Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

Rok Gaber,Andreja Majerle,Roman Jerala,Mojca Benčina

doi:10.3390/s131216330

Abstract

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.

Highlights

We designed a Förster resonance energy transfer (FRET) sensor for the detection of human immunodeficiency virus (HIV) protease activity based on a FRET protein pair (Figure 1A). mCerulean was used as donor protein and was linked to the mCitrine, acceptor protein via polypeptide linker that contained the p17/p24 HIV protease cleavage site of SQVSQNY↓PIVQNLQ [12], which is identical to the one in the commercial HIV Protease
When the FRET-HIV protease-sensitive sensor was excited with a 433-nm light, energy was transferred from mCerulean to mCitrine, and an emission peak shifted from mCerulean (475 nm) to mCitrine (529 nm)
The FRET-HIV sensor was tested for the detection of intracellular HIV protease activity in in situ assays using fluorescence spectroscopy

Summary

Introduction

The human immunodeficiency virus (HIV), which causes the acquired immunodeficiency syndrome (AIDS) was first discovered in 1981. HIV protease is crucial for the maturation of viral particles and represents an important target for therapy. This protease recognizes specific peptide sequences between individual proteins in Gag and Gag-Pol polyproteins [2]. If the HIV protease is inhibited, viral particles still bud from the cellular membrane, but the proteins inside them are not in a mature and active form; the virus is not infective. The rapid mutation rate of the HIV virus results in great numbers of different viral variants in an individual patient. Resistance of the HIV protease to all registered protease inhibitors has already been reported; novel protease inhibitors must be developed in order to continue the fight against the HIV epidemic [2]

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