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Open AccessArticle

Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Division of Structural Biology, University of Oxford, Wellcome Centre for Human Genetics, Headington, Oxford OX3 7BN, UK
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Viruses 2020, 12(2), 206; https://doi.org/10.3390/v12020206
Received: 30 December 2019 / Revised: 6 February 2020 / Accepted: 10 February 2020 / Published: 12 February 2020
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes.
Keywords: HIV-1 fusion; FRET; FLIM; live cell imaging; HIV infection; single cell analysis HIV-1 fusion; FRET; FLIM; live cell imaging; HIV infection; single cell analysis
MDPI and ACS Style

Carlon-Andres, I.; Padilla-Parra, S. Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells. Viruses 2020, 12, 206.

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