ASSESSMENT OF A CYTOPROTECTION ASSAY FOR THE DISCOVERY AND EVALUATIONOF ANTI-HUMAN-IMMUNODEFICIENCY-VIRUS COMPOUNDS UTILIZING A GENETICALLY-IMPAIRED VIRUS

A biologically contained cytoprotection assay was developed to screen inhibitors of the human immunodeficiency virus without the need for hi gh level containment or practices. The virus used has multiple point m utations that have destroyed its ability to produce both Rev and Tat, proteins essential for virus replication in vitro. The original cell l ine employed (CEM-SSTART) contains a genetic construct that allows for the continuous expression of both Rev and Tat, and a subclone (1A2) w as developed that provides for maximum acute cytopathic effect. The Na tional Cancer Institute's AIDS drug screening assay was used to rest k nown drugs with both HIVIIIB virus in the T4 lymphocytic cell line CEM -SS and mutant virus in the 1A2 subclone. This cell-based assay uses t he tetrazolium salt, XTT, as an indicator of cellular metabolism after the cells have been infected with virus. The results of extensive tes ting have shown that the assay using mutant virus is comparable to the current NCI AIDS drug screen. After 42 days in 1A2 or CEM-SS cell cul ture, the virus or the integrated genome did not revert to wild-type, and the virus produced in 1A2 cells was unable to replicate in PBMCs. Mutant viral stocks were devoid of wild-type virus as determined by a PCR assay that would have found 60-600 copies of mutant RNA. These mat erials, which are now available to the scientific community (NIH AIDS Research and Reference Reagent Program), should be useful tools to scr een and test compounds for potential inhibition of HIV in laboratories not equipped to maintain and use wild-type infectious virus.