A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer (original) (raw)
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Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity
Antimicrobial Agents and Chemotherapy, 2001
The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.
Luciferase Time-based, High-throughput Screening Assay for the Discovery of HIV-1 Inhibitors
Abstract With the enhancement of high-throughput screening (HTS) for target-based antiretroviral discovery, fluorescence has been considered the best bioassay. Bioluminescence by genetic reporters has maintained a major place among cell-based virological assay formats. Luminescent bioassays have a wide variety of applications due to their high sensitivity and linearity, even for a range of complex biological samples. In this target-based method, we used an HTS bioluminescence assay and highlighted the detection capabilities of this time-of-addition experiment. Hence, the development of a cell-based assay that uses a phenotypic drug discovery approach based on bioluminescence by stable reporter cells is described. Utilizing this screening method, a bioluminescent, target-based, time-of-addition experiment was performed by adding antiretrovirals with known mechanisms of action to analyze their drug targets by measuring the length of time until the antiretrovirals lost their efficacy in an HIV-1 replication assay. Depending on the viral replication target, the antiretrovirals lost activity at different times: fusion inhibitors acted for 0-2 h; retrotranscriptase inhibitors acted efficiently for the first 4 h; integrase inhibitors acted for 18 h; and protease inhibitors were inactivated after 15 h. The target-based, bioluminescence assay identifies the mode of action of antiretroviral drugs and provides valuable information about drug targets that inhibit HIV replication. This assay delimits the time (hours) for which the addition of an antiviral can be delayed before losing its antiviral activity relative to the replication cycle of HIV. The target of an antiviral compound can be identified both by comparing the relative time until it loses efficacy to that of the reference drugs and by the time elapsed when the antiretroviral loses activity compared to a known control. Our target-based bioluminescence assay is fast, reliable, sensitive and useful for highthroughput drug screening. Keywords HIV; Time-of-addition experiment; Antiretroviral; Bioluminescence assay; Fusion inhibitor; High-throughput screening; Reporter gene; Indicator cell
Biopolymers and Cell, 1995
Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer Synthesis of fluorogenic peptide substrate of HIV-1 protease Dns-SQNYPIVWL which corresponds to the pi?7p24 cleavage site forHIV-J protease havebeen performed. This fluorogenic substrate was based onthe fluorescence resonance energy transfer between donor-Trp residue, and acceptor-dansyl group in the intact peptide. Hydrolysis of substrate by recombinant HIV-1 protease resulted in the time-dependent increase ofTrp fluorescence and decrease of dansyl fluorescence measured at 350 and 500 nm, respectively, due to the break of resonance energy transfer between donor and acceptorfluorophors. Hydrolysis of fluoro genic peptide substrate was studied also by reversed phase HPLC and two peptide fragments after cleavage of substrate have been detected. Kinetic constants of hydrolysis for this fluorogenic peptide substrate by HIV-1 protease were calculated from Lineweaver-Burk plots: K M-29pM, k cat-5.4 s' 1 and k cai lK M-180 000 hf V і .-Introduction* Human immunodeficiency virus protease (HIV-1 protease) per forms the proteolytic processing of viral gag and gag-pol polyproteins precursors during the replication cycle of retrovirus [1, 2]. It has been shown using site directed mutagenesis and also by use of specific inhibitors of HIV-1 protease that disruption of this function produces morphologically immature and noninfectious viral particles [3]. Thus, HIV-1 protease represents an important therapeutic target in the development of treatment for AIDS [4 ]. The number of specific inhibitors of HIV-1 protease have been synthesized and studied and results are summarized in reviews [5, 6 J. The antivirus synergy between HIV-1 protease inhibitors and nucleoside analogue inhibitors of HIV reverse transcrip tase (AZT, ddC) has been also revealed [7 J. • Evaluation of efficiency of novel HIV-1 protease inhibitors requires the rapid and sensitive method of measurement of protease activity. Recently chromophoric peptide substrate for the spectrophotometric assay of HIV-1 protease have been reported [8], which exhibits an absorbance increase upon its hydrolysis by protease. Fluorogenic peptide substrates of HIV-1 protease [9-11 ] are most efficient and sensitive in the monitoring of specific hydrolysis reaction. In this work we report the synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer (FRET). Resonance energy transfer between donor and acceptor chromophores is widely used as a tool for the measurement of intramolecular distances in biopolymers [12] and also to monitor the enzyme activity [9-11, 13]. Our substrate was based on the FRET between two fluorescent probes, tryptophan and dansyl group, introduced into the oligopeptide substrate, which corresponds to the pi 7/p24 cleavage site for HIV-1 protease. Materials and Methods. Purification of HIV-1 protease. The expression system in Escherichia coli strain AR58 was as previously described by Meek et aL [14 ]. Cells (300 g) were thawed in 1500 ml of 50 mM Tris buffer containing
An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells
PLoS ONE, 2010
The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.
Protein Engineering Design and Selection, 2000
The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents. Keywords: cleavage signal sequences/immature virus particle/ mature virus particle/virion incorporation
Virology, 2004
The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we su...
A Sensitive and Versatile Bioluminescence Bioassay for HIV Type 1 Based on Adenoviral Vectors
AIDS Research and Human Retroviruses, 1999
The construction and characterization of a versatile bioassay for the quantification of H IV-1 viral infection and HIV-1 Tat protein activity based on recom binant adenoviral vectors carrying an HIV LTR-driven luciferase reporter gene is described. Th e assay system consists of a set of two adeno-reporter vectors, one of which is responsive to H IV-1 Tat protein activity, and the second of which is not, by virtue of a deletion of the TAR site within the HIV LTR. Th is configuration of the reporter genes allows one to distinguish Tat-specific activation from Tat-non-specific H IV LTR-m ediated gene expression. Th e adenoviral H IV LTR-m ediated luciferase gene expression is highly responsive to Tat and increases linearly with increasing levels of H IV-1 infection, reaching levels of between 3-and 1000-fold induction. The adeno-reporter viruses can be utilized to detect Tat activity and HIV-1 infection in a wide range of cell types, including 293, C EM , HUT-78, Jurkat, and HeLa-derived cell lines. The resulting bioassay is convenient, sensitive, and readily adaptable to autom ated procedures. These characteristics of the adeno-reporter assay m ake it a valuable reagent for studies of H IV infection and for analysis of H IV-inhibitory agents.
J Mol Biol, 1999
The retroviral protease (PR) is absolutely essential for completion of human immunodeficiency virus multiplication cycle, and cannot be replaced by any cellular function. Thus PR, like reverse transcriptase, is an ideal target for the development of anti-AIDS therapy. A large number of human immunodeficiency virus type-1 (HIV-1) PR inhibitors have been developed, and several are currently used as anti-AIDS drugs. These inhibitors are mainly based on the natural PR cleavage sites within the viral Gag and Gag-Pol precursors. The major difficulty encountered while using anti-HIV therapeutic agents in patients has been the rapid emergence of drug-resistant viral strains. Most of the mutations which convert the PR into inhibitor-resistant are located within the substrate binding subsites of the enzyme. Recently, it has been shown that the HIV-1 auxiliary protein Vif, and especially the N-terminal half of Vif (N′-Vif) specifically interacts with the viral PR and inhibits its activity. We now show that efficient inhibition of HIV-1 PR activity can be achieved using Vif-derived peptides. Based on the above model we have performed peptide mapping of N′-Vif in order to find a small peptidic lead compound which inhibits PR activity. The screening revealed that peptides derived from two regions in Vif spanning from residues 30–65 and 78–98 inhibit PR activity in vitro, specifically bind HIV-PR and inhibit HIV-1 production in vivo. Further mapping of these regions revealed the lead compounds Vif81–88 and Vif88–98. These peptides specifically inhibit and bind HIV-1 PR, but do not affect pepsin and rous sarcoma virus protease. In contrast to other known PR inhibitors, these peptides are not substrate-based and their sequences do not resemble the sequences of the natural PR substrates (cleavage sites). Moreover, the Vif-derived peptides themselves are not cleaved by HIV-1 PR. Conversion of the lead peptides into small backbone cyclic peptidomimetics is taking place nowadays in order to turn these lead compounds into metabolically stable selective novel type of HIV-PR non-substrate-based inhibitors.
A Time-Resolved Fluorescence Assay to Identify Small-Molecule Inhibitors of HIV-1 Fusion
Journal of Biomolecular Screening, 2007
Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu 3+ ). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Förster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of timeresolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions. (Journal of Biomolecular Screening 2007:865-874)