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

Irene Carlon-Andres,Sergi Padilla-Parra

doi:10.3390/v12020206

Irene Carlon-Andres, Sergi Padilla-Parra

Open Access

https://doi.org/10.3390/v12020206

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Journal: Viruses	Publication Date: Feb 12, 2020
Citations: 7	License type: CC BY 4.0

Affiliation: Wellcome Centre for Human Genetics, University of Oxford

Abstract

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.

Highlights

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus that fuses with target cells and releases the genome-containing capsid in the cytosol
Viral particles can be manipulated fusing the β-lactamase to the HIV-1 viral protein R, which is incorporated into nascent virions
These apparatuses are often equipped with low-sensitivity analogic detectors (photo-multiplier tubes (PMT)), are used in large populations of cells and are unable to resolve the relative amount of fusogenic particles per cell

Summary

Introduction

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus that fuses with target cells and releases the genome-containing capsid in the cytosol. Several approaches to detect and measure HIV-1 fusion have been developed during the last decades, and they comprise population-based assays (bulk assays) [2] and more precise approaches such as real time single virus tracking (SVT) [3,4]. Both approaches have their strengths and weaknesses; for example, bulk assays average out the fusogenic behavior of viruses for a whole population of cells.

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