title: Assessment of a cytoprotection assay for the discovery and evaluation of anti-human immunodeficiency virus compounds utilizing a genetically-impaired virus.
abstract: A biologically contained cytoprotection assay was developed to screen inhibitors of the human immunodeficiency virus without the need for high level containment or practices. The virus used has multiple point mutations that have destroyed its ability to produce both Rev and Tat, proteins essential for virus replication in vitro. The original cell line employed (CEM-SSTART) contains a genetic construct that allows for the continuous expression of both Rev and Tat, and a subclone (1A2) was developed that provides for maximum acute cytopathic effect. The National Cancer Institute's AIDS drug screening assay was used to test known 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 the tetrazolium salt, XTT, as an indicator of cellular metabolism after the cells have been infected with virus. The results of extensive testing 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 culture, 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 materials, which are now available to the scientific community (NIH AIDS Research and Reference Reagent Program), should be useful tools to screen and test compounds for potential inhibition of HIV in laboratories not equipped to maintain and use wild-type infectious virus.
authors.affiliation: NCI-AIDS Drug Screening and Development Laboratory, SAIC-Frederick, NCI-FCRDC, MD 21702-1201, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kiser
authors.forename: S
authors.initials: S
authors.lastname: Makovsky
authors.forename: S J
authors.initials: SJ
authors.lastname: Terpening
authors.forename: N
authors.initials: N
authors.lastname: Laing
authors.forename: D J
authors.initials: DJ
authors.lastname: Clanton
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004341:Drug Evaluation; D016655:HIV Core Protein p24 / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans
chemical_list: D000998:Antiviral Agents; D016655:HIV Core Protein p24; D054303:HIV Reverse Transcriptase