SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

Abstract Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we u...

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Autores principales:	Roland Remenyi, Grace C. Roberts, Carsten Zothner, Andres Merits, Mark Harris
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cfce921af4d1466292623637d73e7a2c
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spelling	oai:doaj.org-article:cfce921af4d1466292623637d73e7a2c2021-12-02T11:52:36ZSNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy10.1038/s41598-017-05820-02045-2322https://doaj.org/article/cfce921af4d1466292623637d73e7a2c2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-05820-0https://doaj.org/toc/2045-2322Abstract Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP-tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus).Roland RemenyiGrace C. RobertsCarsten ZothnerAndres MeritsMark HarrisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-15 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Roland Remenyi Grace C. Roberts Carsten Zothner Andres Merits Mark Harris SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
description	Abstract Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP-tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus).
format	article
author	Roland Remenyi Grace C. Roberts Carsten Zothner Andres Merits Mark Harris
author_facet	Roland Remenyi Grace C. Roberts Carsten Zothner Andres Merits Mark Harris
author_sort	Roland Remenyi
title	SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
title_short	SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
title_full	SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
title_fullStr	SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
title_full_unstemmed	SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy
title_sort	snap-tagged chikungunya virus replicons improve visualisation of non-structural protein 3 by fluorescence microscopy
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/cfce921af4d1466292623637d73e7a2c
work_keys_str_mv	AT rolandremenyi snaptaggedchikungunyavirusrepliconsimprovevisualisationofnonstructuralprotein3byfluorescencemicroscopy AT gracecroberts snaptaggedchikungunyavirusrepliconsimprovevisualisationofnonstructuralprotein3byfluorescencemicroscopy AT carstenzothner snaptaggedchikungunyavirusrepliconsimprovevisualisationofnonstructuralprotein3byfluorescencemicroscopy AT andresmerits snaptaggedchikungunyavirusrepliconsimprovevisualisationofnonstructuralprotein3byfluorescencemicroscopy AT markharris snaptaggedchikungunyavirusrepliconsimprovevisualisationofnonstructuralprotein3byfluorescencemicroscopy
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SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy

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