title: A rapid, quantitative bioassay based on the human immunodeficiency virus trans-activator.
abstract: We constructed a human immunodeficiency virus (HIV) trans-activator cDNA (tat) encoding the N-terminal 76 amino acids of the viral trans-activator followed by two additional amino acids (val and pro). This cDNA encoded a functional trans-activator (TAT) as shown by cotransfection into murine cells with a HIV promoter-chloramphenicol acetyltransferase DNA construct. The tat cDNA was cloned into an avian retroviral expression vector, a modified spleen necrosis virus (SNV), and high-titer infectious stocks of recombinant virus (SNV-tat) were recovered from dog cells. Hybridization analyses indicated that SNV-tat was stably propagated in these cells for months. We also prepared recombinant cells that stably carry reporter genes, either a human gene encoding a soluble CD4 receptor (sCD4) or the human preprorenin gene, under the transcriptional control of the HIV promoter. Medium obtained from these cell cultures after infection with control viruses or an SNV carrying an antisense tat contained only low background levels of sCD4 or prorenin (HRN) as determined by specific immunoassays (1-10 ng protein per 10(6) cells per ml medium). In contrast, cells infected with SNV carrying tat in the transcriptional sense orientation secreted 75 +/- 7 ng sCD4 and 73 +/- 4 ng HRN per 10(6) cells per ml medium. Moreover, these proteins were constitutively secreted at these levels during months of subculturing. The data indicate that sCD4 and HRN are secreted from these cells because of a TAT-mediated trans-activation of the HIV reporter gene DNA and/or RNA. This combination of recombinant cells, SNV-tat, and specific immunoassays provide a rapid, quantitative, and safe bioassay to seek inhibitors of TAT.
authors.affiliation: Division of Cancer and Infectious Diseases Research, Upjohn Company, Kalamazoo, MI 49001.
authors.forename: J M
authors.initials: JM
authors.lastname: Hasler
authors.forename: T F
authors.initials: TF
authors.lastname: Weighous
authors.forename: T W
authors.initials: TW
authors.lastname: Pitts
authors.forename: D B
authors.initials: DB
authors.lastname: Evans
authors.forename: S K
authors.initials: SK
authors.lastname: Sharma
authors.forename: W G
authors.initials: WG
authors.lastname: Tarpley
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004285:Dogs; D006678:HIV / Q000235:genetics*; D007118:Immunoassay; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000096:biosynthesis*; D014162:Transfection
chemical_list: D015704:CD4 Antigens; D004279:DNA, Viral; D015534:Trans-Activators