A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors.
The Tat function of the human immunodeficiency virus (HIV) represents an important target for the development of new anti-HIV drugs. A rapid, sensitive and simple bioassay was developed for the detection of HIV transactivation inhibitors. A reporter plasmid based on the expression of the green fluorescent protein (GFP) under control of the HIV-1 long terminal repeat (LTR) was constructed. This reporter gene can be quantified by simply measuring the fluorescence irradiated by GFP-producing cells, without the need of extraction procedures or enzymatic assays. Cells, stably expressing HIV-1 Tat protein, were transfected with this plasmid and the inhibitory effect of anti-Tat drugs was assessed by measuring the inhibition of fluorescence. Using this assay system the anti-transactivation activity of several known compounds was confirmed. This is the first HIV transactivation assay using GFP reporter gene in microtiter plates. The assay can be used for the detection and quantification of HIV transactivation, and for the high throughput evaluation of anti-transactivation drugs in different cellular backgrounds.
Daelemans
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Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000, Leuven, Belgium.
De Clercq
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Vandamme
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AM
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D001681:Biological Assay; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D016325:HIV Long Terminal Repeat / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008164:Luminescent Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C081802:9-(2',3'-dihydroxycyclopentan-1'-yl)adenine; D049452:Green Fluorescent Proteins; D000241:Adenosine