A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors

García-Murria, Maria Jesús; Expósito-Domínguez, Neus; Duart, Gerard; Mingarro, Ismael; Martínez-Gil, Luis

doi:10.3390/v11030229

Cited by 13 publications

(9 citation statements)

References 45 publications

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“…Previous reports analyzed syncytia formation between two cell types that express either spike protein or ACE‐2 at their membrane using fluorescence labeling 24‐26 . In another study Jun/Fos and split reporter system was used, which reconstituted when the syncytium, triggered by the Nipah virus F and G protein that interacted with the Ephrin B2 receptor on other cells, was formed 27 . To increase the sensitivity and streamline the analysis, we developed a split luciferase reporter assay based on our previously designed tightly interacting N7/N8 coiled‐coil peptide pair, 19 where each of the N7/N8 peptide of the pair was attached to one fragment of the split firefly luciferase.…”

Section: Resultsmentioning

confidence: 99%

“…[24][25][26] In another study Jun/ Fos and split reporter system was used, which reconstituted when the syncytium, triggered by the Nipah virus F and G protein that interacted with the Ephrin B2 receptor on other cells, was formed. 27 To increase the sensitivity and streamline the analysis, we developed a split luciferase reporter assay based on our previously designed tightly interacting N7/N8 coiled-coil peptide pair, 19 where each of the N7/N8 peptide of the pair was attached to one fragment of the split firefly luciferase. Upon syncytium formation, cLuc:N7 and nLuc:N8, which are initially in different cells, are localized to the same compartment and form a coiled-coil dimer with concomitant luciferase reconstitution.…”

Section: A Sensitive Split Luciferase Assay Enables the Measurement Of Syncytia Formationmentioning

confidence: 99%

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Disruption of disulfides within RBD of SARS‐CoV‐2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs

et al. 2021

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The COVID-19 pandemic is one of the most serious medical emergencies since the last century. SARS-CoV-2, which was first reported at the end of 2019, has affected the entire world, and there are still very few therapeutic options. One of the fastest ways toward therapy would be the repurposing of already approved drugs and dietary compounds. Several drugs have been shown to inhibit viral replication by targeting either viral components, such as inhibitors of viral RNA polymerases, 1,2 or compounds that target the human cell proteins that interact with or process viral components, such as protease or kinase inhibitors [reviewed in 3,4]. Approved compounds that might interfere with the binding of spike proteins to human angiotensin-converting enzyme-2 (ACE2) viral receptors have been proposed. 5 For some compounds, the mechanism of inhibition or molecular target is not clear although the effect on viral replication

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Section: Resultsmentioning

confidence: 99%

Section: A Sensitive Split Luciferase Assay Enables the Measurement Of Syncytia Formationmentioning

confidence: 99%

Disruption of disulfides within RBD of SARS‐CoV‐2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs

et al. 2021

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“…In this case, quantitation is achieved by nuclei and syncytia counting and/or by colorimetric assays. Recently, complementation of a reporter protein, either GFP or luciferase, was introduced to quantify cell-cell fusion (32)(33)(34). To quantitatively assess Syncytin-1-triggered cell-cell fusion, we adapted the NanoLuc Binary Technology complementation assay.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Avian System for Quantitative Analysis of Syncytin-1 Interaction with ASCT2 Receptor

Štafl

Trávnícek

Kučerová

et al. 2021

Preprint

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BackgroundHuman Syncytin-1 is an envelope glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retroviral docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor.ResultsWe have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein production. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the major part of region C did not abolish the interaction of ASCT2 with Syncytin-1.ConclusionsWe present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background, and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C of ASCT2 was essential for protein expression but not for the interaction with Syncytin-1 glycoprotein.

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“…Generation of a signal, which occurs only after the fusion between donor and acceptor cells 27 , provides a good basis for this sensitive method to monitor syncytium formation. Therefore, split fluorescence or luminescence proteins linked to synthetic dimerization domains that form very stable dimers would be a prime choice as reporters.…”

Section: Introductionmentioning

confidence: 99%

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Plaper

Aupič

Dekleva

et al. 2020

Preprint

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Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

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A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors

Cited by 13 publications

References 45 publications

Disruption of disulfides within RBD of SARS‐CoV‐2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs

Disruption of disulfides within RBD of SARS‐CoV‐2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs

Heterologous Avian System for Quantitative Analysis of Syncytin-1 Interaction with ASCT2 Receptor

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

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