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

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An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
Creator	Coyne, Carolyn Bárcena, M Koster, A Melia, C Strating, J Van Bruggen, J Van Der Schaar, H Van Geenen, M Van Kuppeveld, F
Source	PMC
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.
has issue date	2016-07-06(xsd:dateTime)
bibo:doi	10.1128/msphere.00104-16
bibo:pmid	27390781
has license	cc-by
sha1sum (hex)	7146a5eb303bef6ef7c4bc2588a0305fc7b58cd3
schema:url	https://doi.org/10.1128/msphere.00104-16
resource representing a document's title	Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein
has PubMed Central identifier	PMC4935779
has PubMed identifier	27390781
schema:publication	mSphere
resource representing a document's body	covid:7146a5eb303bef6ef7c4bc2588a0305fc7b58cd3#body_text
is schema:about of	named entity 'supplied' named entity 'inserted' named entity 'living cells' named entity 'fluorescent protein' named entity 'van' named entity 'Golgi' named entity 'GFP' named entity 'ENTEROVIRUS' named entity 'SPLIT' named entity 'REPLICATION' named entity 'SPLIT' named entity 'USING' named entity 'LIKE' named entity 'PROTEIN ' named entity 'TOOLS' named entity 'JAC' named entity 'TO EXPRESS' named entity 'CORRELATIVE' named entity 'PRIOR TO' named entity 'REAL TIME' named entity 'THESE' named entity 'LEADS' named entity 'ORGANELLES' named entity 'IMAGING' named entity 'ISOLATION' named entity 'INFECTION' named entity 'MED' named entity 'UNAVAILABLE' named entity 'FRAGMENT' named entity 'SENIOR' named entity 'TRANSIENT' named entity 'OVERCOME' named entity 'POSITIVE' named entity 'ENTEROVIRUSES' named entity 'ESTABLISHED' named entity 'SMALL' named entity 'PROTEINS' named entity 'DYNAMICS' named entity 'INDICATING' named entity 'FAR' named entity 'MARKER' named entity 'DETECTED' named entity 'GENES' named entity 'ILLUMINATION' named entity 'ANCHORED' named entity 'VAN' named entity 'DISINTEGRATION' named entity 'LARGE' named entity 'MCHERRY' named entity 'MELIA' named entity 'CARRY' named entity 'AUTHORS' named entity 'INSERTED INTO' named entity 'MORPHOLOGIES' named entity 'ORIGIN' named entity 'UNIQUE' named entity 'MULTIPLE' named entity 'GOLGI DISASSEMBLY' named entity 'S11' named entity 'LIVING' named entity 'STABLE EXPRESSION' named entity 'RECOMBINANT' named entity 'VIRAL PROTEIN' named entity 'TAGGED' named entity 'GENERATE' named entity 'TAGGED' named entity 'KNOWN' named entity 'CITATION' named entity 'AFFECT' named entity 'WILD-TYPE' named entity 'NEW' named entity 'LIGHT' named entity 'TO FORM'

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