Figure 1.

HIV-1 Gag foci are present in the nucleus of HeLa and U2OS cells regardless of the nature of the tag fused to the C-terminus of Gag, which was expressed in a Rev-dependent fashion using the constructs shown in panels A and B. (A) Schematic of doxycycline-inducible HIV.Gag-GFP rtTA proviral construct expressing Gag fused to GFP, which is stably integrated in HeLa cells (HeLa HIV.Gag-GFP rtTA). The 5′LTR contains a mutated TAR, two NF-κB sites, eight copies of the tetO sites in the promoter, and three SP1 sites. (B) Schematic of pHIV.Gag-CFP/SNAP-tag rtTA doxycycline-inducible construct with the same 5′UTR sequences described in panel A, which expresses Gag fused to either CFP or SNAP-tag. (C) HeLa HIV.Gag-GFP rtTA cell line contains the stably-integrated proviral construct shown in panel A. Numerous Gag foci (green) were seen in the nucleus, which was stained with DAPI (blue). Three-dimensional reconstruction of a confocal z-series is shown with crosshairs through a single Gag focus to demonstrate that Gag was present inside the nucleus (outlined by a dashed white line) in all three planes. (D) HeLa cells co-expressing pHIV.Gag-CFP rtTA with pPB-t-rtTA. (E) U2OS cells transfected with pHIV.Gag-CFP rtTA and pPB-t-rtTA. (F) A HeLa cell line stably expressing rtTA was transfected with pHIV.Gag-SNAP-tag rtTA and incubated with SNAP-tag ligand JF549.