Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recom...

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Autores principales:	H. M. van der Schaar, C. E. Melia, J. A. C. van Bruggen, J. R. P. M. Strating, M. E. D. van Geenen, A. J. Koster, M. Bárcena, F. J. M. van Kuppeveld
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	correlative light electron microscopy enterovirus live-cell imaging viral replication Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d1b2b96f81524978aa1d32d7516d2efd
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spelling	oai:doaj.org-article:d1b2b96f81524978aa1d32d7516d2efd2021-11-15T15:21:14ZIlluminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein10.1128/mSphere.00104-162379-5042https://doaj.org/article/d1b2b96f81524978aa1d32d7516d2efd2016-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00104-16https://doaj.org/toc/2379-5042ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.H. M. van der SchaarC. E. MeliaJ. A. C. van BruggenJ. R. P. M. StratingM. E. D. van GeenenA. J. KosterM. BárcenaF. J. M. van KuppeveldAmerican Society for Microbiologyarticlecorrelative light electron microscopyenteroviruslive-cell imagingviral replicationMicrobiologyQR1-502ENmSphere, Vol 1, Iss 4 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	correlative light electron microscopy enterovirus live-cell imaging viral replication Microbiology QR1-502
spellingShingle	correlative light electron microscopy enterovirus live-cell imaging viral replication Microbiology QR1-502 H. M. van der Schaar C. E. Melia J. A. C. van Bruggen J. R. P. M. Strating M. E. D. van Geenen A. J. Koster M. Bárcena F. J. M. van Kuppeveld Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
description	ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.
format	article
author	H. M. van der Schaar C. E. Melia J. A. C. van Bruggen J. R. P. M. Strating M. E. D. van Geenen A. J. Koster M. Bárcena F. J. M. van Kuppeveld
author_facet	H. M. van der Schaar C. E. Melia J. A. C. van Bruggen J. R. P. M. Strating M. E. D. van Geenen A. J. Koster M. Bárcena F. J. M. van Kuppeveld
author_sort	H. M. van der Schaar
title	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
title_short	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
title_full	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
title_fullStr	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
title_full_unstemmed	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
title_sort	illuminating the sites of enterovirus replication in living cells by using a split-gfp-tagged viral protein
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/d1b2b96f81524978aa1d32d7516d2efd
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Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

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