Fig. 3

Rev-mediated RNA export is necessary for DC maturation but Tat is dispensable. a Optimized 2xTet operator⁴³ was cloned into the 3’LTR of HIV-1-GFPΔtat to generate Tet-HIV-1; the strand-transfer reactions that occur during reverse transcription generate a Tet-regulated 5′-LTR in the provirus. DCs transduced with Tet-HIV-1, rtTA3, or both, were treated for 3 d with 500 ng/mL doxycycline and assayed by flow cytometry for p24, GFP, and CD86. b DCs co-transduced with Tet-HIV-1 and rtTA3 were treated with increasing concentrations of doxycycline. c To generate HIV-1-RTE/CTE, the RTEm26CTE element⁴⁴ was cloned in place of nef in HIV-1-GFPΔrev/ΔRRE. DCs were transduced with the indicated vectors and assessed for p24 and ISG15 by flow cytometry. d DCs were treated with 25 nM leptomycin B, transduced with HIV-1-GFP, and assessed for GFP and ISG15 by flow cytometry. e DCs were treated with 25 nM leptomycin B, transduced with HIV-1-GFP or infected with Sendai virus (SeV), and assessed for ISG15 by flow cytometry. Shown are blood donor data representative of n = 10 (a, c), n = 4 (b), n = 6 (d, e). To determine significance, the MFI of individual flow cytometry samples was calculated as fold-change versus control. When data from each donor replicate within a experiment was combined, the difference in MFI for all experimental vs control conditions was significant in all cases, p < 0.01; one-way ANOVA, Dunnett’s post-test against dox negative control for a, b or HIV-1-GFP for c–e