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A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

The class II membrane fusion induced by flavivirus E proteins is a unique pH-dependent membrane fusion process differently from the class I or III membrane fusion by other enveloped virus proteins. The fusion peptide on the DII of the flavivirus E proteins can insert into the cell membrane as a cell...

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Autores principales: Lin, Hsiao-Han, Huang, Li-Min, Wu, Suh-Chin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553686/
https://www.ncbi.nlm.nih.gov/pubmed/32530355
http://dx.doi.org/10.1080/21645515.2020.1748989
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author Lin, Hsiao-Han
Huang, Li-Min
Wu, Suh-Chin
author_facet Lin, Hsiao-Han
Huang, Li-Min
Wu, Suh-Chin
author_sort Lin, Hsiao-Han
collection PubMed
description The class II membrane fusion induced by flavivirus E proteins is a unique pH-dependent membrane fusion process differently from the class I or III membrane fusion by other enveloped virus proteins. The fusion peptide on the DII of the flavivirus E proteins can insert into the cell membrane as a cell entry process besides the receptor bindings. A traditional assay using C6/36 mosquito cells infected by dengue viruses has been previously reported but did not provide efficient quantitation to measure the virus-triggered membrane fusion. Here we reported the development of a quantitative cell fusion assay for four serotypes of dengue viruses and the recently emerged Zika viruses. We used a pCI-neo vector encoding the prME genes of dengue and Zika viruses and investigated the cell fusion in transfected 293, Vero and CHO cells. Donor cells were co-transfection of the prME genes of dengue and Zika prME gene and T7 RNA polymerase to react with the indicator cells transfected with the luciferase gene under the control of the T7 promoter. Quantification of the virus-induced cell fusion was determined by the luciferase expression levels under a switch of pH from 7.4 to 5.4 in the co-cultured donor and indicator cells. The quantitative luciferase-based assay was applied to measure the anti-fusion activity by two monoclonal antibodies mAb 4G2 and mAb DB42 against dengue virus infections. This assay could quality as a quantitative bioassay for testing the potency of anti-fusion monoclonal antibodies.
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spelling pubmed-75536862020-10-23 A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion Lin, Hsiao-Han Huang, Li-Min Wu, Suh-Chin Hum Vaccin Immunother Research Paper The class II membrane fusion induced by flavivirus E proteins is a unique pH-dependent membrane fusion process differently from the class I or III membrane fusion by other enveloped virus proteins. The fusion peptide on the DII of the flavivirus E proteins can insert into the cell membrane as a cell entry process besides the receptor bindings. A traditional assay using C6/36 mosquito cells infected by dengue viruses has been previously reported but did not provide efficient quantitation to measure the virus-triggered membrane fusion. Here we reported the development of a quantitative cell fusion assay for four serotypes of dengue viruses and the recently emerged Zika viruses. We used a pCI-neo vector encoding the prME genes of dengue and Zika viruses and investigated the cell fusion in transfected 293, Vero and CHO cells. Donor cells were co-transfection of the prME genes of dengue and Zika prME gene and T7 RNA polymerase to react with the indicator cells transfected with the luciferase gene under the control of the T7 promoter. Quantification of the virus-induced cell fusion was determined by the luciferase expression levels under a switch of pH from 7.4 to 5.4 in the co-cultured donor and indicator cells. The quantitative luciferase-based assay was applied to measure the anti-fusion activity by two monoclonal antibodies mAb 4G2 and mAb DB42 against dengue virus infections. This assay could quality as a quantitative bioassay for testing the potency of anti-fusion monoclonal antibodies. Taylor & Francis 2020-06-12 /pmc/articles/PMC7553686/ /pubmed/32530355 http://dx.doi.org/10.1080/21645515.2020.1748989 Text en © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
spellingShingle Research Paper
Lin, Hsiao-Han
Huang, Li-Min
Wu, Suh-Chin
A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title_full A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title_fullStr A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title_full_unstemmed A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title_short A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion
title_sort quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus e protein-triggered membrane fusion
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553686/
https://www.ncbi.nlm.nih.gov/pubmed/32530355
http://dx.doi.org/10.1080/21645515.2020.1748989
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