Fig. 2.

RTX mediates dUTP incorporation into reverse transcribing HIV-1 cDNA and prevents efficient integration and viral protein expression. HIV-1 cDNA levels were measured in RTX-treated HT29 cells at 6 (A), 24 (B), and 48 (C) h after infection. (D) Integrated proviruses were measured at 48 h after infection using an Alu-Gag nested PCR. To determine whether viral DNA contained uracil, PCRs were performed with mock pretreatment of the template (white bars) or pretreatment with 50 nM hUNG and 50 nM APE1 (black bars, A–D), which, when combined, generate strand breaks at uracil sites. (E) Expression of virally encoded eGFP 48 h after infection as analyzed by FACS. (F) Thymidine (1 mM; Thd) was added to the media of infected cells either at the time of infection or 16 h after infection, and the ability to rescue GFP expression was measured by FACS. In all cases, n = 3.