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

Abstract

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.

Retrovirus Drugs-Loaded PEGylated PAMAM for Prolonging Drug Release and Enhancing Efficiency in HIV Treatment

“…HIV-1 proteases is indispensable enzymes in the HIV life cycle and is considered one of the most important targets in anti-HIV drugs development [ 24 , 29 ]. Pepsin and HIV-1 are in the family of aspartic proteases with similar primary structure, the same activating mechanism and are inactivated by pepstatin A inhibitor.…”

Nanoscale flow cytometry reveals interpatient variability in HIV protease activity that correlates with viral infectivity and identifies drug-resistant viruses

“…To analyze HIV protease (PR) activity in single viral particles we chose to employ Förster resonance energy transfer (FRET), a technique previously used to monitor PR activity in cell-free and cell-based assays [18][19][20][21] . We employed the mUKG-mKOκ fluorescent protein pair based on its good spectral separation, compatibility with a wide range of flow cytometry instruments, and high quantum yield that reflects efficient energy transfer between the fluorescent proteins.…”

Function-Based Mutation-Resistant Synthetic Signaling Device Activated by HIV-1 Proteolysis

“…One potential application of our synthetic device could be as a detection assay for screening the library of drug-resistant HIV-1 protease mutants and inhibitors in mammalian culture cells using different reporters, including cell toxic proteins as the selection markers. The advantages of using cell line–based assays for HIV-1 protease activity have already been reported; authors used different HIV-1 protease precursors (GFP-PR or Gal4-PR) or probe proteins, such as Förster resonance energy transfer-based HIV-1 protease-sensitive sensors. , Through experimental implementation of our signaling device, we have found that it is also faster than two-round phenotyping assays for testing HIV-1 isolates …”

“…The advantages of using cell line−based assays for HIV-1 protease activity have already been reported; authors used different HIV-1 protease precursors (GFP-PR 18 or Gal4-PR 23 ) or probe proteins, such as Forster resonance energy transfer-based HIV-1 protease-sensitive sensors. 24,25 Through experimental implementation of our signaling device, we have found that it is also faster than two-round phenotyping assays for testing HIV-1 isolates. 26 The primary purpose of the described device is the targeting of essential viral function, regardless of the specific sequence or spatial structure of the particular viral components.…”