title: Short communication: a single step assay for rapid evaluation of inhibitors targeting HIV type 1 Tat-mediated long terminal repeat transactivation.
abstract: Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter-mediated gene expression is regulated by the viral Tat protein that relieves a block to viral transcription elongation after binding with a viral hairpin loop RNA structure called the trans-activation-responsive region (TAR). Tat protein significantly up-regulates viral genome transcription and hence it has long been considered a potential target for antiretrovirals. Here we report the construction of a plasmid containing an HIV-1 LTR-driven reporter cassette with a colinear tat gene under control of a viral promoter and thus conditionally configured for constitutive expression of reporter genes. Inhibition of luciferase reporter expression in a cell line harboring the plasmid in the presence of tat-targeted shRNA confirmed the specificity of the assay and a dose-dependent reporter activity inhibition by the fluoroquinoline derivative K-37, a class of small RNA binding molecule that inhibits Tat and other RNA-dependent transactivations, further validated the method. Subsequently we also made a lentiviral vector (LV) containing the same transcription units and derived a stable cell line using the said LV and similar dose-dependent inhibition was documented using K-37. This quick and sensitive reporter-based method is the simplest screening assay for putative inhibitors of HIV-1 Tat-induced LTR-driven gene expression requiring test material addition as the only manipulation.
authors: Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
authors: Ajit G
authors: AG
authors: Chande
authors: Masanori
authors: M
authors: Baba
authors: Robin
authors: R
authors: Mukhopadhyaya
mesh_terms: D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008156:Luciferases; D012367:RNA, Viral / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D015533:Transcriptional Activation / Q000187:drug effects*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases