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Int J Mol Sci. 2019 Aug 8;20(16). pii: E3859. doi: 10.3390/ijms20163859.

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

Author information

1
Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, 37077 Göttingen, Germany. mwinkler@dpz.eu.
2
Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, 37077 Göttingen, Germany.
3
Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany.
4
Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany.
5
Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, 37077 Göttingen, Germany. spoehlmann@dpz.eu.
6
Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany. spoehlmann@dpz.eu.

Abstract

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.

KEYWORDS:

FRET; IFITM; influenza virus

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