Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein
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. Method...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-02-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fpls.2018.00070/full |
| _version_ | 1818019811298377728 |
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| author | Baptiste Monsion Marco Incarbone Kamal Hleibieh Vianney Poignavent Ahmed Ghannam Patrice Dunoyer Laurent Daeffler Jens Tilsner Jens Tilsner Christophe Ritzenthaler |
| author_facet | Baptiste Monsion Marco Incarbone Kamal Hleibieh Vianney Poignavent Ahmed Ghannam Patrice Dunoyer Laurent Daeffler Jens Tilsner Jens Tilsner Christophe Ritzenthaler |
| author_sort | Baptiste Monsion |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T07:56:22Z |
| format | Article |
| id | doaj.art-23bb7743c5fd423ab0762cbe568deffe |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-14T07:56:22Z |
| publishDate | 2018-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-23bb7743c5fd423ab0762cbe568deffe2022-12-22T02:05:01ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2018-02-01910.3389/fpls.2018.00070318973Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 ProteinBaptiste Monsion0Marco Incarbone1Kamal Hleibieh2Vianney Poignavent3Ahmed Ghannam4Patrice Dunoyer5Laurent Daeffler6Jens Tilsner7Jens Tilsner8Christophe Ritzenthaler9Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, FranceBiomedical Sciences Research Complex, University of St Andrews, St Andrews, United KingdomCell and Molecular Sciences, The James Hutton Institute, Dundee, United KingdomCentre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg, FranceDouble-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.http://journal.frontiersin.org/article/10.3389/fpls.2018.00070/fulldsRNAvirusviral factoriesreplication complexesdetectionplant |
| spellingShingle | Baptiste Monsion Marco Incarbone Kamal Hleibieh Vianney Poignavent Ahmed Ghannam Patrice Dunoyer Laurent Daeffler Jens Tilsner Jens Tilsner Christophe Ritzenthaler Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein Frontiers in Plant Science dsRNA virus viral factories replication complexes detection plant |
| title | Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein |
| title_full | Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein |
| title_fullStr | Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein |
| title_full_unstemmed | Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein |
| title_short | Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein |
| title_sort | efficient detection of long dsrna in vitro and in vivo using the dsrna binding domain from fhv b2 protein |
| topic | dsRNA virus viral factories replication complexes detection plant |
| url | http://journal.frontiersin.org/article/10.3389/fpls.2018.00070/full |
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