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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay
The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719045/ https://www.ncbi.nlm.nih.gov/pubmed/31398796 http://dx.doi.org/10.3390/ijms20163859 |
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author | Winkler, Michael Wrensch, Florian Bosch, Pascale Knoth, Maike Schindler, Michael Gärtner, Sabine Pöhlmann, Stefan |
author_facet | Winkler, Michael Wrensch, Florian Bosch, Pascale Knoth, Maike Schindler, Michael Gärtner, Sabine Pöhlmann, Stefan |
author_sort | Winkler, Michael |
collection | PubMed |
description | The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions. |
format | Online Article Text |
id | pubmed-6719045 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-67190452019-09-10 Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay Winkler, Michael Wrensch, Florian Bosch, Pascale Knoth, Maike Schindler, Michael Gärtner, Sabine Pöhlmann, Stefan Int J Mol Sci Article The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions. MDPI 2019-08-08 /pmc/articles/PMC6719045/ /pubmed/31398796 http://dx.doi.org/10.3390/ijms20163859 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Winkler, Michael Wrensch, Florian Bosch, Pascale Knoth, Maike Schindler, Michael Gärtner, Sabine Pöhlmann, Stefan Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title | Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title_full | Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title_fullStr | Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title_full_unstemmed | Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title_short | Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay |
title_sort | analysis of ifitm-ifitm interactions by a flow cytometry-based fret assay |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719045/ https://www.ncbi.nlm.nih.gov/pubmed/31398796 http://dx.doi.org/10.3390/ijms20163859 |
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