hygromycin-a and thiazolyl-blue

Since the recognition of its pivotal role in viral replication, Tat activity has become an interesting target for chemotherapeutic intervention of HIV infection. Here, we report a sensitive and simple colorimetric assay for the screening of Tat inhibitors. We have constructed a plasmid that contains the hygromycin B phosphotransferase gene under the control of the HIV-1 long terminal repeat (LTR) and HIV-1 tat gene constitutively expressed from the cytomegalovirus promoter. This plasmid has been stably transfected to the CD4+ T cell line CEM, which is rendered resistant to hygromycin B through the action of Tat. The inhibitory activity of the anti-Tat drugs was assessed by the extent of cytotoxicity in the presence of hygromycin B as a consequence of the suppressed expression of the hygromycin B phosphotransferase gene. Spectrophotometric quantitation of cell viability was done utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye as the indicator. Using this assay system, we have confirmed that known anti-Tat compound Ro5-3335 and its derivative Ro24-7429 could inhibit Tat-mediated gene expression although their selectivities (anti-Tat activity versus nonselective cytotoxicity) were narrow. Since this method offers the advantage of not handling infectious particles or radioactive materials, it can offer wide applicability as a screening system for anti-Tat compounds.

Topics: Antiviral Agents; Base Sequence; Cell Line; Cinnamates; Colorimetry; Coloring Agents; DNA Primers; Drug Evaluation, Preclinical; Gene Products, tat; HIV-1; Humans; Hygromycin B; Molecular Sequence Data; tat Gene Products, Human Immunodeficiency Virus; Tetrazolium Salts; Thiazoles; Virus Replication