A high-throughput drug screening system for HIV-1 transcription inhibitors.
Identification of HIV-1 transcription inhibitors was previously performed using infectivity assays. As de novo HIV-1 infection is highly sensitive to even minor compound toxicities, these assays are plagued by extremely high levels of false-positive hits. Hit identification is further complicated because infectivity assays lack target specificity. The authors demonstrate that it is possible to overcome these limitations by establishing a stable, chronically actively HIV-1-infected reporter cell line that exclusively identifies HIV-1 transcription inhibitors. In the reporter cell line, 2 spectrally separated fluorescence proteins serve as simultaneously accessible quantitative markers of HIV-1 expression and drug toxicity. The combined analysis of these markers drastically reduces the level of false-positive hits. As determination of fluorescence intensity in a plate-based format can be performed in a noninvasive manner, repeated measurements of fluorescence levels over several days after compound addition can be used to define the kinetic and dynamic characteristics of inhibitory drug effects. In addition, because of the stable nature of the reporter cell line, the assay requires no cell manipulation during assay preparation or assay analysis, rendering the system extremely cost-effective and reliable.
Kempf
Mirjam-Colette
MC
Department of Epidemiology, The University of Alabama at Birmingham, 35294, USA.
Jones
Jennifer
J
Heil
Marintha L
ML
Kutsch
Olaf
O
D000818:Animals; D019380:Anti-HIV Agents / Q000032:analysis* / Q000494:pharmacology*; D002460:Cell Line; D002461:Cell Line, Transformed; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000821:virology; D013601:T-Lymphocytes / Q000821:virology; D014158:Transcription, Genetic / Q000187:drug effects*; D014779:Virus Replication / Q000502:physiology
D019380:Anti-HIV Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins