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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany
Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany
Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany
Authors to whom correspondence should be addressed.
Current address: Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France, and Université de Strasbourg, 67000 Strasbourg, France.
Int. J. Mol. Sci. 2019, 20(16), 3859;
Received: 3 July 2019 / Revised: 26 July 2019 / Accepted: 3 August 2019 / Published: 8 August 2019
(This article belongs to the Section Molecular Immunology)
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. View Full-Text
Keywords: IFITM; FRET; influenza virus IFITM; FRET; influenza virus
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MDPI and ACS Style

Winkler, M.; Wrensch, F.; Bosch, P.; Knoth, M.; Schindler, M.; Gärtner, S.; Pöhlmann, S. Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay. Int. J. Mol. Sci. 2019, 20, 3859.

AMA Style

Winkler M, Wrensch F, Bosch P, Knoth M, Schindler M, Gärtner S, Pöhlmann S. Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay. International Journal of Molecular Sciences. 2019; 20(16):3859.

Chicago/Turabian Style

Winkler, Michael, Florian Wrensch, Pascale Bosch, Maike Knoth, Michael Schindler, Sabine Gärtner, and Stefan Pöhlmann. 2019. "Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay" International Journal of Molecular Sciences 20, no. 16: 3859.

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