Evolution of HIV-rtTA after transient dox administration. (A) Schematic of the HIV-rtTA genome. The inactivated Tat-TAR elements (crossed boxes) and the introduced rtTA-tetO elements are indicated. rtTA is a fusion protein of the E. coli Tet repressor (TetR) and the VP16 activation domain (AD) of herpes simplex virus. TetR contains a DNA-binding domain (DNA BD) (amino acids 1–44) and a regulatory core domain (amino acids 75–207) with a dimerization surface. (B-D) Loss of dox-control in cultures of HIV-rtTA after transient activation. SupT1 cells were transfected with HIV-rtTA and cultured at 100 ng/ml dox (B), HIV-rtTAV9I G138D at 10 ng/ml dox (C), and HIV-rtTAG19F E37L at 1000 ng/ml dox (D). Each experiment was started with 12 independent cultures (different symbols represent different cultures). At day 3, dox was washed out and the cultures were continued with dox-free medium. The cultures in which the virus did not lose dox-control were split in two parts at day 64 (C) or day 66 (D) and dox was added to one of the samples. Virus production was monitored by CA-p24 ELISA on culture supernatant samples.