ANTI-TAT MTT ASSAY - A NOVEL ANTI-HIV DRUG SCREENING SYSTEM USING THEVIRAL REGULATORY NETWORK OF REPLICATION

Since the recognition of its pivotal role in viral replication, Tat ac tivity has become an interesting target for chemotherapeutic intervent ion of HIV infection. Here, we report a sensitive and simple colorimet ric assay for the screening of Tat inhibitors. We have constructed a p lasmid that contains the hygromycin B phosphotransferase gene under th e control of the HIV-1 long terminal repeat (LTR) and HIV-1 tat gene c onstitutively expressed from the cytomegalovirus promoter. This plasmi d has been stably transfected to the CD4(+) T cell line GEM, which is rendered resistant to hygromycin B through the action of Tat. The inhi bitory activity of the anti-Tat drugs was assessed by the extent of cy totoxicity in the presence of hygromycin B as a consequence of the sup pressed expression of the hygromycin B phosphotransferase gene. Spectr ophotometric quantitation of cell viability was done utilizing (4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye as the i ndicator. Using this assay system, we have confirmed that known anti-T at compound Ro5-3335 and its derivative Ro24-7429 could inhibit Tat-me diated gene expression although their selectivities (anti-Tat activity versus nonselective cytotoxicity) were narrow. Since this method offe rs the advantage of not handling infectious particles or radioactive m aterials, it can offer wide applicability as a screening system for an ti-Tat compounds.