Figure 5

Evaluation of the transactivation property of the Tat proteins. (A) HEK293 cells (top panel) seeded in 12-well plates were transiently transfected with 0.5 μg of LTR-GFP reporter vector using a standard calcium phosphate protocol. Cells transfected with a blank plasmid were included as a negative control. LTR represents a full-length viral promotor cloned from an Indian primary subtype-C clinical isolate. Twenty four hours after the transfection, cells were incubated with freshly reconstituted Tat protein at a final concentration of 5 μg/ml in complete medium. Twenty four hours following the protein transfection, expression of GFP was documented using the UV-fluorescence microscopy. CEM-GFP cells (bottom panel), containing a stably integrated GFP gene under the control of subtype-B LTR, were transfected with 5 μg/ml of B or C-Tat proteins using a commercial lipid formulation following the directions of the manufacturer (Bioporter, Gene therapy systems, San Diego, CA, USA). or (B) HEK293 cells were transfected with a different reporter vector pLTR-SEAP and treated with Tat as described above. Expression of alkaline phosphate secreted into the medium was estimated at 24, 48 and 72 h using a colorimetric assay. The difference between B- and C-Tat treatments at all the time points was found to be statistically significant by Student's paired t-test. The p value at 72 h is shown. (C) CEM-GFP cells were treated with 5 μg/ml of B- or C-Tat proteins for the duration shown or left without treatment. Cells were harvested, fixed with 2% formaldehyde and evaluated for GFP expression using FACSCalibur flow cytometer (BD Biosciences). The live cells were gated on the basis of forward and side scatter. The number of GFP positive cells was determined by using scattergram of side scatter versus FL-1. Cells with fluorescence intensity greater than 10¹ were considered to be GFP positive and the gating was done accordingly. A total of 10,000 events were scored. The x-axis represents GFP intensity (FL-1) and the y-axis percentage of positive cells. Percent positive cells for the reporter protein are shown. The differences between B- and C-Tat treatments at both the time points were found to be statistically significant by Student's paired t-test. The p values at 24 and 48 h are < 0.0052* and < 0.0025**, respectively. (D) Rescue of a Tat-defective provirus. HLM-1 cells harboring an integrated provirus defective for Tat produce large quantities of virus when complemented with functional Tat protein. The cells were incubated with the Tat protein (5 μg/ml) in complete medium and 24, 48 and 72 h after Tat-transfection, the viral structural protein , p24, secreted into the culture medium was estimated using an antigen-capture assay. The difference between B- and C-Tat treatments at all the time points was found to be statistically significant by Student's paired t-test. The p value at 72 h is shown. (E) HEK293 cells were cotransfected with 0.5 μg of LTR-SEAP reporter vector and 0.1 μg of B-, C-Tat or empty vector. Alkaline phosphatase secreted into the medium was quantified at different time points as shown. All the above experiments were repeated several times and the data presented are representative of these experiments. CMV-β-galactosidase vector was used in all the transfections to control for differences in the transfection efficiency. β-galactosidase levels in the cell extracts were quantified using a colorimetric assay. All the quantitative assays were performed in triplicates and the data are presented as mean of triplicate values ± 1 S.D. The difference between B- and C-Tat treatments was not found to be statistically significant by Student's paired t-test. The p value at 72 h is shown.