Ajit G Chande, Masanori Baba, Robin Mukhopadhyaya
Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
AIDS research and human retroviruses 2012 AugHuman 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.
Ajit G Chande, Masanori Baba, Robin Mukhopadhyaya. Short communication: a single step assay for rapid evaluation of inhibitors targeting HIV type 1 Tat-mediated long terminal repeat transactivation. AIDS research and human retroviruses. 2012 Aug;28(8):902-6
PMID: 21878060
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