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1
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Pathak T, Pal S, Banerjee I. Cathepsins in cellular entry of human pathogenic viruses. J Virol 2025; 99:e0164224. [PMID: 40135892 PMCID: PMC11998514 DOI: 10.1128/jvi.01642-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/27/2025] Open
Abstract
In the life cycle of a virus, host cell entry represents the first step that a virus needs to undertake to gain access to the cell interior for replication. Once a virus attaches itself to its target cell receptor, it activates endogenous cellular responses and exploits host cell factors for its internalization, fusion, and genome release. Among the host factors that critically contribute to the viral entry processes are cathepsins, which are the most abundant endo/lysosomal proteases with diverse physiological functions. This review summarizes previous findings on how different cathepsins contribute to the host cell entry of human pathogenic viruses, focusing on their specific roles in the entry processes of both enveloped and non-enveloped RNA viruses. A comprehensive knowledge of the functions of different cathepsins in viral entry will provide valuable insights into the molecular mechanisms underlying viral infections and can be useful in the development of new antiviral strategies.
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Affiliation(s)
- Tejal Pathak
- Cellular Virology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Punjab, India
| | - Sampurna Pal
- Cellular Virology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Punjab, India
| | - Indranil Banerjee
- Cellular Virology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Punjab, India
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2
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Sevarika B, Capri D, Frey J, Dinamarca MC, Häussinger D, McNeil S. Mannose-6-phosphate-PEG-lipid conjugates improve liposomal uptake. Eur J Pharm Biopharm 2025; 209:114665. [PMID: 39954966 DOI: 10.1016/j.ejpb.2025.114665] [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: 11/19/2024] [Revised: 01/22/2025] [Accepted: 02/12/2025] [Indexed: 02/17/2025]
Abstract
Targeted liposomes are a keystone of nanomedicine, offering a precise and efficient means to deliver therapeutic agents directly to diseased tissues or cells. By incorporating targeting ligands on their surface, liposomes enhance the specificity of drug delivery, improving efficacy and reducing toxicity. Mannose-6-phosphate (M6P) is a crucial molecular tag for internalization and intracellular sorting of macromolecular structures to the lysosome. Taking advantage of this mechanism, we designed and developed liposomal systems to enhance therapeutic delivery to the lysosomes. The synthesized M6P-based targeting molecules were covalently coupled to a phospholipid using a polyethylene glycol (PEG) linker. The prepared ligands were successfully incorporated into the liposomes, yielding a size of roughly 100 nm and a zeta potential of around -40 mV. Incorporating the M6P-based ligand enhances the internalization of liposomes in a concentration-dependent manner, increasing uptake by up to 14-fold in several tested cell lines. In contrast, structurally similar monosaccharides and equally charged ligands failed to replicate this effect, highlighting the specificity of M6P-mediated internalization. Our studies demonstrate that M6P-mediated uptake predominantly occurs via a clathrin-mediated pathway, and once internalized, 72 % of the M6P-coated liposomes are associated with the lysosomal compartment. This study highlights the potential of M6P-based liposomal carriers as a modular platform for targeted lysosomal delivery, offering a promising therapeutic approach for lysosomal storage diseases.
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Affiliation(s)
- Boris Sevarika
- Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50 4056 Basel, Switzerland
| | - Deniz Capri
- Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50 4056 Basel, Switzerland
| | - Joël Frey
- Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50 4056 Basel, Switzerland
| | - Margarita C Dinamarca
- Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50 4056 Basel, Switzerland
| | - Daniel Häussinger
- Department of Chemistry, University of Basel, St. Johanns-Ring 19 4056 Basel, Switzerland
| | - Scott McNeil
- Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50 4056 Basel, Switzerland.
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3
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Asensio-Cob D, Mata CP, Gómez-Blanco J, Vargas J, Rodríguez JM, Luque D. Structural determinants of rotavirus proteolytic activation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.24.644915. [PMID: 40196553 PMCID: PMC11974734 DOI: 10.1101/2025.03.24.644915] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
The infectivity of rotavirus (RV), the leading cause of childhood diarrhea, hinges on the activation of viral particles through the proteolysis of the spike protein by trypsin-like proteases in the host intestinal lumen. Despite comprehensive structural characterization of the virus particle, the structural rationale behind the necessity of trypsin digestion of the VP4 protein for infectivity remains poorly understood. In this study, using cryo-electron microscopy (cryo-EM) and advanced image processing techniques, we compared uncleaved and cleaved RV virions and found that the conformation of the non-proteolyzed spike is constrained by the position of loops that surround its structure, linking the lectin domains of the spike head to its body. The proteolysis of these loops removes this structural constraint, thereby enabling the spike to undergo the necessary conformational changes required for cell membrane penetration. Thus, these loops function as regulatory elements to ensure that the spike protein is activated precisely when and where it is needed to facilitate a successful infection.
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Affiliation(s)
- Dunia Asensio-Cob
- Department of Molecular Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto ON, M5G0A4, Canada
| | - Carlos P. Mata
- Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain
| | | | - Javier Vargas
- Departamento de Óptica, Universidad Complutense de Madrid, Madrid 28040, Spain
| | - Javier M. Rodríguez
- Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain
| | - Daniel Luque
- Electron Microscope Unit, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW 2052, Australia
- School of Biomedical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
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Malka M, Czaczkes I, Kashkash S, Shachar S, Bacharach E, Ehrlich M. Inhibition of early EHDV2-Ibaraki infection steps in bovine cells by endosome alkalinization or ikarugamycin, but not by blockage of individual endocytic pathways. Front Cell Infect Microbiol 2025; 15:1494200. [PMID: 39981379 PMCID: PMC11839642 DOI: 10.3389/fcimb.2025.1494200] [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: 09/10/2024] [Accepted: 01/13/2025] [Indexed: 02/22/2025] Open
Abstract
The Epizootic hemorrhagic disease virus (EHDV), an orbivirus, is the etiological factor of a fatal hemorrhagic disease of wild ruminants. A subset of EHDV serotypes, including the Ibaraki strain of EHDV2 (EHDV2-Ibaraki), infect and cause disease in cattle, thus posing a potential threat to livestock. As a member of the Sedoreoviridae family, the EHDV particle is devoid of a membrane envelope and is predicted to employ endocytic pathways for infection. However, the degree of dependence of EHDV2-Ibaraki on specific internalization pathways while infecting bovine cells (its natural host) is unknown. The endosome alkalinizing agent ammonium chloride blocked EHDV2-Ibaraki infection of Madin-Darby Bovine Kidney (MDBK) cells with dependence on its time of addition, suggesting the criticality of endosomal pH for the completion of early stages of infection. Treatment of cells within the alkalinization-sensitive window (i.e., before endosomal processing) with inhibitors of actin polymerization, macropinocytosis (amiloride), or dynamin GTPase activity (dynasore or dynole), or with the cholesterol-depleting agent methyl-β-cyclodextrin, failed to reduce EHDV2-Ibaraki infection. In contrast, in this same treatment time frame, ikarugamycin potently inhibited infection. Moreover, ikarugamycin inhibited interferon induction in infected cells and induced the accumulation of enlarged Rab7- and lamtor4-decorated vacuoles, suggesting its ability to block viral processing and modify late-endosome compartments. Notably, ikarugamycin treatment at initial infection stages, augmented the infection of MDBK cells with the vesicular stomatitis virus while inhibiting infection with bluetongue virus serotype 8. Together, our results point to differential antiviral effects of ikarugamycin on viruses dependent on distinct sets of endosomes for entry/processing.
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Affiliation(s)
| | | | | | | | | | - Marcelo Ehrlich
- The Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv-Yafo, Israel
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5
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Feng Z. Cathepsins prime hepatitis E virus for cell entry. Hepatology 2024; 80:1009-1011. [PMID: 38776183 DOI: 10.1097/hep.0000000000000936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Accepted: 05/10/2024] [Indexed: 05/24/2024]
Affiliation(s)
- Zongdi Feng
- Center for Vaccines and Immunity, The Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA
- The Ohio State University College of Medicine, Columbus, Ohio, USA
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Temple H, Donnelly B, Mohanty SK, Mowery S, Poling HM, Pasula R, Hartman S, Singh A, Mourya R, Bondoc A, Meller J, Jegga AG, Oyama K, McNeal M, Spearman P, Tiao G. Specific binding sites on Rhesus rotavirus capsid protein dictate the method of endocytosis inducing the murine model of biliary atresia. Am J Physiol Gastrointest Liver Physiol 2024; 327:G267-G283. [PMID: 38860860 PMCID: PMC11687966 DOI: 10.1152/ajpgi.00308.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 05/17/2024] [Accepted: 05/28/2024] [Indexed: 06/12/2024]
Abstract
Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.
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Affiliation(s)
- Haley Temple
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Bryan Donnelly
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Sujit K Mohanty
- Southeast Poultry Research Laboratory, United States National Poultry Research Center, United States Department of Agriculture, Athens, Georgia, United States
| | - Sarah Mowery
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Holly M Poling
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Rajamouli Pasula
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Stephen Hartman
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Akaljot Singh
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Reena Mourya
- Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Alexander Bondoc
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Jaroslaw Meller
- Department of Environmental and Public Health Sciences, University of Cincinnati, Cincinnati, Ohio, United States
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Anil G Jegga
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Kei Oyama
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
| | - Monica McNeal
- Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
| | - Paul Spearman
- Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
| | - Greg Tiao
- Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
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Vetter J, Lee M, Eichwald C. The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms. Viruses 2024; 16:668. [PMID: 38793550 PMCID: PMC11125917 DOI: 10.3390/v16050668] [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: 03/24/2024] [Revised: 04/22/2024] [Accepted: 04/23/2024] [Indexed: 05/26/2024] Open
Abstract
Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid-liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell.
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Affiliation(s)
| | | | - Catherine Eichwald
- Institute of Virology, University of Zurich, 8057 Zurich, Switzerland; (J.V.); (M.L.)
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8
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Hu Y, Jiang L, Wang G, Song Y, Shan Z, Wang X, Deng G, Shi J, Tian G, Zeng X, Liu L, Chen H, Li C. M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes. SCIENCE CHINA. LIFE SCIENCES 2024; 67:579-595. [PMID: 38038885 DOI: 10.1007/s11427-023-2471-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Accepted: 10/18/2023] [Indexed: 12/02/2023]
Abstract
Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.
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Affiliation(s)
- Yuzhen Hu
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Li Jiang
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guangwen Wang
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Yangming Song
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Zhibo Shan
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Xuyuan Wang
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guohua Deng
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Jianzhong Shi
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guobin Tian
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Xianying Zeng
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Liling Liu
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Hualan Chen
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
| | - Chengjun Li
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
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9
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Liu Q, Wang W, Xu L, Zhang Q, Wang H. The host mannose-6-phosphate pathway and viral infection. Front Cell Infect Microbiol 2024; 14:1349221. [PMID: 38357444 PMCID: PMC10865371 DOI: 10.3389/fcimb.2024.1349221] [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: 12/04/2023] [Accepted: 01/11/2024] [Indexed: 02/16/2024] Open
Abstract
Viruses, despite their simple structural composition, engage in intricate and complex interactions with their hosts due to their parasitic nature. A notable demonstration of viral behavior lies in their exploitation of lysosomes, specialized organelles responsible for the breakdown of biomolecules and clearance of foreign substances, to bolster their own replication. The man-nose-6-phosphate (M6P) pathway, crucial for facilitating the proper transport of hydrolases into lysosomes and promoting lysosome maturation, is frequently exploited for viral manipulation in support of replication. Recently, the discovery of lysosomal enzyme trafficking factor (LYSET) as a pivotal regulator within the lysosomal M6P pathway has introduced a fresh perspective on the intricate interplay between viral entry and host factors. This groundbreaking revelation illuminates unexplored dimensions of these interactions. In this review, we endeavor to provide a thorough overview of the M6P pathway and its intricate interplay with viral factors during infection. By consolidating the current understanding in this field, our objective is to establish a valuable reference for the development of antiviral drugs that selectively target the M6P pathway.
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Affiliation(s)
- Qincheng Liu
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
- Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong Higher Education Institutes, Guangzhou, China
| | - Weiqi Wang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
- Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong Higher Education Institutes, Guangzhou, China
| | - Liwei Xu
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
- Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong Higher Education Institutes, Guangzhou, China
| | - Qisheng Zhang
- Shanghai Sino Organoid Lifesciences Co., Ltd., Shanghai, China
| | - Hongna Wang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China
- Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong Higher Education Institutes, Guangzhou, China
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10
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Asensio-Cob D, Rodríguez JM, Luque D. Rotavirus Particle Disassembly and Assembly In Vivo and In Vitro. Viruses 2023; 15:1750. [PMID: 37632092 PMCID: PMC10458742 DOI: 10.3390/v15081750] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 08/11/2023] [Accepted: 08/14/2023] [Indexed: 08/27/2023] Open
Abstract
Rotaviruses (RVs) are non-enveloped multilayered dsRNA viruses that are major etiologic agents of diarrheal disease in humans and in the young in a large number of animal species. The viral particle is composed of three different protein layers that enclose the segmented dsRNA genome and the transcriptional complexes. Each layer defines a unique subparticle that is associated with a different phase of the replication cycle. Thus, while single- and double-layered particles are associated with the intracellular processes of selective packaging, genome replication, and transcription, the viral machinery necessary for entry is located in the third layer. This modular nature of its particle allows rotaviruses to control its replication cycle by the disassembly and assembly of its structural proteins. In this review, we examine the significant advances in structural, molecular, and cellular RV biology that have contributed during the last few years to illuminating the intricate details of the RV particle disassembly and assembly processes.
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Affiliation(s)
- Dunia Asensio-Cob
- Department of Molecular Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G0A4, Canada;
| | - Javier M. Rodríguez
- Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, 28049 Madrid, Spain
| | - Daniel Luque
- Electron Microscopy Unit UCCT/ISCIII, 28220 Majadahonda, Spain
- School of Biomedical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia
- Electron Microscope Unit, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, NSW 2052, Australia
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11
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Liu D, Huang R, Yuan K, Zhao J, Wang Z, Yi Q, Wang J. Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection. FISH & SHELLFISH IMMUNOLOGY 2023; 139:108843. [PMID: 37211330 DOI: 10.1016/j.fsi.2023.108843] [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: 03/12/2023] [Revised: 05/16/2023] [Accepted: 05/18/2023] [Indexed: 05/23/2023]
Abstract
The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.
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Affiliation(s)
- Dan Liu
- College of Fisheries and Life, Dalian Ocean University, Dalian, 116000, China; School of Life Science, Huizhou University, Huizhou, 516000, China
| | - RunQing Huang
- School of Life Science, Huizhou University, Huizhou, 516000, China
| | - Kai Yuan
- School of Life Science, Huizhou University, Huizhou, 516000, China
| | - JiaHao Zhao
- School of Life Science, Huizhou University, Huizhou, 516000, China; College of Fisherie, Tianjin Agricultural University, Tianjin, 300384, China
| | - ZhaoRui Wang
- School of Life Science, Huizhou University, Huizhou, 516000, China
| | - QiLin Yi
- College of Fisheries and Life, Dalian Ocean University, Dalian, 116000, China
| | - JiangYong Wang
- School of Life Science, Huizhou University, Huizhou, 516000, China.
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12
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Jo H, Shim K, Jeoung D. Exosomes: Diagnostic and Therapeutic Implications in Cancer. Pharmaceutics 2023; 15:pharmaceutics15051465. [PMID: 37242707 DOI: 10.3390/pharmaceutics15051465] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 04/25/2023] [Accepted: 05/10/2023] [Indexed: 05/28/2023] Open
Abstract
Exosomes are a subset of extracellular vesicles produced by all cells, and they are present in various body fluids. Exosomes play crucial roles in tumor initiation/progression, immune suppression, immune surveillance, metabolic reprogramming, angiogenesis, and the polarization of macrophages. In this work, we summarize the mechanisms of exosome biogenesis and secretion. Since exosomes may be increased in the cancer cells and body fluids of cancer patients, exosomes and exosomal contents can be used as cancer diagnostic and prognostic markers. Exosomes contain proteins, lipids, and nucleic acids. These exosomal contents can be transferred into recipient cells. Therefore, this work details the roles of exosomes and exosomal contents in intercellular communications. Since exosomes mediate cellular interactions, exosomes can be targeted for developing anticancer therapy. This review summarizes current studies on the effects of exosomal inhibitors on cancer initiation and progression. Since exosomal contents can be transferred, exosomes can be modified to deliver molecular cargo such as anticancer drugs, small interfering RNAs (siRNAs), and micro RNAs (miRNAs). Thus, we also summarize recent advances in developing exosomes as drug delivery platforms. Exosomes display low toxicity, biodegradability, and efficient tissue targeting, which make them reliable delivery vehicles. We discuss the applications and challenges of exosomes as delivery vehicles in tumors, along with the clinical values of exosomes. In this review, we aim to highlight the biogenesis, functions, and diagnostic and therapeutic implications of exosomes in cancer.
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Affiliation(s)
- Hyein Jo
- Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Kyeonghee Shim
- Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Dooil Jeoung
- Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
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13
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Jiang L, Tang A, Song L, Tong Y, Fan H. Advances in the development of antivirals for rotavirus infection. Front Immunol 2023; 14:1041149. [PMID: 37006293 PMCID: PMC10063883 DOI: 10.3389/fimmu.2023.1041149] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2022] [Accepted: 02/28/2023] [Indexed: 03/19/2023] Open
Abstract
Rotavirus (RV) causes 200,000 deaths per year and imposes a serious burden to public health and livestock farming worldwide. Currently, rehydration (oral and intravenous) remains the main strategy for the treatment of rotavirus gastroenteritis (RVGE), and no specific drugs are available. This review discusses the viral replication cycle in detail and outlines possible therapeutic approaches including immunotherapy, probiotic-assisted therapy, anti-enteric secretory drugs, Chinese medicine, and natural compounds. We present the latest advances in the field of rotavirus antivirals and highlights the potential use of Chinese medicine and natural compounds as therapeutic agents. This review provides an important reference for rotavirus prevention and treatment.
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Affiliation(s)
| | | | - Lihua Song
- *Correspondence: Huahao Fan, ; Yigang Tong, ; Lihua Song,
| | - Yigang Tong
- *Correspondence: Huahao Fan, ; Yigang Tong, ; Lihua Song,
| | - Huahao Fan
- *Correspondence: Huahao Fan, ; Yigang Tong, ; Lihua Song,
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14
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Zhang C, Chen W, Pan S, Zhang S, Xie H, Zhang Z, Lei W, Bao L, You Y. SEVs-mediated miR-6750 transfer inhibits pre-metastatic niche formation in nasopharyngeal carcinoma by targeting M6PR. Cell Death Dis 2023; 9:2. [PMID: 36609569 PMCID: PMC9823008 DOI: 10.1038/s41420-022-01262-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2022] [Revised: 11/14/2022] [Accepted: 11/17/2022] [Indexed: 01/07/2023]
Abstract
Reliable detection of circulating small extracellular vesicles (SEVs) and their miRNA cargo has been needed to develop potential specific non-invasive diagnostic and therapeutic marker for cancer metastasis. Here, we detected miR-6750, the precise molecular function of which was largely unknown, was significantly enriched in serum-SEVs from normal volunteers vs. patients with nasopharyngeal carcinoma (NPC). And we determined that miR-6750-SEVs attenuated NPC metastasis. Subsequently, miR-6750-SEVs was proven to inhibit angiogenesis and activate macrophage toward to M1 phenotype to inhibit pre-metastatic niche formation. After analyzing the expression level of miR-6750 in NPC cells, HUVECs and macrophage, we found that once miR-6750 level in NPC cells was close to or higher than normal nasopharyngeal epithelial cells (NP69), miR-6750-SEVs would be transferred from NPC cells to macrophage and then to HUVECs to modulate metastatic niche. Moreover, in vitro assays and BALB/c mouse tumor models revealed that miR-6750 directly targeted mannose 6-phosphate receptor (M6PR). Taken together, our findings revealed that miR-6750-M6PR axis can mediate NPC metastasis by remodeling tumor microenvironment (TME) via SEVs, which give novel sights to pathogenesis of NPC.
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Affiliation(s)
- Caiming Zhang
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Wenhui Chen
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Si Pan
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Siyu Zhang
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Haijing Xie
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Zixiang Zhang
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Wei Lei
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Lili Bao
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
| | - Yiwen You
- grid.440642.00000 0004 0644 5481Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China ,grid.440642.00000 0004 0644 5481Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, China
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15
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Patra U, Mukhopadhyay U, Mukherjee A, Dutta S, Chawla-Sarkar M. Treading a HOSTile path: Mapping the dynamic landscape of host cell-rotavirus interactions to explore novel host-directed curative dimensions. Virulence 2021; 12:1022-1062. [PMID: 33818275 PMCID: PMC8023246 DOI: 10.1080/21505594.2021.1903198] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Revised: 01/20/2021] [Accepted: 03/10/2021] [Indexed: 12/27/2022] Open
Abstract
Viruses are intracellular pathogens and are dependent on host cellular resources to carry out their cycles of perpetuation. Obtaining an integrative view of host-virus interaction is of utmost importance to understand the complex and dynamic interplay between viral components and host machineries. Besides its obvious scholarly significance, a comprehensive host-virus interaction profile also provides a platform where from host determinants of pro-viral and antiviral importance can be identified and further be subjected to therapeutic intervention. Therefore, adjunct to conventional methods of prophylactic vaccination and virus-directed antivirals, this host-targeted antiviral approach holds promising therapeutic potential. In this review, we present a comprehensive landscape of host cellular reprogramming in response to infection with rotavirus (RV) which causes profuse watery diarrhea in neonates and infants. In addition, an emphasis is given on how host determinants are either usurped or subverted by RV in course of infection and how therapeutic manipulation of specific host factors can effectively modulate the RV life cycle.
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Affiliation(s)
- Upayan Patra
- Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India
| | - Urbi Mukhopadhyay
- Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India
| | - Arpita Mukherjee
- Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India
| | - Shanta Dutta
- Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India
| | - Mamta Chawla-Sarkar
- Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India
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16
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Swevers L, Kontogiannatos D, Kolliopoulou A, Ren F, Feng M, Sun J. Mechanisms of Cell Entry by dsRNA Viruses: Insights for Efficient Delivery of dsRNA and Tools for Improved RNAi-Based Pest Control. Front Physiol 2021; 12:749387. [PMID: 34858204 PMCID: PMC8632066 DOI: 10.3389/fphys.2021.749387] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 10/11/2021] [Indexed: 12/18/2022] Open
Abstract
While RNAi is often heralded as a promising new strategy for insect pest control, a major obstacle that still remains is the efficient delivery of dsRNA molecules within the cells of the targeted insects. However, it seems overlooked that dsRNA viruses already have developed efficient strategies for transport of dsRNA molecules across tissue barriers and cellular membranes. Besides protecting their dsRNA genomes in a protective shell, dsRNA viruses also display outer capsid layers that incorporate sophisticated mechanisms to disrupt the plasma membrane layer and to translocate core particles (with linear dsRNA genome fragments) within the cytoplasm. Because of the perceived efficiency of the translocation mechanism, it is well worth analyzing in detail the molecular processes that are used to achieve this feat. In this review, the mechanism of cell entry by dsRNA viruses belonging to the Reoviridae family is discussed in detail. Because of the large amount of progress in mammalian versus insect models, the mechanism of infections of reoviruses in mammals (orthoreoviruses, rotaviruses, orbiviruses) will be treated as a point of reference against which infections of reoviruses in insects (orbiviruses in midges, plant viruses in hemipterans, insect-specific cypoviruses in lepidopterans) will be compared. The goal of this discussion is to uncover the basic principles by which dsRNA viruses cross tissue barriers and translocate their cargo to the cellular cytoplasm; such knowledge subsequently can be incorporated into the design of dsRNA virus-based viral-like particles for optimal delivery of RNAi triggers in targeted insect pests.
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Affiliation(s)
- Luc Swevers
- Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos”, Athens, Greece
| | - Dimitrios Kontogiannatos
- Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos”, Athens, Greece
| | - Anna Kolliopoulou
- Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research “Demokritos”, Athens, Greece
| | - Feifei Ren
- Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Min Feng
- Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Jingchen Sun
- Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China
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17
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Caddy S, Papa G, Borodavka A, Desselberger U. Rotavirus research: 2014-2020. Virus Res 2021; 304:198499. [PMID: 34224769 DOI: 10.1016/j.virusres.2021.198499] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Revised: 06/24/2021] [Accepted: 06/25/2021] [Indexed: 02/09/2023]
Abstract
Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.
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Affiliation(s)
- Sarah Caddy
- Cambridge Institute for Therapeutic Immunology and Infectious Disease Jeffery Cheah Biomedical Centre, Cambridge, CB2 0AW, UK.
| | - Guido Papa
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus Francis Crick Avenue, Cambridge, CB2 0QH, UK.
| | - Alexander Borodavka
- Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK.
| | - Ulrich Desselberger
- Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
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18
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Listeria exploits IFITM3 to suppress antibacterial activity in phagocytes. Nat Commun 2021; 12:4999. [PMID: 34404769 PMCID: PMC8371165 DOI: 10.1038/s41467-021-24982-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Accepted: 07/18/2021] [Indexed: 12/20/2022] Open
Abstract
The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3-/- mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.
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19
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Recent advances in rotavirus reverse genetics and its utilization in basic research and vaccine development. Arch Virol 2021; 166:2369-2386. [PMID: 34216267 PMCID: PMC8254061 DOI: 10.1007/s00705-021-05142-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Accepted: 04/27/2021] [Indexed: 11/29/2022]
Abstract
Rotaviruses are segmented double-stranded RNA viruses with a high frequency of gene reassortment, and they are a leading cause of global diarrheal deaths in children less than 5 years old. Two-thirds of rotavirus-associated deaths occur in low-income countries. Currently, the available vaccines in developing countries have lower efficacy in children than those in developed countries. Due to added safety concerns and the high cost of current vaccines, there is a need to develop cost-effective next-generation vaccines with improved safety and efficacy. The reverse genetics system (RGS) is a powerful tool for investigating viral protein functions and developing novel vaccines. Recently, an entirely plasmid-based RGS has been developed for several rotaviruses, and this technological advancement has significantly facilitated novel rotavirus research. Here, we review the recently developed RGS platform and discuss its application in studying infection biology, gene reassortment, and development of vaccines against rotavirus disease.
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20
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Viral Interactions with Adaptor-Protein Complexes: A Ubiquitous Trait among Viral Species. Int J Mol Sci 2021; 22:ijms22105274. [PMID: 34067854 PMCID: PMC8156722 DOI: 10.3390/ijms22105274] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 05/10/2021] [Accepted: 05/12/2021] [Indexed: 12/22/2022] Open
Abstract
Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.
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21
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Abstract
Group A rotaviruses (RVAs) are the major cause of severe acute gastroenteritis (AGE) in children under 5 years of age, annually resulting in nearly 130,000 deaths worldwide. Social conditions in developing countries that contribute to decreased oral rehydration and vaccine efficacy and the lack of approved antiviral drugs position RVA as a global health concern. In this minireview, we present an update in the field of antiviral compounds, mainly in relation to the latest findings in RVA virion structure and the viral replication cycle. In turn, we attempt to provide a perspective on the possible treatments for RVA-associated AGE, with special focus on novel approaches, such as those representing broad-spectrum therapeutic options. In this context, the modulation of host factors, lipid droplets, and the viral polymerase, which is highly conserved among AGE-causing viruses, are analyzed as possible drug targets.
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22
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Arnold MM, Dijk A, López S. Double‐stranded RNA Viruses. Virology 2021. [DOI: 10.1002/9781119818526.ch2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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23
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Arias CF, López S. Rotavirus cell entry: not so simple after all. Curr Opin Virol 2021; 48:42-48. [PMID: 33887683 DOI: 10.1016/j.coviro.2021.03.011] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 03/28/2021] [Accepted: 03/29/2021] [Indexed: 01/25/2023]
Abstract
Rotaviruses are important agents of severe gastroenteritis in young children, and show a very selective cell and tissue tropism, as well as significant age and host restriction. In the last few years, these properties have been associated with the initial interaction of the virus with histo-blood group antigens on the cell surface, although post-attachment interactions have also been found to define the susceptibility to infection of human enteroids. These initial interactions seem also to determine the virus entry pathway, as well as the induction of signaling cascades that influence the virus intracellular vesicular traffic and escape from endosomes. Here we review the current knowledge of the different stages of the virus entry journey.
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Affiliation(s)
- Carlos F Arias
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos, Mexico.
| | - Susana López
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos, Mexico
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24
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Wang R, Yu R, Chen B, Si F, Wang J, Xie C, Men C, Dong S, Li Z. Identification of host cell proteins that interact with the M protein of porcine epidemic diarrhea virus. Vet Microbiol 2020; 246:108729. [PMID: 32605758 PMCID: PMC7241372 DOI: 10.1016/j.vetmic.2020.108729] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 05/13/2020] [Accepted: 05/17/2020] [Indexed: 12/03/2022]
Abstract
Interaction of PEDV M protein with host cellular proteins eIF3L, CDC42 and Rab11A was confirmed. PEDV replication may be regulated by eIF3L expression. 218 host cell proteins were designated putative PEDV M protein interacting proteins. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes severe diarrhea in pigs of all ages and a high fatality rate in neonates. The PEDV membrane protein (M) plays crucial roles in viral assembly, viral budding and host immune regulation, most likely by interacting with host cell proteins that have yet to be identified. In this study, co-immunoprecipitation (Co-IP) using an M-specific monoclonal antibody, coupled with LC-MS/MS, was employed to identify M protein-interacting proteins in PEDV-infected cells. Three viral proteins (S, E and ORF3) and 218 host cell proteins were identified as putative M-interacting partners. Bioinformatic analysis showed that the identified host cell proteins were related to 131 signal pathways and 10 biological processes. In addition, interaction between translation initiation factor 3(eIF3L) and M protein was validated by Co-IP. Down-regulation of eIF3L expression significantly increased viral production, which suggests that eIF3L could be a negative regulator in PEDV replication. This interactome study of the PEDV M protein will serve to clarify its function during viral replication.
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Affiliation(s)
- Ruiyang Wang
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Ruisong Yu
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Bingqing Chen
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Fusheng Si
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Jian Wang
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Chunfang Xie
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Chengfang Men
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China
| | - Shijuan Dong
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China.
| | - Zhen Li
- Institute of Animal Husbandry and Veterinary Science, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Engineering Research Center of Breeding Pig, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201106 China.
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25
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Nasr ML. Large nanodiscs going viral. Curr Opin Struct Biol 2020; 60:150-156. [PMID: 32066086 PMCID: PMC10712563 DOI: 10.1016/j.sbi.2020.01.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 01/08/2020] [Accepted: 01/09/2020] [Indexed: 12/29/2022]
Abstract
Covalently circularized and DNA-corralled nanodisc technologies have enabled engineering of large-sized bilayer nanodiscs up to 90nm. These large nanodiscs have the potential to extend the applicability of nanodisc technology from studying small and medium-sized membrane proteins to acting as surrogate membranes to investigate functional and structural aspects of viral entry. Here, we discuss the recent technical developments leading to construction of large circularized and DNA-corralled nanodiscs and examine their application in viral entry.
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Affiliation(s)
- Mahmoud L Nasr
- Division of Renal Medicine, Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
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26
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Rodríguez JM, Luque D. Structural Insights into Rotavirus Entry. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1215:45-68. [PMID: 31317495 DOI: 10.1007/978-3-030-14741-9_3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
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27
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Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells. J Virol 2018; 92:JVI.01674-18. [PMID: 30282712 PMCID: PMC6258943 DOI: 10.1128/jvi.01674-18] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 09/22/2018] [Indexed: 12/24/2022] Open
Abstract
Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.
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Oceguera A, Peralta AV, Martínez-Delgado G, Arias CF, López S. Rotavirus RNAs sponge host cell RNA binding proteins and interfere with their subcellular localization. Virology 2018; 525:96-105. [PMID: 30253276 DOI: 10.1016/j.virol.2018.09.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Revised: 09/13/2018] [Accepted: 09/17/2018] [Indexed: 10/28/2022]
Abstract
Cellular mRNAs cycle between translating and non-translating pools, polysomes compose the translating pool, while RNA granules contain translationally-silenced mRNAs, where the RNAs are either stored in stress granules, or accumulate in processing bodies (PBs) or GW-bodies, which have an important role in RNA degradation. Viruses have developed measures to prevent the deleterious effects of these structures during their replication. Rotavirus, the most common agent of viral gastroenteritis, is capable of establishing a successful infection by counteracting several of the antiviral responses of its host. Here, we describe that in rotavirus-infected cells the distribution of several RNA binding proteins is changed causing the disaggregation of PBs, the relocalization of GW-body proteins, and the cytoplasmic accumulation of HuR, a predominantly nuclear protein. We show that this redistribution of proteins is more likely caused by the accumulation of viral RNA in the cytoplasm of infected-cells, where it might be acting as an RBP sponge.
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Affiliation(s)
- Alfonso Oceguera
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico
| | - Andrea V Peralta
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico
| | - Gustavo Martínez-Delgado
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico
| | - Carlos F Arias
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico
| | - Susana López
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico.
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Soliman M, Kim DS, Kim C, Seo JY, Kim JY, Park JG, Alfajaro MM, Baek YB, Cho EH, Park SI, Kang MI, Chang KO, Goodfellow I, Cho KO. Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II. Vet Res 2018; 49:92. [PMID: 30223898 PMCID: PMC6142377 DOI: 10.1186/s13567-018-0584-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Accepted: 08/27/2018] [Indexed: 11/22/2022] Open
Abstract
Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.
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Affiliation(s)
- Mahmoud Soliman
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Deok-Song Kim
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Chonsaeng Kim
- Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea
| | - Ja-Young Seo
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Ji-Yun Kim
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Jun-Gyu Park
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Mia Madel Alfajaro
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Yeong-Bin Baek
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Eun-Hyo Cho
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Sang-Ik Park
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Mun-Il Kang
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
| | - Kyeong-Ok Chang
- Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS USA
| | - Ian Goodfellow
- Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK
| | - Kyoung-Oh Cho
- Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea
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Civra A, Francese R, Gamba P, Testa G, Cagno V, Poli G, Lembo D. 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes. Redox Biol 2018; 19:318-330. [PMID: 30212801 PMCID: PMC6138790 DOI: 10.1016/j.redox.2018.09.003] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Revised: 08/29/2018] [Accepted: 09/03/2018] [Indexed: 11/21/2022] Open
Abstract
A novel innate immune strategy, involving specific cholesterol oxidation products as effectors, has begun to reveal connections between cholesterol metabolism and immune response against viral infections. Indeed, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC), physiologically produced by enzymatic oxidation of cholesterol, act as inhibitors of a wide spectrum of enveloped and non-enveloped human viruses. However, the mechanisms underlying their protective effects against non-enveloped viruses are almost completely unexplored. To get insight into this field, we investigated the antiviral activity of 25HC and 27HC against a non-enveloped virus causing acute gastroenteritis in children, the human rotavirus (HRV). We found that 25HC and 27HC block the infectivity of several HRV strains at 50% inhibitory concentrations in the low micromolar range in the absence of cell toxicity. Both molecules affect the final step of virus penetration into cells by preventing the association of two cellular proteins: the oxysterol binding protein (OSBP) and the vesicle-associated membrane protein-associated protein-A (VAP-A). By altering the activity of these cellular mediators, 25HC and 27HC disturb the recycling of cholesterol between the endoplasmic reticulum and the late endosomes which are exploited by HRV to penetrate into the cell. The substantial accumulation of cholesterol in the late endosomal compartment results in sequestering viral particles inside these vesicles thereby preventing cytoplasmic virus replication. These findings suggest that cholesterol oxidation products of enzymatic origin might be primary effectors of host restriction strategies to counteract HRV infection and point to redox active lipids involvement in viral infections as a research area of focus to better focus in order to identify novel antiviral agents targets.
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Affiliation(s)
- Andrea Civra
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy
| | - Rachele Francese
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy
| | - Paola Gamba
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy
| | - Gabriella Testa
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy
| | - Valeria Cagno
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy
| | - Giuseppe Poli
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy.
| | - David Lembo
- Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, TO, Italy.
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Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection. J Virol 2018; 92:JVI.00612-18. [PMID: 29769336 DOI: 10.1128/jvi.00612-18] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Accepted: 05/08/2018] [Indexed: 12/21/2022] Open
Abstract
Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.
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Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication. J Virol 2018. [PMID: 29540593 DOI: 10.1128/jvi.01964-17] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.
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Makaraci P, Kim K. trans-Golgi network-bound cargo traffic. Eur J Cell Biol 2018; 97:137-149. [PMID: 29398202 DOI: 10.1016/j.ejcb.2018.01.003] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Revised: 12/15/2017] [Accepted: 01/16/2018] [Indexed: 12/19/2022] Open
Abstract
Cargo following the retrograde trafficking are sorted at endosomes to be targeted the trans-Golgi network (TGN), a central receiving organelle. Though molecular requirements and their interaction networks have been somewhat established, the complete understanding of the intricate nature of their action mechanisms in every step of the retrograde traffic pathway remains unachieved. This review focuses on elucidating known functions of key regulators, including scission factors at the endosome and tethering/fusion mediators at the receiving dock, TGN, as well as a diverse range of cargo.
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Affiliation(s)
- Pelin Makaraci
- Department of Biology, Missouri State University, 901 S National Ave., Springfield, MO 65807, USA
| | - Kyoungtae Kim
- Department of Biology, Missouri State University, 901 S National Ave., Springfield, MO 65807, USA.
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Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating. PLoS Pathog 2018; 14:e1006820. [PMID: 29352319 PMCID: PMC5792019 DOI: 10.1371/journal.ppat.1006820] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Revised: 01/31/2018] [Accepted: 12/15/2017] [Indexed: 11/19/2022] Open
Abstract
The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections. Viral particles must transport their genome into the cytoplasm or the nucleus of host cells to initiate successful infection. Knowledge of how viruses may pirate host cell signaling cascades or molecules to promote their own replication can facilitate the development of antiviral drugs. Group A rotavirus (RVA) is a major etiological agent of acute gastroenteritis in young children and the young of various mammals. RVA enters cells by a complex multistep process. However, the cellular signaling cascades or molecules that facilitate these processes are incompletely understood. Here, we demonstrate that infection with late-penetration RVA strains results in phosphorylation of PI3K, Akt, and ERK signaling molecules at an early stage of infection, a process mediated by the multistep binding of RVAs outer capsid proteins. Specific inhibitors for PI3K/Akt and MEK/ERK signaling pathways trap the viral particles in late endosome, and acidic replenishment restores and releases them. Moreover, the RVA-induced phosphorylated PI3K, Akt, and ERK directly interact with the subunit E of the V-ATPase proton pump, required for endosomal acidification and RVA uncoating. Understanding how RVA-induced early activation of cellular signaling molecules mediates the V-ATPase-dependent endosomal acidification required for uncoating of viral particles opens up opportunities for targeted interventions against rotavirus entry.
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Díaz-Salinas MA, Casorla LA, López T, López S, Arias CF. Most rotavirus strains require the cation-independent mannose-6-phosphate receptor, sortilin-1, and cathepsins to enter cells. Virus Res 2017; 245:44-51. [PMID: 29275103 PMCID: PMC7173016 DOI: 10.1016/j.virusres.2017.12.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2017] [Revised: 12/04/2017] [Accepted: 12/06/2017] [Indexed: 12/27/2022]
Abstract
Rotaviruses require the TGN to LE transporter CI-M6PR for cell entry. Sortilin-1 was identified as a cell factor involved in rotavirus replication. Rotaviruses require cathepsins also to enter Caco-2 cells. Cathepsins, endosomal acid proteases, are transported from the trans-Golgi network to late endosomes by the mannose-6-phosphate receptor (M6PR). We have previously demonstrated that some rotavirus strains, like UK, Wa, WI61, DS-1, and YM, require the cation-dependent (CD-) M6PR and cathepsins to enter from late endosomes to the cytoplasm in MA104 cells, while other strains, like the simian strain RRV, which enter cells from maturing endosomes, do not. However, the role of other trans-Golgi network-late endosome transporters, such as the cation-independent (CI-) M6PR and sortillin-1, has not been evaluated. In this work, we found that several rotavirus strains that require the CD-M6PR for cell entry are also dependent on CI-M6PR and sortilin-1. Furthermore, we showed that the infectivity of all these rotavirus strains also requires cathepsins to enter not only MA104 cells, but also human intestinal Caco-2 cells. This study identifies sortilin-1 as a novel cell factor necessary for the infectivity of a virus; in addition, our results strongly suggest that cathepsins could be common cell factors needed for the infectivity of most rotavirus strains.
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Affiliation(s)
- Marco A Díaz-Salinas
- Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico
| | - Luis A Casorla
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico
| | - Tomás López
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico
| | - Susana López
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico
| | - Carlos F Arias
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico.
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Breach: Host Membrane Penetration and Entry by Nonenveloped Viruses. Trends Microbiol 2017; 26:525-537. [PMID: 29079499 DOI: 10.1016/j.tim.2017.09.010] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2017] [Revised: 09/06/2017] [Accepted: 09/26/2017] [Indexed: 11/22/2022]
Abstract
Disruption of host membranes by nonenveloped viruses, which allows the nucleocapsid or genome to enter the cytosol, is a mechanistically diverse process. Although the membrane-penetrating agents are usually small, hydrophobic or amphipathic peptides deployed from the capsid interior during entry, their manner of membrane interaction varies substantially. In this review, we discuss recent data about the molecular pathways for externalization of viral peptides amidst conformational alterations in the capsid, as well as mechanisms of membrane penetration, which is influenced by structural features of the peptides themselves as well as physicochemical properties of membranes, and other host factors. The membrane-penetrating components of nonenveloped viruses constitute an interesting class of cell-penetrating peptides, and may have potential therapeutic value for gene transfer.
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Pramanik A, Jones S, Gao Y, Sweet C, Begum S, Shukla MK, Buchanan JP, Moser RD, Ray PC. A bio-conjugated chitosan wrapped CNT based 3D nanoporous architecture for separation and inactivation of Rotavirus and Shigella waterborne pathogens. J Mater Chem B 2017; 5:9522-9531. [DOI: 10.1039/c7tb02815f] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
The multifunctional bio-conjugated 3D architecture reported here represents huge advances in the fields of environmental remediation and sustainable remediation.
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Affiliation(s)
- Avijit Pramanik
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
| | - Stacy Jones
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
| | - Ye Gao
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
| | - Carrie Sweet
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
| | - Salma Begum
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
| | - Manoj K. Shukla
- US Army Engineer Research and Development Center
- Vicksburg
- USA
| | | | - Robert D. Moser
- US Army Engineer Research and Development Center
- Vicksburg
- USA
| | - Paresh Chandra Ray
- Department of Chemistry and Biochemistry
- Jackson State University
- Jackson
- USA
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Cevallos Porta D, López S, Arias CF, Isa P. Polarized rotavirus entry and release from differentiated small intestinal cells. Virology 2016; 499:65-71. [PMID: 27639572 DOI: 10.1016/j.virol.2016.09.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Revised: 09/06/2016] [Accepted: 09/08/2016] [Indexed: 01/26/2023]
Abstract
Rotaviruses infect mature enterocytes from small intestine, however most data about their cellular entry are from studies carried out in non-intestinal polarized or non-polarized cell lines. In this work the entry of porcine rotavirus YM strain into small intestinal cell line IPEC-J2 was studied. It was found that YM and the human rotavirus Wa strain infect preferentially from the basolateral cell surface. Cell infection from the apical and basolateral surfaces was dependent on the presence of cholesterol. The treatment with neuraminidase, sucrose, and bafilomycin suggests that there are differences in the receptor usage and entry mechanism of the virus from the apical and basolateral surface. While cell entry is more efficient from basolateral surface, the viruses egressed mainly from the apical cell side.
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Affiliation(s)
- Diego Cevallos Porta
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico.
| | - Susana López
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico.
| | - Carlos F Arias
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico.
| | - Pavel Isa
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico.
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Abstract
Rotavirus, a member of the family Reoviridae, was identified as the leading etiological agent of severe gastroenteritis in infants and young children in 1973. The rotavirus genome is composed of 11 gene segments of double-stranded (ds)RNA. During the last 40 years, a large amount of basic research on rotavirus structure, genome, antigen, replication, pathogenesis, epidemiology, immune responses, and evolution has been accumulated. This article reviews the fundamental aspects of rotavirology including recent important achievements in research.
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41
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Guerrero CA, Acosta O. Inflammatory and oxidative stress in rotavirus infection. World J Virol 2016; 5:38-62. [PMID: 27175349 PMCID: PMC4861870 DOI: 10.5501/wjv.v5.i2.38] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Revised: 10/23/2015] [Accepted: 01/29/2016] [Indexed: 02/05/2023] Open
Abstract
Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines.
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Patel A, Mohl BP, Roy P. Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid. J Biol Chem 2016; 291:12408-19. [PMID: 27036941 PMCID: PMC4933286 DOI: 10.1074/jbc.m115.700856] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Indexed: 12/03/2022] Open
Abstract
The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.
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Affiliation(s)
- Avnish Patel
- From the Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom
| | - Bjorn-Patrick Mohl
- From the Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom
| | - Polly Roy
- From the Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom
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43
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Arias C, Silva-Ayala D, Isa P, Díaz-Salinas M, López S. Rotavirus Attachment, Internalization, and Vesicular Traffic. VIRAL GASTROENTERITIS 2016:103-119. [DOI: 10.1016/b978-0-12-802241-2.00006-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2025]
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44
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Swaine T, Dittmar MT. CDC42 Use in Viral Cell Entry Processes by RNA Viruses. Viruses 2015; 7:6526-36. [PMID: 26690467 PMCID: PMC4690878 DOI: 10.3390/v7122955] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Revised: 11/03/2015] [Accepted: 11/30/2015] [Indexed: 01/08/2023] Open
Abstract
The cellular actin cytoskeleton presents a barrier that must be overcome by many viruses, and it has become increasingly apparent many viral species have developed a diverse repertoire of mechanisms to hijack cellular actin-regulating signalling pathways as part of their cell entry processes. The Rho family GTPase Cdc42 is appreciated as a key moderator of cellular actin dynamics, and the development of specific Cdc42-inhibiting agents has given us an unprecedented ability to investigate its individual role in signalling pathways. However, investigative use of said agents, and the subsequent characterisation of the role Cdc42 plays in viral entry processes has been lacking. Here, we describe the current literature on the role of Cdc42 in human immunodeficiency virus (HIV)-1 cell entry, which represents the most investigated instance of Cdc42 function in viral cell entry processes, and also review evidence of Cdc42 use in other RNA virus cell entries, demonstrating prime areas for more extensive research using similar techniques.
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Affiliation(s)
- Thomas Swaine
- Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, 4 Newark Street, London E1 2AT, UK.
| | - Matthias T Dittmar
- Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, 4 Newark Street, London E1 2AT, UK.
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45
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López T, López S, Arias CF. The tyrosine kinase inhibitor genistein induces the detachment of rotavirus particles from the cell surface. Virus Res 2015. [PMID: 26216271 DOI: 10.1016/j.virusres.2015.07.020] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Group A rotaviruses are a major cause of severe gastroenteritis in young infants. In this work we evaluated the potential role of protein tyrosine kinases on rotavirus infectivity and viral progeny production. From the broad-spectrum inhibitors tested, only genistein, a flavonoid, inhibited rotavirus infectivity. The inhibition observed was dose and strain dependent, with more than 10-fold IC50 differences for some rotavirus strains, and the effect of the drug was shown to be dependent of their activity as a protein tyrosine kinase inhibitor, since the inactive analogue of genistein, daidzein, had no effect on virus infection. Investigation of the stage of virus replication blocked by the drug showed that it interferes with the early interactions of the virus with receptors and/or co-receptors, since treatment of the cells with genistein promoted the detachment of the virus from the cell surface.
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Affiliation(s)
- Tomás López
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Susana López
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Carlos F Arias
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
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46
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Romero-Maraccini OC, Shisler JL, Nguyen TH. Solar and temperature treatments affect the ability of human rotavirus wa to bind to host cells and synthesize viral RNA. Appl Environ Microbiol 2015; 81:4090-7. [PMID: 25862222 PMCID: PMC4524135 DOI: 10.1128/aem.00027-15] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 04/01/2015] [Indexed: 12/13/2022] Open
Abstract
Rotavirus, the leading cause of diarrheal diseases in children under the age of five, is often resistant to conventional wastewater treatment and thus can remain infectious once released into the aquatic environment. Solar and heat treatments can inactivate rotavirus, but it is unknown how these treatments inactivate the virus on a molecular level. To answer this question, our approach was to correlate rotavirus inactivation with the inhibition of portions of the virus life cycle as a means to identify the mechanisms of solar or heat inactivation. Specifically, the integrity of the rotavirus NSP3 gene, virus-host cell interaction, and viral RNA synthesis were examined after heat (57°C) or solar treatment of rotavirus. Only the inhibition of viral RNA synthesis positively correlated with a loss of rotavirus infectivity; 57°C treatment of rotavirus resulted in a decrease of rotavirus RNA synthesis at the same rate as rotavirus infectivity. These data suggest that heat treatment neutralized rotaviruses primarily by targeting viral transcription functions. In contrast, when using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute to inactivation. Nevertheless, both solar and heat inactivation of rotaviruses disrupted viral RNA synthesis as a mechanism for inactivation.
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Affiliation(s)
- Ofelia C Romero-Maraccini
- Department of Civil and Environmental Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
| | - Joanna L Shisler
- Department of Microbiology, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
| | - Thanh H Nguyen
- Department of Civil and Environmental Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
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47
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Civra A, Giuffrida MG, Donalisio M, Napolitano L, Takada Y, Coulson BS, Conti A, Lembo D. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition. J Biol Chem 2015; 290:12403-14. [PMID: 25814665 PMCID: PMC4424369 DOI: 10.1074/jbc.m114.620500] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2014] [Revised: 03/24/2015] [Indexed: 11/06/2022] Open
Abstract
Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding.
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Affiliation(s)
- Andrea Civra
- From the Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, Regione Gonzole, 10, 10043 Orbassano, Torino, Italy
| | - Maria Gabriella Giuffrida
- the Institute of Sciences of Food Production-National Research Council (ISPA-CNR), c/o Bioindustry Park "SilvanoFumero", 10100 CollerettoGiacosa, Torino, Italy
| | - Manuela Donalisio
- From the Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, Regione Gonzole, 10, 10043 Orbassano, Torino, Italy
| | - Lorenzo Napolitano
- the Institute of Sciences of Food Production-National Research Council (ISPA-CNR), c/o Bioindustry Park "SilvanoFumero", 10100 CollerettoGiacosa, Torino, Italy
| | - Yoshikazu Takada
- the Department of Dermatology and Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, California 95817, and
| | - Barbara S Coulson
- the Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia
| | - Amedeo Conti
- the Institute of Sciences of Food Production-National Research Council (ISPA-CNR), c/o Bioindustry Park "SilvanoFumero", 10100 CollerettoGiacosa, Torino, Italy
| | - David Lembo
- From the Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, Regione Gonzole, 10, 10043 Orbassano, Torino, Italy,
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48
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Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step. J Virol 2014; 89:2931-43. [PMID: 25552710 DOI: 10.1128/jvi.03398-14] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
UNLABELLED Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.
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49
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Torres-Flores JM, Silva-Ayala D, Espinoza MA, López S, Arias CF. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells. Virology 2014; 475:172-8. [PMID: 25481868 DOI: 10.1016/j.virol.2014.11.016] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2014] [Revised: 09/12/2014] [Accepted: 11/12/2014] [Indexed: 01/19/2023]
Abstract
Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.
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Affiliation(s)
- Jesús M Torres-Flores
- Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Daniela Silva-Ayala
- Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Marco A Espinoza
- Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Susana López
- Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
| | - Carlos F Arias
- Instituto de Biotecnología, Universidad Nacional Autónoma de México. Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
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50
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Abstract
Rotaviruses are the leading etiological agents of acute gastroenteritis in infants and young children worldwide. These nonenveloped viruses enter cells using different types of endocytosis and, depending on the virus strain, travel to different endosomal compartments before exiting to the cytosolic space. In this Gem, we review the viral and cellular factors involved in the different stages of a productive virus cell entry and share with the readers the journey that we have taken into the cell to learn about virus entry.
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