Figure 2
Specific expression of reporter by the Rev-dependent lentivirus in HIV infected T cells. CEM-SS cells were not infected (A) or were infected (B, C) with HIV-1 NL4-3.HSA.R+E-, an HIV-1 NL4-3 clone with the murine heat stable antigen (HSA; CD24) gene inserted into the nef gene. Both uninfected (A) and infected (C) cells were then transduced with the Rev-dependent reporter virus vNL-GFP-RRE(SA) (5 ng p24/106 cells; VSV-G envelope) or not transduced (B). At 72 hrs post transduction, cells were stained with R-phycoerythrin conjugated rat-anti-mouse CD24 antibody and analyzed by flow cytometer for CD24 and GPF expression. GFP was expressed only in HIV-1 infected (C), but not in HIV-1 uninfected (A) cells. Fifty thousand cells were examined in each run. In this run 61% of total cells were CD24-positive and of the CD24-positive population 17% were GFP-positive. Thresholds for CD24-positive and GFP-positive were set at a fluorescent intensity of 10 on the x- and y-axis respectively. Two independent analyses yielded the same result. D. Percent HIV-infected cells that express GFP versus reporter vector input. A concentrated preparation of reporter particles was used at varied dosages in HIV-infected CEM cells, as above. Flow cytometry studies yielded percent CD24-positive cells that became GFP-positive after 3 days (y-axis) versus reporter virus input (ng p24). Non-linear curve fitting (rectangular hyperbola) yielded R2 = 99.4; maximum GFP-positive = 86.1 ± 6.3% (best-fit values ± std. error); KD = 128.1 ± 21.5 ng p24. Dashed lines are 95% confidence bands.