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J Virol Methods ; 249: 1-9, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28807730

RESUMO

Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Doxiciclina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Regulação Viral da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Purinas/farmacologia , RNA Viral/genética , Roscovitina , Sensibilidade e Especificidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
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