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1
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Shrestha R, Johnson PM, Ghimire R, Whitley CJ, Channappanavar R. Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages. Pathogens 2024; 13:1033. [PMID: 39770293 PMCID: PMC11676137 DOI: 10.3390/pathogens13121033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2024] [Revised: 11/13/2024] [Accepted: 11/19/2024] [Indexed: 01/11/2025] Open
Abstract
RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as "dysregulated immunity" is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.
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Affiliation(s)
- Rakshya Shrestha
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA; (R.S.); (P.M.J.); (R.G.); (C.J.W.)
| | - Paige Marie Johnson
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA; (R.S.); (P.M.J.); (R.G.); (C.J.W.)
| | - Roshan Ghimire
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA; (R.S.); (P.M.J.); (R.G.); (C.J.W.)
| | - Cody John Whitley
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA; (R.S.); (P.M.J.); (R.G.); (C.J.W.)
| | - Rudragouda Channappanavar
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA; (R.S.); (P.M.J.); (R.G.); (C.J.W.)
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, OK 74078, USA
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2
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Nishikiori M, den Boon JA, Unchwaniwala N, Ahlquist P. Crowning Touches in Positive-Strand RNA Virus Genome Replication Complex Structure and Function. Annu Rev Virol 2022; 9:193-212. [PMID: 35610038 DOI: 10.1146/annurev-virology-092920-021307] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Masaki Nishikiori
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Johan A den Boon
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Nuruddin Unchwaniwala
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Current affiliation: Assembly Biosciences, Inc., South San Francisco, California, USA
| | - Paul Ahlquist
- John and Jeanne Rowe Center for Research in Virology, Morgridge Institute for Research, Madison, Wisconsin, USA; .,Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA
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3
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Fernández de Castro I, Tenorio R, Ortega-González P, Knowlton JJ, Zamora PF, Lee CH, Fernández JJ, Dermody TS, Risco C. A modified lysosomal organelle mediates nonlytic egress of reovirus. J Cell Biol 2020; 219:e201910131. [PMID: 32356864 PMCID: PMC7337502 DOI: 10.1083/jcb.201910131] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Revised: 02/20/2020] [Accepted: 04/06/2020] [Indexed: 12/20/2022] Open
Abstract
Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.
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Affiliation(s)
- Isabel Fernández de Castro
- Cell Structure Laboratory, National Center for Biotechnology, Spanish National Research Council, Madrid, Spain
| | - Raquel Tenorio
- Cell Structure Laboratory, National Center for Biotechnology, Spanish National Research Council, Madrid, Spain
| | - Paula Ortega-González
- Cell Structure Laboratory, National Center for Biotechnology, Spanish National Research Council, Madrid, Spain
| | - Jonathan J. Knowlton
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Paula F. Zamora
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA
| | - Christopher H. Lee
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA
- Center for Microbial Pathogenesis, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA
| | - José J. Fernández
- Department of Macromolecular Structures, National Center for Biotechnology, Spanish National Research Council, Madrid, Spain
| | - Terence S. Dermody
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA
- Center for Microbial Pathogenesis, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA
| | - Cristina Risco
- Cell Structure Laboratory, National Center for Biotechnology, Spanish National Research Council, Madrid, Spain
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4
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Replication of Hepatitis E Virus (HEV) in Primary Human-Derived Monocytes and Macrophages In Vitro. Vaccines (Basel) 2020; 8:vaccines8020239. [PMID: 32455708 PMCID: PMC7349946 DOI: 10.3390/vaccines8020239] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 05/15/2020] [Accepted: 05/18/2020] [Indexed: 12/16/2022] Open
Abstract
HEV is the most causative agent of acute viral hepatitis globally. HEV causes acute, chronic, and extrahepatic manifestations. Chronic HEV infection develops in immunocompromised patients such as organ transplant patients, HIV-infected patients, and leukemic patients. The source of chronic HEV infection is not known. Also, the source of extrahepatic manifestations associated with HEV infection is still unclear. Hepatotropic viruses such as HCV and HBV replicate in peripheral blood mononuclear cells (PBMCs) and these cells become a source of chronic reactivation of the infections in allograft organ transplant patients. Herein, we reported that PBMCs and bone marrow-derived macrophages (BMDMs), isolated from healthy donors (n = 3), are susceptible to HEV in vitro. Human monocytes (HMOs), human macrophages (HMACs), and human BMDMs were challenged with HEV-1 and HEV-3 viruses. HEV RNA was measured by qPCR, the marker of the intermediate replicative form (ds-RNA) was assessed by immunofluorescence, and HEV capsid protein was assessed by flow cytometry and ELISA. HEV infection was successfully established in primary HMOs, HMACs, and human BMDMs, but not in the corresponding cells of murine origin. Intermediate replicative form (ds RNA) was detected in HMOs and HMACs challenged with HEV. The HEV load was increased over time, and the HEV capsid protein was detected intracellularly in the HEV-infected cells and accumulated extracellularly over time, confirming that HEV completes the life cycle inside these cells. The HEV particles produced from the infected BMDMs were infectious to naive HMOs in vitro. The HEV viral load was comparable in HEV-1- and HEV-3-infected cells, but HEV-1 induced more inflammatory responses. In conclusion, HMOs, HMACs, and human BMDMs are permissive to HEV infection and these cells could be the source of chronic and recurrent infection, especially in immunocompromised patients. Replication of HEV in human BMDMs could be related to hematological disorders associated with extrahepatic manifestations.
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5
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George S, Viswanathan R, Sapkal GN. Molecular aspects of the teratogenesis of rubella virus. Biol Res 2019; 52:47. [PMID: 31455418 PMCID: PMC6712747 DOI: 10.1186/s40659-019-0254-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Accepted: 08/12/2019] [Indexed: 11/10/2022] Open
Abstract
Rubella or German measles is an infection caused by rubella virus (RV). Infection of children and adults is usually characterized by a mild exanthematous febrile illness. However, RV is a major cause of birth defects and fetal death following infection in pregnant women. RV is a teratogen and is a major cause of public health concern as there are more than 100,000 cases of congenital rubella syndrome (CRS) estimated to occur every year. Several lines of evidence in the field of molecular biology of RV have provided deeper insights into the teratogenesis process. The damage to the growing fetus in infected mothers is multifactorial, arising from a combination of cellular damage, as well as its effect on the dividing cells. This review focuses on the findings in the molecular biology of RV, with special emphasis on the mitochondrial, cytoskeleton and the gene expression changes. Further, the review addresses in detail, the role of apoptosis in the teratogenesis process.
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Affiliation(s)
- Suji George
- Diagnostic Virology Group, ICMR-National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune, Maharashtra 411001 India
| | - Rajlakshmi Viswanathan
- Diagnostic Virology Group, ICMR-National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune, Maharashtra 411001 India
| | - Gajanan N. Sapkal
- Diagnostic Virology Group, ICMR-National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune, Maharashtra 411001 India
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6
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Sachse M, Fernández de Castro I, Tenorio R, Risco C. The viral replication organelles within cells studied by electron microscopy. Adv Virus Res 2019; 105:1-33. [PMID: 31522702 PMCID: PMC7112055 DOI: 10.1016/bs.aivir.2019.07.005] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Transmission electron microscopy (TEM) has been crucial to study viral infections. As a result of recent advances in light and electron microscopy, we are starting to be aware of the variety of structures that viruses assemble inside cells. Viruses often remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. Here we revise the most relevant imaging technologies to study the biogenesis of viral replication organelles. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ in two and three dimensions are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral life cycle events with a detail never seen before.
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Affiliation(s)
- Martin Sachse
- Unité Technologie et service BioImagerie Ultrastructurale, Institut Pasteur, Paris, France.
| | | | - Raquel Tenorio
- Cell Structure Laboratory, National Center for Biotechnology, CSIC, Madrid, Spain
| | - Cristina Risco
- Cell Structure Laboratory, National Center for Biotechnology, CSIC, Madrid, Spain.
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7
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The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication. Virology 2018; 515:11-20. [DOI: 10.1016/j.virol.2017.11.019] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Revised: 11/19/2017] [Accepted: 11/23/2017] [Indexed: 01/12/2023]
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8
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Monsion B, Incarbone M, Hleibieh K, Poignavent V, Ghannam A, Dunoyer P, Daeffler L, Tilsner J, Ritzenthaler C. Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein. FRONTIERS IN PLANT SCIENCE 2018; 9:70. [PMID: 29449856 PMCID: PMC5799278 DOI: 10.3389/fpls.2018.00070] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/10/2017] [Accepted: 01/12/2018] [Indexed: 05/17/2023]
Abstract
Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.
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Affiliation(s)
- Baptiste Monsion
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Marco Incarbone
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Kamal Hleibieh
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Vianney Poignavent
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Ahmed Ghannam
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Patrice Dunoyer
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
| | - Laurent Daeffler
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Jens Tilsner
- Biomedical Sciences Research Complex, University of St Andrews, St Andrews, United Kingdom
- Cell and Molecular Sciences, The James Hutton Institute, Dundee, United Kingdom
| | - Christophe Ritzenthaler
- Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, France
- *Correspondence: Christophe Ritzenthaler
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9
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Ertel KJ, Benefield D, Castaño-Diez D, Pennington JG, Horswill M, den Boon JA, Otegui MS, Ahlquist P. Cryo-electron tomography reveals novel features of a viral RNA replication compartment. eLife 2017; 6. [PMID: 28653620 PMCID: PMC5515581 DOI: 10.7554/elife.25940] [Citation(s) in RCA: 75] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Accepted: 06/20/2017] [Indexed: 12/18/2022] Open
Abstract
Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule’s necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control. DOI:http://dx.doi.org/10.7554/eLife.25940.001
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Affiliation(s)
- Kenneth J Ertel
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, United States
| | - Desirée Benefield
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Morgridge Institute for Research, University of Wisconsin-Madison, Madison, United States
| | | | - Janice G Pennington
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, United States
| | - Mark Horswill
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Morgridge Institute for Research, University of Wisconsin-Madison, Madison, United States
| | - Johan A den Boon
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Morgridge Institute for Research, University of Wisconsin-Madison, Madison, United States
| | - Marisa S Otegui
- Department of Botany, University of Wisconsin-Madison, Madison, United States.,Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, United States
| | - Paul Ahlquist
- Institute for Molecular Virology, University of Wisconsin-Madison, Madison, United States.,Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, United States.,Morgridge Institute for Research, University of Wisconsin-Madison, Madison, United States
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10
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Infectious Bursal Disease Virus Subverts Autophagic Vacuoles To Promote Viral Maturation and Release. J Virol 2017; 91:JVI.01883-16. [PMID: 27974565 DOI: 10.1128/jvi.01883-16] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2016] [Accepted: 12/07/2016] [Indexed: 01/30/2023] Open
Abstract
Autophagy functions as an intrinsic antiviral defense. However, some viruses can subvert or even enhance host autophagic machinery to increase viral replication and pathogenesis. The role of autophagy during avibirnavirus infection, especially late stage infection, remains unclear. In this study, infectious bursal disease virus (IBDV) was used to investigate the role of autophagy in avibirnavirus replication. We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes, endogenous levels of LC3-II, and ultrastructural characteristics typical of autophagosomes during the late stage of infection. Induction of autophagy enhances IBDV replication, whereas inhibition of autophagy impairs viral replication. We also demonstrated that IBDV infection induced autophagosome-lysosome fusion, but without active degradation of their contents. Moreover, inhibition of fusion or of lysosomal hydrolysis activity significantly reduced viral replication, indicating that virions utilized the low-pH environment of acidic organelles to facilitate viral maturation. Using immuno-transmission electron microscopy (TEM), we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins, some of which lay between virions. Additionally, many virions were encapsulated within the vesicular membranes, with an obvious release stage observed by TEM. The autophagic endosomal pathway facilitates low-pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.IMPORTANCE IBDV is the most extensively studied virus in terms of molecular characteristics and pathogenesis; however, mechanisms underlying the IBDV life cycle require further exploration. The present study demonstrated that autophagy enhances viral replication at the late stage of infection, and the autophagy pathway facilitates IBDV replication complex function and virus assembly, which is critical to completion of the virus life cycle. Moreover, the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner. This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation, release, and reinternalization. This report presents a concordance in exit strategies among some RNA and DNA viruses, which exploit autophagy pathway for their release from cells.
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11
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Ávila-Pérez G, Rejas MT, Rodríguez D. Ultrastructural characterization of membranous torovirus replication factories. Cell Microbiol 2016; 18:1691-1708. [PMID: 27218226 PMCID: PMC7162420 DOI: 10.1111/cmi.12620] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2016] [Revised: 04/27/2016] [Accepted: 05/19/2016] [Indexed: 12/24/2022]
Abstract
Plus‐stranded RNA viruses replicate in the cytosol of infected cells, in membrane‐bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double‐membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV‐infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double‐stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.
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Affiliation(s)
- Ginés Ávila-Pérez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, 28049, Madrid, Spain
| | - María Teresa Rejas
- Electron Microscopy Facility, Centro de Biología Molecular Severo Ochoa, CSIC, C/Nicolás Cabrera 1, 28049, Madrid, Spain
| | - Dolores Rodríguez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, 28049, Madrid, Spain
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12
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Oh SW, Onomoto K, Wakimoto M, Onoguchi K, Ishidate F, Fujiwara T, Yoneyama M, Kato H, Fujita T. Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules. PLoS Pathog 2016; 12:e1005444. [PMID: 26862753 PMCID: PMC4749238 DOI: 10.1371/journal.ppat.1005444] [Citation(s) in RCA: 59] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2015] [Accepted: 01/19/2016] [Indexed: 12/22/2022] Open
Abstract
RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.
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Affiliation(s)
- Seong-Wook Oh
- Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan
| | - Koji Onomoto
- Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan
| | - Mai Wakimoto
- Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan
- Laboratory of Molecular and Cellular Immunology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Kazuhide Onoguchi
- Department of Microbiology and Immunology, University of California, San Francisco, California, United States of America
| | - Fumiyoshi Ishidate
- Carl Zeiss MicroImaging Co., Ltd., Tokyo, Japan
- Center for Meso-Bio Single-Molecule Imaging (CeMI), Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
| | - Takahiro Fujiwara
- Center for Meso-Bio Single-Molecule Imaging (CeMI), Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
| | - Mitsutoshi Yoneyama
- Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan
| | - Hiroki Kato
- Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan
- Laboratory of Molecular and Cellular Immunology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Takashi Fujita
- Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan
- Laboratory of Molecular and Cellular Immunology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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13
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Role of Mitochondrial Membrane Spherules in Flock House Virus Replication. J Virol 2016; 90:3676-83. [PMID: 26792749 DOI: 10.1128/jvi.03080-15] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2015] [Accepted: 01/14/2016] [Indexed: 01/20/2023] Open
Abstract
UNLABELLED Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCE It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.
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14
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Perelygina L, Adebayo A, Metcalfe M, Icenogle J. Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells. PLoS One 2015; 10:e0133267. [PMID: 26177032 PMCID: PMC4503567 DOI: 10.1371/journal.pone.0133267] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2015] [Accepted: 06/24/2015] [Indexed: 12/05/2022] Open
Abstract
Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1–4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV.
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Affiliation(s)
- Ludmila Perelygina
- Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Adebola Adebayo
- Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Maureen Metcalfe
- Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Joseph Icenogle
- Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
- * E-mail:
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15
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Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses. J Virol 2015; 89:9383-92. [PMID: 26136565 DOI: 10.1128/jvi.01299-15] [Citation(s) in RCA: 119] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Accepted: 06/23/2015] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.
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16
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Vachon VK, Conn GL. Adenovirus VA RNA: An essential pro-viral non-coding RNA. Virus Res 2015; 212:39-52. [PMID: 26116898 DOI: 10.1016/j.virusres.2015.06.018] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Revised: 06/15/2015] [Accepted: 06/17/2015] [Indexed: 12/25/2022]
Abstract
Adenovirus (AdV) 'virus-associated' RNAs (VA RNAs) are exceptionally abundant (up to 10(8)copies/cell), heterogeneous, non-coding RNA transcripts (∼ 150-200 nucleotides). The predominant species, VA RNAI, is best recognized for its essential function in relieving the cellular anti-viral blockade of protein synthesis through inhibition of the double-stranded RNA-activated protein kinase (PKR). More recent evidence has revealed that VA RNAs also interfere with several other host cell processes, in part by virtue of the high level to which they accumulate. Following transcription by cellular RNA polymerase III, VA RNAs saturate the nuclear export protein Exportin 5 (Exp5) and the cellular endoribonculease Dicer, interfering with pre-micro (mi)RNA export and miRNA biogenesis, respectively. Dicer-processed VA RNA fragments are incorporated into the RNA-induced silencing complex (RISC) as 'mivaRNAs', where they may specifically target cellular genes. VA RNAI also interacts with other innate immune proteins, including OAS1. While intact VA RNAI has the paradoxical effect of activating OAS1, a non-natural VA RNAI construct lacking the entire Terminal Stem has been reported to be a pseudoinhibitor of OAS1. Here, we show that a VA RNAI construct corresponding to an authentic product of Dicer processing similarly fails to activate OAS1 but also retains only a modest level of inhibitory activity against PKR in contrast to the non-natural deletion construct. These findings underscore the complexity of the arms race between virus and host, and highlight the need for further exploration of the impact of VA RNAI interactions with host defenses on the outcome of AdV infection beyond that of well-established PKR inhibition. Additional contributions of VA RNAI heterogeneity resulting from variations in transcription initiation and termination to each of these functions remain open questions that are discussed here.
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Affiliation(s)
- Virginia K Vachon
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA; Microbiology and Molecular Genetics (MMG) Program, Graduate Division of Biological and Biomedical Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Graeme L Conn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
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17
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Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology 2015; 479-480:418-33. [PMID: 25746936 PMCID: PMC7111692 DOI: 10.1016/j.virol.2015.02.029] [Citation(s) in RCA: 114] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Revised: 01/06/2015] [Accepted: 02/16/2015] [Indexed: 12/13/2022]
Abstract
Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication.
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Affiliation(s)
- Christian Harak
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany.
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18
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Fernández-de-Castro I, Risco C. Imaging RNA virus replication assemblies: bunyaviruses and reoviruses. Future Virol 2014. [DOI: 10.2217/fvl.14.95] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
ABSTRACT RNA viruses replicate in the cytoplasm in close association with host cell membranes. Both viral and cellular factors generate organelle-like structures termed viral factories, viral inclusions or viroplasms. Biochemical, light and electron microscopy analyses, including 3D models, have improved our understanding of the architecture and function of RNA virus replication factories. In these structures, the virus compartmentalizes genome replication and transcription, thus enhancing replication efficiency and protection from host defenses. Recent studies with diverse RNA viruses have elucidated the ultrastructure of replication organelles and shown how these structures act in close coordination with virion assembly. This review focuses on a general description of RNA virus factories and summarizes recent progress in the characterization of those assembled by bunyaviruses and reoviruses. We describe how these viruses modify intracellular membranes; we highlight similarities with the structures induced by viruses of other families, and discuss how these structures might be formed.
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Affiliation(s)
| | - Cristina Risco
- Cell Structure Laboratory, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
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19
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Barroso-González J, García-Expósito L, Puigdomènech I, de Armas-Rillo L, Machado JD, Blanco J, Valenzuela-Fernández A. Viral infection. Commun Integr Biol 2014. [DOI: 10.4161/cib.16716] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
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20
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Membranous replication factories induced by plus-strand RNA viruses. Viruses 2014; 6:2826-57. [PMID: 25054883 PMCID: PMC4113795 DOI: 10.3390/v6072826] [Citation(s) in RCA: 209] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2014] [Revised: 06/02/2014] [Accepted: 06/24/2014] [Indexed: 12/13/2022] Open
Abstract
In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.
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21
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Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells. J Virol 2014; 88:4687-97. [PMID: 24522909 DOI: 10.1128/jvi.00118-14] [Citation(s) in RCA: 152] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
UNLABELLED During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. IMPORTANCE Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.
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22
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Hsu WL, Chen CL, Huang SW, Wu CC, Chen IH, Nadar M, Su YP, Tsai CH. The untranslated regions of classic swine fever virus RNA trigger apoptosis. PLoS One 2014; 9:e88863. [PMID: 24533157 PMCID: PMC3923050 DOI: 10.1371/journal.pone.0088863] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2013] [Accepted: 01/13/2014] [Indexed: 11/23/2022] Open
Abstract
Classical swine fever virus (CSFV) causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3' UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5' and 3' UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5'UTR-Luc-3'UTR) triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5' or 3' UTR RNA; even when the 5' UTR and 3' UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5'UTR-Luc-3'UTR RNA. Interestingly, RNA composed of the 5'UTR and of stem-loop I of the 3'UTR triggered much stronger apoptosis than the 5' or 3'UTR alone. These results indicate that the 5' and 3' UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.
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Affiliation(s)
- Wei-Li Hsu
- Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan
| | - Chung-Lun Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Shi-Wei Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Chia-Chen Wu
- Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Muthukumar Nadar
- Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya University, Coimbatore, Tamil Nadu, India
| | - Yin-Peng Su
- Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
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23
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[The life cycle of Rubella Virus]. Uirusu 2014; 64:137-46. [PMID: 26437836 DOI: 10.2222/jsv.64.137] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Rubella virus (RV), an infectious agent of rubella, is the sole member of the genus Rubivirus in the family of Togaviridae. RV has a positive-stranded sense RNA as a genome. A natural host of RV is limited to human, and rubella is considered to be a childhood disease in general. When woman is infected with RV during early pregnancy, her fetus may develop severe birth defects known as congenital rubella syndrome. In this review, the RV life cycle from the virus entry to budding is illustrated in comparison with those of member viruses of the genus alphavirus in the same family. The multiple functions of the RV capsid protein are also introduced.
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24
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Delgui LR, Rodríguez JF, Colombo MI. The endosomal pathway and the Golgi complex are involved in the infectious bursal disease virus life cycle. J Virol 2013; 87:8993-9007. [PMID: 23741000 PMCID: PMC3754037 DOI: 10.1128/jvi.03152-12] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2012] [Accepted: 05/30/2013] [Indexed: 12/22/2022] Open
Abstract
Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.
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Affiliation(s)
- Laura R. Delgui
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología de Mendoza, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina
- Instituto de Ciencias Básicas, Universidad Nacional de Cuyo, Mendoza, Argentina
| | - José F. Rodríguez
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología (CSIC), Madrid, Spain
| | - María I. Colombo
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología de Mendoza, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina
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25
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Paul D, Bartenschlager R. Architecture and biogenesis of plus-strand RNA virus replication factories. World J Virol 2013; 2:32-48. [PMID: 24175228 PMCID: PMC3785047 DOI: 10.5501/wjv.v2.i2.32] [Citation(s) in RCA: 217] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/18/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virus-induced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.
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26
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Risco C, Sanmartín-Conesa E, Tzeng WP, Frey TK, Seybold V, de Groot RJ. Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy. Structure 2012; 20:759-66. [PMID: 22579245 DOI: 10.1016/j.str.2012.04.001] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2012] [Revised: 03/14/2012] [Accepted: 04/05/2012] [Indexed: 02/09/2023]
Abstract
More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.
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Affiliation(s)
- Cristina Risco
- Cell Structure Lab, Centro Nacional de Biotecnología/CSIC, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.
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27
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Moser TS, Schieffer D, Cherry S. AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog 2012; 8:e1002661. [PMID: 22532801 PMCID: PMC3330235 DOI: 10.1371/journal.ppat.1002661] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2011] [Accepted: 03/07/2012] [Indexed: 11/18/2022] Open
Abstract
The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism. RNA viruses represent an important worldwide source of infection and disease in both humans and animals. While individual viruses have unique characteristics, some stages of infection are conserved and must be completed in order to successfully infect and grow. Viruses must undergo genome replication, protein synthesis, and assembly of new virus particles. In particular, numerous RNA viruses manipulate cellular membranes to create new complex structures required for viral replication in a process that is often dependent on fatty acid biosynthesis. This is a process that is tightly regulated by the energy sensor AMPK. We have found that energy-mediated activation of AMPK restricts infection of the Bunyavirus Rift Valley fever virus by decreasing levels of fatty acid synthesis. Furthermore, several additional RNA viruses from disparate families that share this dependence of membrane modification and fatty acid synthesis are also restricted by AMPK. Thus AMPK likely represents a novel component of the cell intrinsic immune response to RNA viruses, and may be a good target for the development of antiviral therapeutics against a range of medically important viruses.
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Affiliation(s)
| | | | - Sara Cherry
- Department of Microbiology, Penn Genome Frontiers Institute, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America
- * E-mail:
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28
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Determinants in the maturation of rubella virus p200 replicase polyprotein precursor. J Virol 2012; 86:6457-69. [PMID: 22491463 DOI: 10.1128/jvi.06132-11] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5' and 3' cis-acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.
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Liao JY, Thakur SA, Zalinger ZB, Gerrish KE, Imani F. Inosine-containing RNA is a novel innate immune recognition element and reduces RSV infection. PLoS One 2011; 6:e26463. [PMID: 22028885 PMCID: PMC3196583 DOI: 10.1371/journal.pone.0026463] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2011] [Accepted: 09/27/2011] [Indexed: 12/24/2022] Open
Abstract
During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p<0.01). Flow cytometry data revealed a corresponding 4-fold increase in influx of neutrophils into the lungs by ss-Ino-RNA treatment. In our in vitro experiments, treatment of epithelial cells with ss-Ino-RNA reduced replication of respiratory syncytial virus (RSV). Interestingly, RNA structural analysis showed that ss-Ino-RNA had increased formation of secondary structures. Our data further revealed that extracellular ss-Ino-RNA was taken up by scavenger receptor class-A (SR-A) which activated downstream MAP Kinase pathways through Toll-like receptor 3 (TLR3) and dsRNA-activated protein kinase (PKR). Our data suggests that ss-Ino-RNA is an as yet undescribed virus-associated innate immune stimulus.
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Affiliation(s)
- Jie-ying Liao
- Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States of America
| | - Sheetal A. Thakur
- Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States of America
| | - Zachary B. Zalinger
- Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States of America
| | - Kevin E. Gerrish
- Gene Array Core Facility, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States of America
| | - Farhad Imani
- ViraSource Laboratories, Durham, North Carolina, United States of America
- * E-mail:
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Matthews JD, Frey TK. Analysis of subcellular G3BP redistribution during rubella virus infection. J Gen Virol 2011; 93:267-274. [PMID: 21994324 DOI: 10.1099/vir.0.036780-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Rubella virus (RUBV) replicates slowly and to low titre in vertebrate cultured cells, with minimal cytopathology. To determine whether a cellular stress response is induced during such an infection, the formation of Ras-GAP-SH3 domain-binding protein (G3BP)-containing stress granules (SGs) in RUBV-infected cells was examined. Late in infection, accumulation of G3BP granules was detected, albeit in fewer than half of infected cells. Active virus RNA replication was required for induction of these granules, but they were found to differ from SGs induced by arsenite treatment both in composition (they did not uniformly contain other SG proteins, such as PABP and TIA-1) and in resistance to cycloheximide treatment. Thus, bona fide SGs do not appear to be induced during RUBV infection. The distribution of G3BP, either on its own or in granules, did not overlap with that of dsRNA-containing replication complexes, indicating that it played no role in virus RNA synthesis. However, G3BP did co-localize with viral ssRNAs in perinuclear clusters, suggesting an interaction that could possibly be important in a post-replicative role in virus replication, such as encapsidation.
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Affiliation(s)
- Jason D Matthews
- Georgia State University, Department of Biology, Atlanta, GA 30303, USA
| | - Teryl K Frey
- Georgia State University, Department of Biology, Atlanta, GA 30303, USA
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Barroso-González J, García-Expósito L, Puigdomènech I, de Armas-Rillo L, Machado JD, Blanco J, Valenzuela-Fernández A. Viral infection: Moving through complex and dynamic cell-membrane structures. Commun Integr Biol 2011; 4:398-408. [PMID: 21966556 DOI: 10.4161/cib.4.4.16716] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2011] [Accepted: 05/31/2011] [Indexed: 01/19/2023] Open
Abstract
Viruses have developed different survival strategies in host cells by crossing cell-membrane compartments, during different steps of their viral life cycle. In fact, the non-regenerative viral membrane of enveloped viruses needs to encounter the dynamic cell-host membrane, during early steps of the infection process, in which both membranes fuse, either at cell-surface or in an endocytic compartment, to promote viral entry and infection. Once inside the cell, many viruses accomplish their replication process through exploiting or modulating membrane traffic, and generating specialized compartments to assure viral replication, viral budding and spreading, which also serve to evade the immune responses against the pathogen. In this review, we have attempted to present some data that highlight the importance of membrane dynamics during viral entry and replicative processes, in order to understand how viruses use and move through different complex and dynamic cell-membrane structures and how they use them to persist.
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Affiliation(s)
- Jonathan Barroso-González
- Laboratorio de Inmunología Celular y Viral; Laboratorio de Neurosecreción; Unidad de Farmacología; Departamento de Medicina Física y Farmacología; Facultad de Medicina; Instituto de Tecnologías Biomédicas (ITB); Universidad de La Laguna (ULL)
| | - Laura García-Expósito
- Laboratorio de Inmunología Celular y Viral; Laboratorio de Neurosecreción; Unidad de Farmacología; Departamento de Medicina Física y Farmacología; Facultad de Medicina; Instituto de Tecnologías Biomédicas (ITB); Universidad de La Laguna (ULL)
| | - Isabel Puigdomènech
- Fundació irsiCaixa-HIVACAT; Institut de Recerca en Ciències de la Salut Germans Trias i Pujol (IGTP); Hospital Germans Trias i Pujol; Universitat Autònoma de Barcelona; Barcelona, Catalonia Spain
| | - Laura de Armas-Rillo
- Laboratorio de Inmunología Celular y Viral; Laboratorio de Neurosecreción; Unidad de Farmacología; Departamento de Medicina Física y Farmacología; Facultad de Medicina; Instituto de Tecnologías Biomédicas (ITB); Universidad de La Laguna (ULL)
| | - José-David Machado
- Laboratorio de Inmunología Celular y Viral; Laboratorio de Neurosecreción; Unidad de Farmacología; Departamento de Medicina Física y Farmacología; Facultad de Medicina; Instituto de Tecnologías Biomédicas (ITB); Universidad de La Laguna (ULL)
| | - Julià Blanco
- Fundació irsiCaixa-HIVACAT; Institut de Recerca en Ciències de la Salut Germans Trias i Pujol (IGTP); Hospital Germans Trias i Pujol; Universitat Autònoma de Barcelona; Barcelona, Catalonia Spain
| | - Agustín Valenzuela-Fernández
- Laboratorio de Inmunología Celular y Viral; Laboratorio de Neurosecreción; Unidad de Farmacología; Departamento de Medicina Física y Farmacología; Facultad de Medicina; Instituto de Tecnologías Biomédicas (ITB); Universidad de La Laguna (ULL)
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Bamunusinghe D, Seo JK, Rao ALN. Subcellular localization and rearrangement of endoplasmic reticulum by Brome mosaic virus capsid protein. J Virol 2011; 85:2953-63. [PMID: 21209103 PMCID: PMC3067956 DOI: 10.1128/jvi.02020-10] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2010] [Accepted: 12/29/2010] [Indexed: 01/04/2023] Open
Abstract
Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, as well as in other positive-strand RNA viruses pathogenic to humans (e.g., poliovirus) and animals (e.g., Flock House virus), is functionally coupled to replication. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. In this study, the application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a green fluorescent protein (GFP) fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV-infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP itself has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. The significance of CP-induced vesicles in relation to CP-organized viral functions that are linked to replication-coupled packaging is discussed.
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Affiliation(s)
- Devinka Bamunusinghe
- Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521-0122
| | - Jang-Kyun Seo
- Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521-0122
| | - A. L. N. Rao
- Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521-0122
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Boonyaratanakornkit J, Bartlett E, Schomacker H, Surman S, Akira S, Bae YS, Collins P, Murphy B, Schmidt A. The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R. J Virol 2011; 85:1495-506. [PMID: 21123378 PMCID: PMC3028907 DOI: 10.1128/jvi.01297-10] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2010] [Accepted: 11/23/2010] [Indexed: 12/25/2022] Open
Abstract
Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C-)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.
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Affiliation(s)
- Jim Boonyaratanakornkit
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Emmalene Bartlett
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Henrick Schomacker
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Sonja Surman
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Shizuo Akira
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Yong-Soo Bae
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Peter Collins
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Brian Murphy
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
| | - Alexander Schmidt
- Laboratory of Infectious Diseases, RNA Viruses Section, NIAID, NIH, Bethesda, Maryland 20892, Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan, Department of Biological Sciences, Sungkyunkwan University, Choenchoen-Dong, Jangan-Gu, Suwon, Gyeonggi-Do 440-746, South Korea
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Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus. J Virol 2011; 85:3881-92. [PMID: 21248045 DOI: 10.1128/jvi.02492-10] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.
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Jurgeit A, Moese S, Roulin P, Dorsch A, Lötzerich M, Lee WM, Greber UF. An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections. Virol J 2010; 7:264. [PMID: 20937137 PMCID: PMC2958916 DOI: 10.1186/1743-422x-7-264] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2010] [Accepted: 10/11/2010] [Indexed: 01/17/2023] Open
Abstract
Background Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA. Results Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections. Conclusions We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes without the risk of assay specific artifacts.
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Affiliation(s)
- Andreas Jurgeit
- Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
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The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex. J Virol 2010; 84:10438-47. [PMID: 20686019 DOI: 10.1128/jvi.00986-10] [Citation(s) in RCA: 290] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV(KUN)) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV(KUN) replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV(KUN) replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.
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Fontana J, López-Iglesias C, Tzeng WP, Frey TK, Fernández JJ, Risco C. Three-dimensional structure of Rubella virus factories. Virology 2010; 405:579-91. [PMID: 20655079 PMCID: PMC7111912 DOI: 10.1016/j.virol.2010.06.043] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2010] [Revised: 06/07/2010] [Accepted: 06/24/2010] [Indexed: 12/14/2022]
Abstract
Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.
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Affiliation(s)
- Juan Fontana
- Cell Structure Lab, Centro Nacional de Biotecnología, CSIC, Darwin, Madrid, Spain
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Abstract
Innate immunity is the first line of defense against viral infections. It is based on a mechanism of sensing pathogen-associated molecular patterns through host germline-encoded pattern recognition receptors. dsRNA is arguably the most important viral pathogen-associated molecular pattern due to its expression by almost all viruses at some point during their replicative cycle. Viral dsRNA has been studied for over 55 years, first as a toxin, then as a type I interferon inducer, a viral mimetic and an immunomodulator for therapeutic purposes. This article will focus on dsRNA, its structure, generation (both endogenous and viral), host sensing mechanisms and induction of type I interferons. The possible therapeutic applications of these findings will also be discussed. The goal of this article is to give an overview of these mechanisms, highlighting novel findings, while providing a historical perspective.
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Affiliation(s)
- Stephanie J DeWitte-Orr
- McMaster University, Department of Pathology & Molecular Medicine, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada
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Fei Z, Liu Y, Yan Z, Fan D, Alexander A, Yang JH. Targeting viral dsRNA for antiviral prophylaxis. FASEB J 2009; 25:1767-74. [PMID: 19880628 DOI: 10.1096/fj.09-144915] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Double-stranded (ds)RNA in the infected cells is a trait shared by most if not all viruses. While humans have developed variable immune responses, viruses have also developed countermeasures to defeat dsRNA-induced antiviral strategies. Thus, we proposed a broad antiviral strategy to antagonize the countermeasures of viruses and bypass the dsRNA-induced signals that are readily defeated by viruses. By rewiring the dsRNA-binding proteins in the dsRNA complex and reconnecting them to apoptosis signaling, we created several dsRNA-dependent caspase recruiters, termed dsCAREs, to bypass dsRNA-induced antiviral signals that would otherwise be targeted by viruses. Adenovirus and vesicular stomatitis virus, representing viruses of the dsDNA and negative-stranded RNA viral groups, were used to infect HEK293 cells. The dsCARE chimera was added in medium to evaluate its antiviral activity. The truncated dsCAREs were used as controls. We demonstrate that dsCARE suppresses viral infection starting at 0.1 μg/ml and reaches the peak at 2 μg/ml. The EC(50) was ∼0.2 μg/ml. However, it had an undetectable effect on uninfected cells. Further data show that both dsRNA binding and apoptosis activation of dsCARE are essential for its antiviral activity. We conclude that dsRNA is a practical virus-associated molecular pattern that can be targeted for broad and rapid antiviral prophylaxis.
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Affiliation(s)
- Zhou Fei
- Department of Surgery, Boston Veterans Affairs Healthcare System, Boston University School of Medicine, Boston, Massachusetts 02130, USA
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40
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Dauber B, Wolff T. Activation of the Antiviral Kinase PKR and Viral Countermeasures. Viruses 2009; 1:523-44. [PMID: 21994559 PMCID: PMC3185532 DOI: 10.3390/v1030523] [Citation(s) in RCA: 89] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2009] [Revised: 10/26/2009] [Accepted: 10/26/2009] [Indexed: 12/25/2022] Open
Abstract
The interferon-induced double-stranded (ds)RNA-dependent protein kinase (PKR) limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5′-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR.
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Affiliation(s)
- Bianca Dauber
- Department of Medical Microbiology & Immunology, University of Alberta, 632 Heritage Medical Research Center, Edmonton, AB, T6G 2S2, Canada
- Authors to whom correspondence should be addressed; E-Mails: (B.D.); (T.W.)
| | - Thorsten Wolff
- P15, Robert Koch-Institute/Nordufer 20, 13353 Berlin, Germany
- Authors to whom correspondence should be addressed; E-Mails: (B.D.); (T.W.)
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Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway. J Virol 2009; 83:9709-19. [PMID: 19587041 DOI: 10.1128/jvi.00600-09] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.
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Matthews JD, Tzeng WP, Frey TK. Determinants of subcellular localization of the rubella virus nonstructural replicase proteins. Virology 2009; 390:315-23. [PMID: 19539969 DOI: 10.1016/j.virol.2009.05.019] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2009] [Revised: 04/18/2009] [Accepted: 05/12/2009] [Indexed: 10/20/2022]
Abstract
The rubella virus (RUBV) nonstructural replicase proteins (NSPs), P150 and P90, are proteolytically processed from a P200 precursor. To understand the NSPs' function in the establishment of virus RNA replication complexes (RCs), the NSPs were analyzed in virus-infected cells or cells transfected with NSP-expressing plasmids. In infected cells, P150 was localized in cytoplasmic foci at 24 hpi and in cytoplasmic fibers, unique to RUBV, by 48 hpi. RCs, marked by dsRNA, colocalized with P150-foci, but only occasionally with the endosome/lysosome marker LAMP-2, indicating that RNA synthesis occurs at other sites rather than exclusively in endosomes/lysosomes as was previously thought. An expressed cleavage-deficient form of P200 also localized to cytoplasmic foci, suggesting that the precursor is required for targeting to sites of RC establishment. P150 was found to be the determinant of fiber formation and the NSP membrane-binding domain was mapped to the N-terminus of P150.
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Affiliation(s)
- Jason D Matthews
- Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302-4010, USA
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Abstract
Plus-stranded RNA viruses induce large membrane structures that might support the replication of their genomes. Similarly, cytoplasmic replication of poxviruses (large DNA viruses) occurs in associated membranes. These membranes originate from the endoplasmic reticulum (ER) or endosomes. Membrane vesicles that support viral replication are induced by a number of RNA viruses. Similarly, the poxvirus replication site is surrounded by a double-membraned cisterna that is derived from the ER. Analogies to autophagy have been proposed since the finding that autophagy cellular processes involve the formation of double-membrane vesicles. However, molecular evidence to support this hypothesis is lacking. Membrane association of the viral replication complex is mediated by the presence of one or more viral proteins that contain sequences which associate with, or integrate into, membranes. Replication-competent membranes might contain viral or cellular proteins that contain amphipathic helices, which could mediate the membrane bending that is required to form spherical vesicles. Whereas poxvirus DNA replication occurs inside the ER-enclosed site, for most RNA viruses the topology of replication is not clear. Preliminary results for some RNA viruses suggest that their replication could also occur inside double-membrane vesicles. We speculate that cytoplasmic replication might occur inside sites that are 'enwrapped' by an ER-derived cisterna, and that these cisternae are open to the cytoplasm. Thus, RNA and DNA viruses could use a common mechanism for replication that involves membrane wrapping by cellular cisternal membranes. We propose that three-dimensional analyses using high-resolution electron-microscopy techniques could be useful for addressing this issue. High-throughput small-interfering-RNA screens should also shed light on molecular requirements for virus-induced membrane modifications. Many viruses induce the formation of altered membrane structures upon replication in host cells. This Review examines how viruses modify intracellular membranes, highlights similarities between the structures that are induced by viruses from different families and discusses how these structures could be formed. Viruses are intracellular parasites that use the host cell they infect to produce new infectious progeny. Distinct steps of the virus life cycle occur in association with the cytoskeleton or cytoplasmic membranes, which are often modified during infection. Plus-stranded RNA viruses induce membrane proliferations that support the replication of their genomes. Similarly, cytoplasmic replication of some DNA viruses occurs in association with modified cellular membranes. We describe how viruses modify intracellular membranes, highlight similarities between the structures that are induced by viruses of different families and discuss how these structures could be formed.
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Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol 2007; 5:e220. [PMID: 17696647 PMCID: PMC1945040 DOI: 10.1371/journal.pbio.0050220] [Citation(s) in RCA: 229] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2007] [Accepted: 06/15/2007] [Indexed: 12/11/2022] Open
Abstract
Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions. Whereas cells store and replicate their genomes as DNA, most viruses have RNA genomes that replicate by using virus-specific pathways in the host cell. The largest class of RNA viruses, the positive-strand RNA viruses, replicate their genomes on intracellular membranes. However, little is understood about how and why these viruses use membranes in RNA replication. The well-studied flock house virus (FHV) replicates its RNA on mitochondrial membranes. We found that the single FHV RNA replication factor and newly synthesized FHV RNA localized predominantly in numerous infection-specific membrane vesicles inside the outer mitochondrial membrane. We used electron microscope tomography to image these membranes in three dimensions and found that the interior of each vesicle was connected to the cytoplasm by a single necked channel large enough to import ribonucleotide substrates and to export product RNA. The results suggest that FHV uses these vesicles as replication compartments, which may also protect replicating RNA from competing processes and host defenses. These findings complement results from other viruses to support possible parallels between genome replication by positive-strand RNA viruses and two distinct virus classes, double-stranded RNA and reverse-transcribing viruses. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements; here, a three-dimensional analysis of these complexes in flock house nodavirus-infected Drosophila cells provides insight into the replication process.
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Affiliation(s)
- Benjamin G Kopek
- Institute for Molecular Virology, University of Wisconsin–Madison, Madison, Wisconsin, United States of America
| | - Guy Perkins
- National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California, United States of America
- Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America
| | - David J Miller
- Department of Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
- Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
| | - Mark H Ellisman
- National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California, United States of America
- Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America
| | - Paul Ahlquist
- Institute for Molecular Virology, University of Wisconsin–Madison, Madison, Wisconsin, United States of America
- Howard Hughes Medical Institute, University of Wisconsin–Madison, Madison, Wisconsin, United States of America
- * To whom correspondence should be addressed. E-mail:
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Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol 2007; 82:2182-95. [PMID: 18094154 DOI: 10.1128/jvi.01565-07] [Citation(s) in RCA: 143] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.
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Netherton C, Moffat K, Brooks E, Wileman T. A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv Virus Res 2007; 70:101-82. [PMID: 17765705 PMCID: PMC7112299 DOI: 10.1016/s0065-3527(07)70004-0] [Citation(s) in RCA: 164] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.
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Affiliation(s)
- Christopher Netherton
- Vaccinology Group, Pirbright Laboratories, Institute for Animal Health, Surrey, United Kingdom
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Limmon GV, Arredouani M, McCann KL, Corn Minor RA, Kobzik L, Imani F. Scavenger receptor class-A is a novel cell surface receptor for double-stranded RNA. FASEB J 2007; 22:159-67. [PMID: 17709607 DOI: 10.1096/fj.07-8348com] [Citation(s) in RCA: 110] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Double-stranded RNA (dsRNA) is a potent signal to the host immune system for the presence of an ongoing viral infection. The presence of dsRNA, intracellularly or extracellularly, leads to the induction of innate inflammatory cytokines in many cell types including epithelial cells. However, the cell surface receptor for recognition of extracellular dsRNA is not yet determined. Here, we report that extracellular dsRNA is recognized and internalized by scavenger receptor class-A (SR-A). Treatment of human epithelial cells with specific antagonists of SR-A or with an anti-SR-A antibody significantly inhibited dsRNA induction of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, and regulated on activation normal T-cell expressed and secreted (RANTES). Furthermore, intranasal dsRNA treatment of SR-A-deficient (SR-A(-/-)) mice showed a significant decrease in the expression of inflammatory cytokines and a corresponding decrease in the accumulation of polymorphonuclear leukocytes (PMNs) in lungs. These data provide direct evidence that SR-A is a novel cell surface receptor for dsRNA, and therefore, SR-A may play a role in antiviral immune responses.
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Affiliation(s)
- Gino V Limmon
- NIEHS/NIH, Laboratory of Respiratory Biology, Durham, North Carolina 27709, USA
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Chiang ET, Persaud-Sawin DA, Kulkarni S, Garcia JGN, Imani F. Bluetongue virus and double-stranded RNA increase human vascular permeability: role of p38 MAPK. J Clin Immunol 2006; 26:406-16. [PMID: 16786433 DOI: 10.1007/s10875-006-9024-4] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in trans-endothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases.
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Affiliation(s)
- Eddie T Chiang
- University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637, USA
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Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 2006; 80:5059-64. [PMID: 16641297 PMCID: PMC1472073 DOI: 10.1128/jvi.80.10.5059-5064.2006] [Citation(s) in RCA: 749] [Impact Index Per Article: 39.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.
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Affiliation(s)
- Friedemann Weber
- Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany.
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Ahlquist P. Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol 2006; 4:371-82. [PMID: 16582931 PMCID: PMC7097367 DOI: 10.1038/nrmicro1389] [Citation(s) in RCA: 215] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Viruses are exceptionally diverse and are grouped by genome replication and encapsidation strategies into seven distinct classes: two classes of DNA viruses (encapsidating single-stranded (ss)DNA or double-stranded (ds)DNA), three classes of RNA viruses (encapsidating mRNA-sense ssRNA, antisense ssRNA or dsRNA) and two classes of reverse-transcribing viruses (encapsidating RNA or DNA). Despite substantial life-cycle differences, positive-strand RNA ((+)RNA) viruses, dsRNA viruses and reverse-transcribing viruses share multiple similarities in genome replication. All replicate their genomes through RNA intermediates that also serve as mRNAs. Moreover, the intracellular RNA-replication complexes of (+)RNA viruses share similarities in structure, assembly and function with the polymerase-containing virion cores of dsRNA and reverse transcribing viruses. Brome mosaic virus (BMV) RNA-replication factors 1a and 2apol and cis-acting template-recruitment signals parallel retrovirus Gag, Pol and RNA-packaging signals in virion assembly: 1a localizes to specific membranes, self-interacts and induces ∼60-nm membrane invaginations to which it recruits 2apol and viral RNAs for replication. Therefore, like retroviruses and dsRNA viruses, BMV sequesters its genomic RNA and polymerase in a virus-induced compartment for replication. BMV and some other alphavirus-like (+)RNA viruses also parallel retroviruses in using tRNA-related sequences to initiate genome replication, and share with dsRNA reoviruses aspects of the function and interaction of their RNA polymerase and RNA-capping enzymes. Emerging results indicate that the genome-replication machineries of these viruses might share other mechanistic features. Whereas (+)RNA alphavirus-like viruses, dsRNA reoviruses and retroviruses are linked by the above similarities, (+)RNA picornaviruses, dsRNA birnaviruses and reverse-transcribing hepadnaviruses share some distinct features, including protein-primed nucleic-acid synthesis. Such parallels suggest that at least some (+)RNA viruses, dsRNA viruses and reverse-transcribing viruses might have evolved from common ancestors. The transitions required for such evolution can be readily envisioned and some have precedents. These underlying parallels in genome replication by four of the seven main virus classes might provide a basis for more generalizable or broader-spectrum approaches for virus control. Despite major differences in the life cycles of the seven different classes of known viruses, the genome-replication processes of certain positive-strand RNA viruses, double-stranded RNA viruses and reverse-transcribing viruses show striking parallels. Paul Ahlquist highlights these similarities and discusses their intriguing evolutionary implications. Viruses are divided into seven classes on the basis of differing strategies for storing and replicating their genomes through RNA and/or DNA intermediates. Despite major differences among these classes, recent results reveal that the non-virion, intracellular RNA-replication complexes of some positive-strand RNA viruses share parallels with the structure, assembly and function of the replicative cores of extracellular virions of reverse-transcribing viruses and double-stranded RNA viruses. Therefore, at least four of seven principal virus classes share several underlying features in genome replication and might have emerged from common ancestors. This has implications for virus function, evolution and control.
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Affiliation(s)
- Paul Ahlquist
- Institute for Molecular Virology and Howard Hughes Medical Institute, University of Wisconsin--Madison, Madison, Wisconsin 53706, USA.
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