title: Anti-Tat MTT assay: a novel anti-HIV drug screening system using the viral regulatory network of replication.
abstract: 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.
authors.affiliation: Department of Microbiology, Fukushima Medical College, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kira
authors.forename: J P
authors.initials: JP
authors.lastname: Merin
authors.forename: M
authors.initials: M
authors.lastname: Baba
authors.forename: S
authors.initials: S
authors.lastname: Shigeta
authors.forename: T
authors.initials: T
authors.lastname: Okamoto
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002934:Cinnamates; D003124:Colorimetry; D004396:Coloring Agents; D017931:DNA Primers; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015696:Gene Products, tat / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D013778:Tetrazolium Salts; D013844:Thiazoles; D014779:Virus Replication / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D000998:Antiviral Agents; D002934:Cinnamates; D004396:Coloring Agents; D017931:DNA Primers; D015696:Gene Products, tat; D013778:Tetrazolium Salts; D013844:Thiazoles; D054322:tat Gene Products, Human Immunodeficiency Virus; D006921:Hygromycin B; C026273:hygromycin A; C022616:thiazolyl blue