing alterations to multiple physiological responses of SARS-CoV-2-infected cells.
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Affiliation(s)
- Wejdan Albalawi
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
- Department Clinical Laboratory Sciences, College of Applied Medical Sciences, University of Aljouf, Sakakah 72388, Saudi Arabia
| | - Jordan Thomas
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
| | - Farah Mughal
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
| | - Aurelia Kotsiri
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
| | - Kelly J. Roper
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
- Virology Department, Animal and Plant Health Agency (APHA-Weybridge), Addlestone KT15 3NB, UK
| | - Abdullateef Alshehri
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
- Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, P.O. Box 1988, Najran 61441, Saudi Arabia
| | - Matthew Kelbrick
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
| | - Georgios Pollakis
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
| | - William A. Paxton
- Department of Clinical Infection, Microbiology and Immunology (CIMI), Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, Liverpool L69 7BE, UK; (W.A.); (J.T.); (F.M.); (A.K.); (K.J.R.); (A.A.); (M.K.)
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22
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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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23
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Hirabayashi A, Muramoto Y, Takenaga T, Tsunoda Y, Wakazaki M, Sato M, Fujita-Fujiharu Y, Nomura N, Yamauchi K, Onishi C, Nakano M, Toyooka K, Noda T. Coatomer complex I is required for the transport of SARS-CoV-2 progeny virions from the endoplasmic reticulum-Golgi intermediate compartment. mBio 2025; 16:e0333124. [PMID: 39611845 PMCID: PMC11708035 DOI: 10.1128/mbio.03331-24] [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: 10/24/2024] [Accepted: 11/07/2024] [Indexed: 11/30/2024] Open
Abstract
SARS-CoV-2 undergoes budding within the lumen of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and the progeny virions are delivered to the cell surface via vesicular transport. However, the molecular mechanisms remain poorly understood. Using three-dimensional electron microscopic analysis, such as array tomography and electron tomography, we found that virion-transporting vesicles possessed protein coats on their membrane and demonstrated that the protein coat was coatomer complex I (COPI). During the later stages of SARS-CoV-2 infection, we observed a notable alteration in the distribution of COPI and ERGIC throughout the cytoplasm, suggesting their potential involvement in virus replication. Depletion of COPB2, a key component of COPI, led to the confinement of SARS-CoV-2 progeny virions within the ERGIC at the perinuclear region. While the expression levels of viral proteins within cells were comparable, this depletion significantly reduced the efficiency of virion release, leading to the significant reduction of viral replication. Hence, our findings suggest COPI as a critical player in facilitating the transport of SARS-CoV-2 progeny virions from the ERGIC. Thus, COPI could be a promising target for the development of antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2 virions are synthesized within the ERGIC and are transported to the cell surface via vesicular transport for release. However, the precise mechanisms remain unclear. Through various electron microscopic techniques, we identified the presence of COPI on virion-transporting vesicles. Alterations in the distribution of COPI and ERGIC in SARS-CoV-2 infected cells are evident, suggesting their involvement in virus replication. When COPB2, a component of COPI, is depleted, progeny virions become trapped within the ERGIC, leading to a reduction in the efficiency of virion release. These findings highlight COPI's crucial role in mediating SARS-CoV-2 vesicular transport from the ERGIC and suggest it as a potential antiviral target.
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Affiliation(s)
- Ai Hirabayashi
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
| | - Yukiko Muramoto
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
| | - Toru Takenaga
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
| | - Yugo Tsunoda
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
| | - Mayumi Wakazaki
- RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa Prefecture, Japan
| | - Mayuko Sato
- RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa Prefecture, Japan
| | - Yoko Fujita-Fujiharu
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
| | - Norimichi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, , Kyoto, Kyoto Prefecture, Japan
| | - Koji Yamauchi
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
| | - Chiho Onishi
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
| | - Masahiro Nakano
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
| | - Kiminori Toyooka
- RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa Prefecture, Japan
| | - Takeshi Noda
- Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- CREST, Japan Science and Technology Agency, Kawaguchi, Saitama Prefecture, Japan
- Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto Prefecture, Japan
- Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Kyoto Prefecture, Japan
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24
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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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25
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Milojević L, Si Z, Xia X, Chen L, He Y, Tang S, Luo M, Zhou ZH. Capturing intermediates and membrane remodeling in class III viral fusion. SCIENCE ADVANCES 2024; 10:eadn8579. [PMID: 39630917 PMCID: PMC11616707 DOI: 10.1126/sciadv.adn8579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Accepted: 10/29/2024] [Indexed: 12/07/2024]
Abstract
Enveloped viruses enter cells by fusing their envelopes to host cell membranes. Vesicular stomatitis virus (VSV) glycoprotein (G) is a prototype for class III fusion proteins. Although structures of the stable pre- and postfusion ectodomain of G are known, its fusogenic intermediates are insufficiently characterized. Here, we incubated VSV virions with late endosome-mimicking liposomes at pH 5.5 and used cryo-electron tomography (cryo-ET) to visualize stages of VSV's membrane fusion pathway, capture refolding intermediates of G, and reconstruct a sequence of G conformational changes. We observe that the G trimer disassembles into monomers and parallel dimers that explore a broad conformational space. Extended intermediates engage target membranes and mediate fusion, resulting in viral uncoating and linearization of the ribonucleoprotein genome. These viral fusion intermediates provide mechanistic insights into class III viral fusion processes, opening avenues for future research and structure-based design of fusion inhibition-based antiviral therapeutics.
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Affiliation(s)
- Lenka Milojević
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
| | - Zhu Si
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Xian Xia
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Lauren Chen
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
| | - Yao He
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
| | - Sijia Tang
- Department of Chemistry, Centre for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA
| | - Ming Luo
- Department of Chemistry, Centre for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA
- Department of Chemistry, Institute of Biomedical Sciences, Georgia State University, Atlanta, GA 30302, USA
| | - Z. Hong Zhou
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
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26
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Hoffmann T, Michel J, Nitsche A, Mache C, Schulze J, Wolff T, Laue M. Electron microscopy images and morphometric data of SARS-CoV-2 variants in ultrathin plastic sections. Sci Data 2024; 11:1322. [PMID: 39632915 PMCID: PMC11618623 DOI: 10.1038/s41597-024-04182-3] [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: 08/12/2024] [Accepted: 11/28/2024] [Indexed: 12/07/2024] Open
Abstract
Conventional thin section electron microscopy of viral pathogens, such as the pandemic SARS-CoV-2, can provide structural information on the virus particle phenotype and its evolution. We recorded about 900 transmission electron microscopy images of different SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron BA.2 (B.1.1.529) and determined various morphometric parameters, such as maximal diameter and spike number, using a previously published measurement method. The datasets of the evolved virus variants were supplemented with images and measurements of the early SARS-CoV-2 isolates Munich929 and Italy-INMI1 to allow direct comparison. Infected Vero cell cultures were cultivated under comparable conditions to produce the viruses for imaging and morphometric analysis. The images and measurements can be used as a basis to analyse the morphometric changes of further evolving viruses at the particle level or for developing automated image processing workflows and analysis.
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Affiliation(s)
- Tobias Hoffmann
- Advanced Light and Electron Microscopy, Centre for Biological Threats and Special Pathogens 4 (ZBS 4), Robert Koch Institute, Berlin, Germany
| | - Janine Michel
- Highly Pathogenic Viruses, Centre for Biological Threats and Special Pathogens 1 (ZBS 1), Robert Koch Institute, Berlin, Germany
| | - Andreas Nitsche
- Highly Pathogenic Viruses, Centre for Biological Threats and Special Pathogens 1 (ZBS 1), Robert Koch Institute, Berlin, Germany
| | - Christin Mache
- Influenza and Other Respiratory Viruses (Unit 17), Robert Koch Institute, Berlin, Germany
| | - Jessica Schulze
- Influenza and Other Respiratory Viruses (Unit 17), Robert Koch Institute, Berlin, Germany
| | - Thorsten Wolff
- Influenza and Other Respiratory Viruses (Unit 17), Robert Koch Institute, Berlin, Germany
| | - Michael Laue
- Advanced Light and Electron Microscopy, Centre for Biological Threats and Special Pathogens 4 (ZBS 4), Robert Koch Institute, Berlin, Germany.
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27
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Ke Z, Peacock TP, Brown JC, Sheppard CM, Croll TI, Kotecha A, Goldhill DH, Barclay WS, Briggs JAG. Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants. EMBO J 2024; 43:6469-6495. [PMID: 39543395 PMCID: PMC11649927 DOI: 10.1038/s44318-024-00303-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 10/23/2024] [Accepted: 10/25/2024] [Indexed: 11/17/2024] Open
Abstract
The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins, which are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S protein from SARS-CoV-2 variants has revealed this structural adaptation at high resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures, and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Taken together, our data are consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of the S protein.
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Affiliation(s)
- Zunlong Ke
- Department of Cell and Virus Structure, Max Planck Institute of Biochemistry, Martinsried, Germany
- Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA
| | - Thomas P Peacock
- Department of Infectious Disease, Imperial College London, London, UK
- The Pirbright Institute, Woking, UK
| | - Jonathan C Brown
- Department of Infectious Disease, Imperial College London, London, UK
| | - Carol M Sheppard
- Department of Infectious Disease, Imperial College London, London, UK
| | - Tristan I Croll
- Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
- Altos Labs, Cambridge, UK
| | - Abhay Kotecha
- Materials and Structural Analysis, Thermo Fisher Scientific, Eindhoven, The Netherlands
| | - Daniel H Goldhill
- Department of Infectious Disease, Imperial College London, London, UK
- Department of Pathobiology and Population Sciences, Royal Veterinary College, London, UK
| | - Wendy S Barclay
- Department of Infectious Disease, Imperial College London, London, UK
| | - John A G Briggs
- Department of Cell and Virus Structure, Max Planck Institute of Biochemistry, Martinsried, Germany.
- Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
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28
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Esler M, Belica C, Rollie J, Brown W, Moghadasi SA, Shi K, Harki D, Harris R, Aihara H. A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain. Nucleic Acids Res 2024; 52:13138-13151. [PMID: 39380503 PMCID: PMC11602162 DOI: 10.1093/nar/gkae874] [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: 01/27/2024] [Revised: 08/29/2024] [Accepted: 09/24/2024] [Indexed: 10/10/2024] Open
Abstract
SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5'-TCGGAT-3') of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.
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Affiliation(s)
- Morgan A Esler
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - Christopher A Belica
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - Joseph A Rollie
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - William L Brown
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - Seyed Arad Moghadasi
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - Ke Shi
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
| | - Daniel A Harki
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
| | - Reuben S Harris
- Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA
- Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Hideki Aihara
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
- Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
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29
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Zhang J, Xu Y, Chen M, Wang S, Lin G, Huang Y, Yang C, Yang Y, Song Y. Spatial Engineering of Heterotypic Antigens on a DNA Framework for the Preparation of Mosaic Nanoparticle Vaccines with Enhanced Immune Activation against SARS-CoV-2 Variants. Angew Chem Int Ed Engl 2024; 63:e202412294. [PMID: 39030890 DOI: 10.1002/anie.202412294] [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: 07/01/2024] [Revised: 07/11/2024] [Accepted: 07/17/2024] [Indexed: 07/22/2024]
Abstract
Mosaic nanoparticle vaccines with heterotypic antigens exhibit broad-spectrum antiviral capabilities, but the impact of antigen proportions and distribution patterns on vaccine-induced immunity remains largely unexplored. Here, we present a DNA nanotechnology-based strategy for spatially assembling heterotypic antigens to guide the rational design of mosaic nanoparticle vaccines. By utilizing two aptamers with orthogonal selectivity for the original SARS-CoV-2 spike trimer and Omicron receptor-binding domain (RBD), along with a DNA soccer-ball framework, we precisely manipulate the spacing, stoichiometry, and overall distribution of heterotypic antigens to create mosaic nanoparticles with average, bipolar, and unipolar antigen distributions. Systematic in vitro and in vivo immunological investigations demonstrate that 30 heterotypic antigens in equivalent proportions, with an average distribution, lead to higher production of broad-spectrum neutralizing antibodies compared to the bipolar and unipolar distributions. Furthermore, the precise assembly utilizing our developed methodology reveals that a mere increment of five Omicron RBD antigens on a nanoparticle (from 15 to 20) not only diminishes neutralization against the Omicron variant but also triggers excessive inflammation. This work provides a unique perspective on the rational design of mosaic vaccines by highlighting the significance of the spatial placement and proportion of heterotypic antigens in their structure-activity mechanisms.
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Affiliation(s)
- Jialu Zhang
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
| | - Yunyun Xu
- Institute of Molecular Medicine and Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Mingying Chen
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
| | - Shengwen Wang
- Institute of Molecular Medicine and Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
- College of Chemistry and Materials Science, Shanghai Normal University, Shanghai, 200234, China
| | - Guihong Lin
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
| | - Yihao Huang
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
| | - Chaoyong Yang
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
- Institute of Molecular Medicine and Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Yang Yang
- Institute of Molecular Medicine and Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, State Key Laboratory of Oncogenes and Related Genes, Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Yanling Song
- The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen, Fujian, 361005, China
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30
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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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31
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Dutta NS, Carroll GM, Neale NR, Han SD, Al-Jassim M, Jungjohann K. Operando Freezing Cryogenic Electron Microscopy of Active Battery Materials. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2024; 30:844-852. [PMID: 39373722 DOI: 10.1093/mam/ozae097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Revised: 09/06/2024] [Accepted: 09/06/2024] [Indexed: 10/08/2024]
Abstract
Understanding structural and chemical evolution of battery materials during operation is critical to achieving safe, efficient, and long-lasting energy storage. Cryogenic electron microscopy (cryo-EM) has become a valuable tool in battery characterization, leveraging low temperatures to improve stability of sensitive materials under electron beam irradiation. However, typical cryo-EM sample preparations leave extended time between the electrochemical point of interest and ex situ freezing of samples, during which active structures may relax, degrade, or otherwise evolve. Here, we detail a method for operando freezing cryo-EM to preserve and characterize native electrode and interfacial structures that arise during battery cycling, based on an operando plunge freezer and cold sample removal process. We validate the method on multiple electrode materials and quantify and discuss the freezing rate achieved. Operando freezing cryo-EM can be used to directly visualize transient features that arise at active electrochemical interfaces, to enable deeper understanding of structural evolution and interfacial chemistry in batteries and other electrochemical systems.
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Affiliation(s)
- Nikita S Dutta
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
| | - Gerard Michael Carroll
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
| | - Nathan R Neale
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
| | - Sang-Don Han
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
- Department of Chemistry, Sejong University, 209 Neungdong-ro, Seoul 05006, Republic of Korea
| | - Mowafak Al-Jassim
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
| | - Katherine Jungjohann
- Materials, Chemical, and Computational Science Directorate, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
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32
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Martinez-Sanchez A, Lamm L, Jasnin M, Phelippeau H. Simulating the Cellular Context in Synthetic Datasets for Cryo-Electron Tomography. IEEE TRANSACTIONS ON MEDICAL IMAGING 2024; 43:3742-3754. [PMID: 38717878 DOI: 10.1109/tmi.2024.3398401] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2024]
Abstract
Cryo-electron tomography (cryo-ET) allows to visualize the cellular context at macromolecular level. To date, the impossibility of obtaining a reliable ground truth is limiting the application of deep learning-based image processing algorithms in this field. As a consequence, there is a growing demand of realistic synthetic datasets for training deep learning algorithms. In addition, besides assisting the acquisition and interpretation of experimental data, synthetic tomograms are used as reference models for cellular organization analysis from cellular tomograms. Current simulators in cryo-ET focus on reproducing distortions from image acquisition and tomogram reconstruction, however, they can not generate many of the low order features present in cellular tomograms. Here we propose several geometric and organization models to simulate low order cellular structures imaged by cryo-ET. Specifically, clusters of any known cytosolic or membrane-bound macromolecules, membranes with different geometries as well as different filamentous structures such as microtubules or actin-like networks. Moreover, we use parametrizable stochastic models to generate a high diversity of geometries and organizations to simulate representative and generalized datasets, including very crowded environments like those observed in native cells. These models have been implemented in a multiplatform open-source Python package, including scripts to generate cryo-tomograms with adjustable sizes and resolutions. In addition, these scripts provide also distortion-free density maps besides the ground truth in different file formats for efficient access and advanced visualization. We show that such a realistic synthetic dataset can be readily used to train generalizable deep learning algorithms.
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33
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Zhang J, Fan X, Wang P, Liang R, Wang D, Xu J, Zhang D, Xie Y, Liao Q, Jiao Z, Shi Y, Peng G. Identification of novel broad-spectrum antiviral drugs targeting the N-terminal domain of the FIPV nucleocapsid protein. Int J Biol Macromol 2024; 279:135352. [PMID: 39242012 DOI: 10.1016/j.ijbiomac.2024.135352] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 08/25/2024] [Accepted: 09/03/2024] [Indexed: 09/09/2024]
Abstract
Coronaviruses pose serious threats to human and animal health worldwide, of which their structural nucleocapsid (N) proteins play multiple key roles in viral replication. However, the structures of animal coronavirus N proteins are poorly understood, posing challenges for research on their functions and pathogenic mechanisms as well as the development of N protein-based antiviral drugs. Therefore, N proteins must be further explored as potential antiviral targets. We determined the structure of the NNTD of feline infectious peritonitis virus (FIPV) and identified 3,6-dihydroxyflavone (3,6- DHF) as an effective N protein inhibitor. 3,6-DHF successfully inhibited FIPV replication in CRFK cells, showing broad-spectrum activity and effectiveness against drugresistant strains. Our study provides important insights for developing novel broadspectrum anti-coronavirus drugs and treating infections caused by drug-resistant mutant strains.
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Affiliation(s)
- Jintao Zhang
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Xinyu Fan
- Department of Biotechnology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Pengpeng Wang
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Rui Liang
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Donghan Wang
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Juan Xu
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Ding Zhang
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Yunfei Xie
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Qi Liao
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China
| | - Zhe Jiao
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China.
| | - Yuejun Shi
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China.
| | - Guiqing Peng
- National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China; Hongshan Laboratory, Wuhan, China.
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34
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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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35
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Tants JN, Schlundt A. The role of structure in regulatory RNA elements. Biosci Rep 2024; 44:BSR20240139. [PMID: 39364891 PMCID: PMC11499389 DOI: 10.1042/bsr20240139] [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: 05/23/2024] [Revised: 10/02/2024] [Accepted: 10/04/2024] [Indexed: 10/05/2024] Open
Abstract
Regulatory RNA elements fulfill functions such as translational regulation, control of transcript levels, and regulation of viral genome replication. Trans-acting factors (i.e., RNA-binding proteins) bind the so-called cis elements and confer functionality to the complex. The specificity during protein-RNA complex (RNP) formation often exploits the structural plasticity of RNA. Functional integrity of cis-trans pairs depends on the availability of properly folded RNA elements, and RNA conformational transitions can cause diseases. Knowledge of RNA structure and the conformational space is needed for understanding complex formation and deducing functional effects. However, structure determination of RNAs under in vivo conditions remains challenging. This review provides an overview of structured eukaryotic and viral RNA cis elements and discusses the effect of RNA structural equilibria on RNP formation. We showcase implications of RNA structural changes for diseases, outline strategies for RNA structure-based drug targeting, and summarize the methodological toolbox for deciphering RNA structures.
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Affiliation(s)
- Jan-Niklas Tants
- Institute for Molecular Biosciences and Biomolecular Resonance Center (BMRZ), Goethe University Frankfurt, Max-von-Laue-Str. 7-9, 60438 Frankfurt, Germany
| | - Andreas Schlundt
- Institute for Molecular Biosciences and Biomolecular Resonance Center (BMRZ), Goethe University Frankfurt, Max-von-Laue-Str. 7-9, 60438 Frankfurt, Germany
- University of Greifswald, Institute of Biochemistry, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany
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36
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Comas-Garcia M. How structural biology has changed our understanding of icosahedral viruses. J Virol 2024; 98:e0111123. [PMID: 39291975 PMCID: PMC11495149 DOI: 10.1128/jvi.01111-23] [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: 09/19/2024] Open
Abstract
Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.
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Affiliation(s)
- Mauricio Comas-Garcia
- Science Department, Autonomous University of San Luis Potosi, San Luis Potosí, Mexico
- High-Resolution Microscopy Section, Research Center for Health Sciences and Biomedicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico
- Translational and Molecular Medicine Section, Research Center for Health Sciences and Biomedicine, Autonomous University of San Luis Potosi, San Luis Potosí, Mexico
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Katiyar H, Arduini A, Li Y, Liang C. SARS-CoV-2 Assembly: Gaining Infectivity and Beyond. Viruses 2024; 16:1648. [PMID: 39599763 PMCID: PMC11598957 DOI: 10.3390/v16111648] [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: 10/02/2024] [Revised: 10/12/2024] [Accepted: 10/21/2024] [Indexed: 11/29/2024] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was responsible for causing the COVID-19 pandemic. Intensive research has illuminated the complex biology of SARS-CoV-2 and its continuous evolution during and after the COVID-19 pandemic. While much attention has been paid to the structure and functions of the viral spike protein and the entry step of viral infection, partly because these are targets for neutralizing antibodies and COVID-19 vaccines, the later stages of SARS-CoV-2 replication, including the assembly and egress of viral progenies, remain poorly characterized. This includes insight into how the activities of the viral structural proteins are orchestrated spatially and temporally, which cellular proteins are assimilated by the virus to assist viral assembly, and how SARS-CoV-2 counters and evades the cellular mechanisms antagonizing virus assembly. In addition to becoming infectious, SARS-CoV-2 progenies also need to survive the hostile innate and adaptive immune mechanisms, such as recognition by neutralizing antibodies. This review offers an updated summary of the roles of SARS-CoV-2 structural proteins in viral assembly, the regulation of assembly by viral and cellular factors, and the cellular mechanisms that restrict this process. Knowledge of these key events often reveals the vulnerabilities of SARS-CoV-2 and aids in the development of effective antiviral therapeutics.
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Affiliation(s)
- Harshita Katiyar
- Lady Davis Institute, Jewish General Hospital, Montreal, QC H3T 1E2, Canada; (H.K.); (A.A.); (Y.L.)
- Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada
| | - Ariana Arduini
- Lady Davis Institute, Jewish General Hospital, Montreal, QC H3T 1E2, Canada; (H.K.); (A.A.); (Y.L.)
- Department of Medicine, McGill University, Montreal, QC H3G 2M1, Canada
| | - Yichen Li
- Lady Davis Institute, Jewish General Hospital, Montreal, QC H3T 1E2, Canada; (H.K.); (A.A.); (Y.L.)
- Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada
| | - Chen Liang
- Lady Davis Institute, Jewish General Hospital, Montreal, QC H3T 1E2, Canada; (H.K.); (A.A.); (Y.L.)
- Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada
- Department of Medicine, McGill University, Montreal, QC H3G 2M1, Canada
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Favetta B, Wang H, Cubuk J, Barai M, Ramirez C, Gormley AJ, Murthy S, Soranno A, Shi Z, Schuster BS. Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between Two Membrane-Associated Condensate States. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.17.618867. [PMID: 39464032 PMCID: PMC11507936 DOI: 10.1101/2024.10.17.618867] [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: 10/29/2024]
Abstract
The SARS-CoV-2 Nucleocapsid protein (N) performs several functions during the viral lifecycle, including transcription regulation and viral genome encapsulation. We hypothesized that N toggles between these functions via phosphorylation-induced conformational change, thereby altering N interactions with membranes and RNA. We found that phosphorylation changes how biomolecular condensates composed of N and RNA interact with membranes: phosphorylated N (pN) condensates form thin films, while condensates with unmodified N are engulfed. This partly results from changes in material properties, with pN forming less viscous and elastic condensates. The weakening of protein-RNA interaction in condensates upon phosphorylation is driven by a decrease in binding between pN and unstructured RNA. We show that phosphorylation induces a conformational change in the serine/arginine-rich region of N that increases interaction between pN monomers and decreases nonspecific interaction with RNA. These findings connect the conformation, material properties, and membrane-associated states of N, with potential implications for COVID-19 treatment.
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Affiliation(s)
- Bruna Favetta
- Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Huan Wang
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Jasmine Cubuk
- Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, St. Louis, MO 63110
| | - Mayur Barai
- Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Cesar Ramirez
- Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Adam J Gormley
- Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Sanjeeva Murthy
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Andrea Soranno
- Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, St. Louis, MO 63110
| | - Zheng Shi
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
| | - Benjamin S Schuster
- Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
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Tang Y, Tang K, Hu Y, Ye ZW, Luo W, Luo C, Cao H, Wang R, Yue X, Liu D, Liu C, Ge X, Liu T, Chen Y, Yuan S, Deng L. M protein ectodomain-specific immunity restrains SARS-CoV-2 variants replication. Front Immunol 2024; 15:1450114. [PMID: 39416782 PMCID: PMC11480003 DOI: 10.3389/fimmu.2024.1450114] [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: 06/16/2024] [Accepted: 09/05/2024] [Indexed: 10/19/2024] Open
Abstract
Introduction The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear. Methods We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay. Results Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30. Discussion The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections.
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Affiliation(s)
- Yibo Tang
- Hunan Provincial Key Laboratory of Medical Virology, College of Biology, Hunan University, Changsha, China
| | - Kaiming Tang
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China
| | - Yunqi Hu
- School of Public Health, Sun Yat-sen University, Shenzhen, China
| | - Zi-Wei Ye
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China
| | - Wanyu Luo
- School of Public Health, Sun Yat-sen University, Shenzhen, China
| | - Cuiting Luo
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China
| | - Hehe Cao
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China
| | - Ran Wang
- Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
| | - Xinyu Yue
- School of Public Health, Sun Yat-sen University, Shenzhen, China
| | - Dejian Liu
- Hunan Provincial Key Laboratory of Medical Virology, College of Biology, Hunan University, Changsha, China
| | - Cuicui Liu
- Hunan Provincial Key Laboratory of Medical Virology, College of Biology, Hunan University, Changsha, China
| | - Xingyi Ge
- Hunan Provincial Key Laboratory of Medical Virology, College of Biology, Hunan University, Changsha, China
| | - Tianlong Liu
- National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Yaoqing Chen
- School of Public Health, Sun Yat-sen University, Shenzhen, China
- National Medical Products Administration Key Laboratory for Quality Monitoring and Evaluation of Vaccines and Biological Products, Sun Yat-sen University, Guangzhou, China
| | - Shuofeng Yuan
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China
- Department of Clinical Microbiology and Infection Control, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
| | - Lei Deng
- Hunan Provincial Key Laboratory of Medical Virology, College of Biology, Hunan University, Changsha, China
- Research and Development Department, Beijing Weimiao Biotechnology Co. Ltd., Beijing, China
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Cetinkaya PG, Abras IF, Evcili I, Yildirim T, Ceylan Y, Kara Eroglu F, Kayaoglu B, İpekoglu EM, Akarsu A, Yıldırım M, Kahraman T, Cengiz AB, Sahiner UM, Sekerel BE, Ozsurekci Y, Soyer O, Gursel I. Plasma Extracellular Vesicles Derived from Pediatric COVID-19 Patients Modulate Monocyte and T Cell Immune Responses Based on Disease Severity. Immunol Invest 2024; 53:1141-1175. [PMID: 39115924 DOI: 10.1080/08820139.2024.2385992] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/02/2024]
Abstract
BACKGROUND The COVID-19 pandemic has caused significant morbidity and mortality globally. The role of plasma-derived extracellular vesicles (EVs) in pediatric COVID-19 patients remains unclear. METHODS We isolated EVs from healthy controls (n = 13) and pediatric COVID-19 patients (n = 104) with varying severity during acute and convalescent phases using serial ultracentrifugation. EV effects on healthy PBMCs, naïve CD4+ T cells, and monocytes were assessed through in vitro assays, flow cytometry, and ELISA. RESULTS Our findings indicate that COVID-19 severity correlates with diverse immune responses. Severe acute cases exhibited increased cytokine levels, decreased IFNγ levels, and lower CD4+ T cell and monocyte counts, suggesting immunosuppression. EVs from severe acute patients stimulated healthy cells to express higher PDL1, increased Th2 and Treg cells, reduced IFNγ secretion, and altered Th1/Th17 ratios. Patient-derived EVs significantly reduced proinflammatory cytokine production by monocytes (p < .001 for mild, p = .0025 for severe cases) and decreased CD4+ T cell (p = .043) and monocyte (p = .033) populations in stimulated healthy PBMCs. CONCLUSION This study reveals the complex relationship between immunological responses and EV-mediated effects, emphasizing the impact of COVID-19 severity. We highlight the potential role of plasma-derived EVs in early-stage immunosuppression in severe COVID-19 patients.
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Affiliation(s)
- Pınar Gur Cetinkaya
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Irem Fatma Abras
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Irem Evcili
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Tugçe Yildirim
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
- Basic and Translational Research Program, Izmir Biomedicine and Genome Center, Izmir, Turkey
| | - Yasemin Ceylan
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Fehime Kara Eroglu
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Başak Kayaoglu
- Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
| | - Emre Mert İpekoglu
- Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
| | - Aysegul Akarsu
- Division of Pediatric Allergy and Asthma Unit, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Muzaffer Yıldırım
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
- Basic and Translational Research Program, Izmir Biomedicine and Genome Center, Izmir, Turkey
| | - Tamer Kahraman
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
| | - Ali Bülent Cengiz
- Division of Pediatric Infectious Diseases, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Umit Murat Sahiner
- Division of Pediatric Allergy and Asthma, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Bulent Enis Sekerel
- Division of Pediatric Allergy and Asthma, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Yasemin Ozsurekci
- Division of Pediatric Infectious Diseases, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Ozge Soyer
- Division of Pediatric Allergy and Asthma, Faculty of Medicine, Hacettepe University, Ankara, Turkey
| | - Ihsan Gursel
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
- Basic and Translational Research Program, Izmir Biomedicine and Genome Center, Izmir, Turkey
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Nazir F, John Kombe Kombe A, Khalid Z, Bibi S, Zhang H, Wu S, Jin T. SARS-CoV-2 replication and drug discovery. Mol Cell Probes 2024; 77:101973. [PMID: 39025272 DOI: 10.1016/j.mcp.2024.101973] [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: 01/11/2024] [Revised: 07/14/2024] [Accepted: 07/15/2024] [Indexed: 07/20/2024]
Abstract
The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to wreak havoc across the globe. This sudden and deadly pandemic emphasizes the necessity for anti-viral drug development that can be rapidly administered to reduce morbidity, mortality, and virus propagation. Thus, lacking efficient anti-COVID-19 treatment, and especially given the lengthy drug development process as well as the critical death tool that has been associated with SARS-CoV-2 since its outbreak, drug repurposing (or repositioning) constitutes so far, the ideal and ready-to-go best approach in mitigating viral spread, containing the infection, and reducing the COVID-19-associated death rate. Indeed, based on the molecular similarity approach of SARS-CoV-2 with previous coronaviruses (CoVs), repurposed drugs have been reported to hamper SARS-CoV-2 replication. Therefore, understanding the inhibition mechanisms of viral replication by repurposed anti-viral drugs and chemicals known to block CoV and SARS-CoV-2 multiplication is crucial, and it opens the way for particular treatment options and COVID-19 therapeutics. In this review, we highlighted molecular basics underlying drug-repurposing strategies against SARS-CoV-2. Notably, we discussed inhibition mechanisms of viral replication, involving and including inhibition of SARS-CoV-2 proteases (3C-like protease, 3CLpro or Papain-like protease, PLpro) by protease inhibitors such as Carmofur, Ebselen, and GRL017, polymerases (RNA-dependent RNA-polymerase, RdRp) by drugs like Suramin, Remdesivir, or Favipiravir, and proteins/peptides inhibiting virus-cell fusion and host cell replication pathways, such as Disulfiram, GC376, and Molnupiravir. When applicable, comparisons with SARS-CoV inhibitors approved for clinical use were made to provide further insights to understand molecular basics in inhibiting SARS-CoV-2 replication and draw conclusions for future drug discovery research.
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Affiliation(s)
- Farah Nazir
- Center of Disease Immunity and Investigation, College of Medicine, Lishui University, Lishui, 323000, China
| | - Arnaud John Kombe Kombe
- Laboratory of Structural Immunology, Key Laboratory of Immune Response and Immunotherapy, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230027, China
| | - Zunera Khalid
- Laboratory of Structural Immunology, Key Laboratory of Immune Response and Immunotherapy, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230027, China
| | - Shaheen Bibi
- Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Anhui, China
| | - Hongliang Zhang
- Center of Disease Immunity and Investigation, College of Medicine, Lishui University, Lishui, 323000, China
| | - Songquan Wu
- Center of Disease Immunity and Investigation, College of Medicine, Lishui University, Lishui, 323000, China.
| | - Tengchuan Jin
- Center of Disease Immunity and Investigation, College of Medicine, Lishui University, Lishui, 323000, China; Laboratory of Structural Immunology, Key Laboratory of Immune Response and Immunotherapy, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230027, China; Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Anhui, China; Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, Anhui, China; Biomedical Sciences and Health Laboratory of Anhui Province, University of Science & Technology of China, Hefei, 230027, China; Clinical Research Hospital of Chinese Academy of Sciences (Hefei), University of Science and Technology of China, Hefei, 230001, China.
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42
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Saratov GA, Belogurov AA, Kudriaeva AA. Myelin basic protein antagonizes the SARS-CoV-2 protein ORF3a-induced autophagy inhibition. Biochimie 2024; 225:1-9. [PMID: 38703943 DOI: 10.1016/j.biochi.2024.04.011] [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: 01/17/2024] [Revised: 04/06/2024] [Accepted: 04/26/2024] [Indexed: 05/06/2024]
Abstract
Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome-lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS.
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Affiliation(s)
- George A Saratov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia; Moscow Institute of Physics and Technology (national Research University), Phystech School of Biological and Medical Physics, 141701, Dolgoprudny, Moscow Region, Russia
| | - Alexey A Belogurov
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia; Russian University of Medicine, Department of Biological Chemistry, Ministry of Health of Russian Federation, 127473, Moscow, Russia.
| | - Anna A Kudriaeva
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia.
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Sokolova OS, Trifonova TS, Derkacheva NI, Moiseenko AV. Visualization of Nucleic Acids in Microand Nanometer-Scale Biological Objects Using Analytical Electron Microscopy. Acta Naturae 2024; 16:38-47. [PMID: 39877006 PMCID: PMC11771847 DOI: 10.32607/actanaturae.27483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Accepted: 10/21/2024] [Indexed: 01/31/2025] Open
Abstract
Analytical electron microscopy techniques, including energy-dispersive X-ray spectroscopy (EDX) and electron energy-loss spectroscopy (EELS), are employed in materials science and biology to visualize and chemically map diverse elements. This review presents cases of successful identification of nucleic acids in cells and in DNA- and RNA-containing viruses that use the chemical element phosphorus as a marker.
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Affiliation(s)
- O. S. Sokolova
- Lomonosov Moscow State University, Faculty of Biology, Moscow, 119234 Russian Federation
| | - T. S. Trifonova
- Lomonosov Moscow State University, Faculty of Biology, Moscow, 119234 Russian Federation
| | - N. I. Derkacheva
- Russian University of Medicine, Department of Biochemistry, Moscow, 127473 Russian Federation
| | - A. V. Moiseenko
- Lomonosov Moscow State University, Faculty of Biology, Moscow, 119234 Russian Federation
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Huang Q, Zhou Y, Bartesaghi A. MiLoPYP: self-supervised molecular pattern mining and particle localization in situ. Nat Methods 2024; 21:1863-1872. [PMID: 39251798 PMCID: PMC11468773 DOI: 10.1038/s41592-024-02403-6] [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: 12/26/2023] [Accepted: 08/05/2024] [Indexed: 09/11/2024]
Abstract
Cryo-electron tomography allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography, it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with cryo-electron tomography/single-particle tomography are the automatic identification and localization of proteins, tasks that are hindered by the molecular crowding inside cells, imaging distortions characteristic of cryo-electron tomography tomograms and the sheer size of tomographic datasets. Current methods suffer from low accuracy, demand extensive and time-consuming manual labeling or are limited to the detection of specific types of proteins. Here, we present MiLoPYP, a two-step dataset-specific contrastive learning-based framework that enables fast molecular pattern mining followed by accurate protein localization. MiLoPYP's ability to effectively detect and localize a wide range of targets including globular and tubular complexes as well as large membrane proteins, will contribute to streamline and broaden the applicability of high-resolution workflows for in situ structure determination.
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Affiliation(s)
- Qinwen Huang
- Department of Computer Science, Duke University, Durham, NC, USA
| | - Ye Zhou
- Department of Computer Science, Duke University, Durham, NC, USA
| | - Alberto Bartesaghi
- Department of Computer Science, Duke University, Durham, NC, USA.
- Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA.
- Department of Electrical and Computer Engineering, Duke University, Durham, NC, USA.
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Laughlin PM, Young K, Gonzalez-Gutierrez G, Wang JCY, Zlotnick A. A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation. J Biol Chem 2024; 300:107831. [PMID: 39343003 PMCID: PMC11541828 DOI: 10.1016/j.jbc.2024.107831] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Revised: 09/16/2024] [Accepted: 09/19/2024] [Indexed: 10/01/2024] Open
Abstract
SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30 kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.
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Affiliation(s)
- Patrick M Laughlin
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA
| | - Kimberly Young
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA
| | | | - Joseph C-Y Wang
- Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Adam Zlotnick
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
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Lyu CA, Shen Y, Zhang P. Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods. Viruses 2024; 16:1504. [PMID: 39339980 PMCID: PMC11437419 DOI: 10.3390/v16091504] [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: 08/29/2024] [Revised: 09/19/2024] [Accepted: 09/22/2024] [Indexed: 09/30/2024] Open
Abstract
RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats.
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Affiliation(s)
- Cheng-An Lyu
- Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK;
- Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford OX3 7BN, UK
| | - Yao Shen
- Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK;
| | - Peijun Zhang
- Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK;
- Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford OX3 7BN, UK
- Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK
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Purves K, Reynolds LJ, Sala-Comorera L, Martin NA, Dahly DL, Meijer WG, Fletcher NF. Decay of RNA and infectious SARS-CoV-2 and murine hepatitis virus in wastewater. THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 944:173877. [PMID: 38871327 DOI: 10.1016/j.scitotenv.2024.173877] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 06/06/2024] [Accepted: 06/07/2024] [Indexed: 06/15/2024]
Abstract
Wastewater-based epidemiology (WBE) has been an important tool for population surveillance during the COVID-19 pandemic and continues to play a key role in monitoring SARS-CoV-2 infection levels following reductions in national clinical testing schemes. Studies measuring decay profiles of SARS-CoV-2 in wastewater have underscored the value of WBE, however investigations have been hampered by high biosafety requirements for SARS-CoV-2 infection studies. Therefore, surrogate viruses with lower biosafety standards have been used for SARS-CoV-2 decay studies, such as murine hepatitis virus (MHV), but few studies have directly compared decay rates of both viruses. We compared the persistence of SARS-CoV-2 and MHV in wastewater, using 50 % tissue culture infectious dose (TCID50) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays to assess infectious virus titre and viral gene markers, respectively. Infectious SARS-CoV-2 and MHV indicate similar endpoints, however observed early decay characteristics differed, with infectious SARS-CoV-2 decaying more rapidly than MHV. We find that MHV is an appropriate infectious virus surrogate for viable SARS-CoV-2, however inconsistencies exist in viral RNA decay parameters, indicating MHV may not be a suitable nucleic acid surrogate across certain temperature regimes. This study highlights the importance of sample preparation and the potential for decay rate overestimation in wastewater surveillance for SARS-CoV-2 and other pathogens.
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Affiliation(s)
- Kevin Purves
- UCD School of Veterinary Medicine and UCD Conway Institute, University College Dublin, Ireland
| | - Liam J Reynolds
- UCD School of Biomolecular and Biomedical Science, UCD Earth Institute and UCD Conway Institute, University College Dublin, Ireland
| | - Laura Sala-Comorera
- Section of Microbiology, Virology and Biotechnology, Department of Genetics, Microbiology and Statistics, University of Barcelona, Spain
| | - Niamh A Martin
- UCD School of Biomolecular and Biomedical Science, UCD Earth Institute and UCD Conway Institute, University College Dublin, Ireland
| | - Darren L Dahly
- Health Research Board Clinical Research Facility, University College Cork, Ireland; School of Public Health, University College Cork, Ireland
| | - Wim G Meijer
- UCD School of Biomolecular and Biomedical Science, UCD Earth Institute and UCD Conway Institute, University College Dublin, Ireland
| | - Nicola F Fletcher
- UCD School of Veterinary Medicine and UCD Conway Institute, University College Dublin, Ireland.
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48
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Eisenreich W, Leberfing J, Rudel T, Heesemann J, Goebel W. Interactions of SARS-CoV-2 with Human Target Cells-A Metabolic View. Int J Mol Sci 2024; 25:9977. [PMID: 39337465 PMCID: PMC11432161 DOI: 10.3390/ijms25189977] [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: 08/15/2024] [Revised: 09/13/2024] [Accepted: 09/13/2024] [Indexed: 09/30/2024] Open
Abstract
Viruses are obligate intracellular parasites, and they exploit the cellular pathways and resources of their respective host cells to survive and successfully multiply. The strategies of viruses concerning how to take advantage of the metabolic capabilities of host cells for their own replication can vary considerably. The most common metabolic alterations triggered by viruses affect the central carbon metabolism of infected host cells, in particular glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. The upregulation of these processes is aimed to increase the supply of nucleotides, amino acids, and lipids since these metabolic products are crucial for efficient viral proliferation. In detail, however, this manipulation may affect multiple sites and regulatory mechanisms of host-cell metabolism, depending not only on the specific viruses but also on the type of infected host cells. In this review, we report metabolic situations and reprogramming in different human host cells, tissues, and organs that are favorable for acute and persistent SARS-CoV-2 infection. This knowledge may be fundamental for the development of host-directed therapies.
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Affiliation(s)
- Wolfgang Eisenreich
- Structural Membrane Biochemistry, Bavarian NMR Center (BNMRZ), Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Lichtenbergstr. 4, 85747 Garching, Germany;
| | - Julian Leberfing
- Structural Membrane Biochemistry, Bavarian NMR Center (BNMRZ), Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Lichtenbergstr. 4, 85747 Garching, Germany;
| | - Thomas Rudel
- Chair of Microbiology, Biocenter, University of Würzburg, 97074 Würzburg, Germany;
| | - Jürgen Heesemann
- Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, 80336 München, Germany; (J.H.); (W.G.)
| | - Werner Goebel
- Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, 80336 München, Germany; (J.H.); (W.G.)
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Tiano SML, Landi N, Marano V, Ragucci S, Bianco G, Cacchiarelli D, Swuec P, Silva M, De Cegli R, Sacco F, Di Maro A, Cortese M. Quinoin, type 1 ribosome inactivating protein alters SARS-CoV-2 viral replication organelle restricting viral replication and spread. Int J Biol Macromol 2024:135700. [PMID: 39288862 DOI: 10.1016/j.ijbiomac.2024.135700] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2024] [Revised: 09/09/2024] [Accepted: 09/13/2024] [Indexed: 09/19/2024]
Abstract
SARS-CoV-2 pandemic clearly demonstrated the lack of preparation against novel and emerging viral diseases. This prompted an enormous effort to identify antiviral to curb viral spread and counteract future pandemics. Ribosome Inactivating Proteins (RIPs) and Ribotoxin-Like Proteins (RL-Ps) are toxin enzymes isolated from edible plants and mushrooms, both able to inactivate protein biosynthesis. In the present study, we combined imaging analyses, transcriptomic and proteomic profiling to deeper investigate the spectrum of antiviral activity of quinoin, type 1 RIP from quinoa seeds. Here, we show that RIPs, but not RL-Ps, acts on a post-entry step and impair SARS-CoV-2 replication, potentially by direct degradation of viral RNA. Interestingly, the inhibitory activity of quinoin was conserved also against other members of the Coronaviridae family suggesting a broader antiviral effect. The integration of mass spectrometry (MS)-based proteomics with transcriptomics, provided a comprehensive picture of the quinoin dependent remodeling of crucial biological processes, highlighting an unexpected impact on lipid metabolism. Thus, direct and indirect mechanisms can contribute to the inhibitory mechanism of quinoin, making RIPs family a promising candidate not only for their antiviral activity, but also as an effective tool to better understand the cellular functions and factors required during SARS-CoV-2 replication.
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Affiliation(s)
- Sofia Maria Luigia Tiano
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Scuola Superiore Meridionale (SSM, School of Advanced Studies), Genomics and Experimental Medicine Program, Naples, Italy
| | - Nicola Landi
- Institute of Crystallography, National Research Council, Caserta, Italy; Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy
| | - Valentina Marano
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; PhD Program in Cellular and Molecular Biology, Department of Biology, University of Rome "Tor Vergata", Rome, Italy
| | - Sara Ragucci
- Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy
| | - Gennaro Bianco
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy
| | - Davide Cacchiarelli
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Scuola Superiore Meridionale (SSM, School of Advanced Studies), Genomics and Experimental Medicine Program, Naples, Italy; Department of Translational Medicine, University of Naples "Federico II", Naples, Italy
| | - Paolo Swuec
- Cryo-Electron Microscopy Unit, National Facility for Structural Biology, Human Technopole, Milan, Italy
| | - Malan Silva
- Cryo-Electron Microscopy Unit, National Facility for Structural Biology, Human Technopole, Milan, Italy
| | - Rossella De Cegli
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy
| | - Francesca Sacco
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Department of Biology, University of Rome Tor Vergata, Rome, Italy
| | - Antimo Di Maro
- Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy.
| | - Mirko Cortese
- Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Scuola Superiore Meridionale (SSM, School of Advanced Studies), Genomics and Experimental Medicine Program, Naples, Italy; Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy.
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50
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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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