FIG. 6.
Tat- and Rev/CRM1-dependent HIV-1 gene expression is robust in rabbit cells, including primary T cells. (A) The indicated cell lines were cotransfected with expression constructs encoding the Tat- and/or Rev-dependent RFP reporter, HIV-1 Tat and/or HIV-1 Rev, and GFP (to identify transfected cells). The product of the percentage of GFP/RFP-positive cells and the MFI of RFP, measured in the absence of Tat and Rev coexpression, was set to 1 for each cell line. Shown are arithmetic means ± SEM of results from triplicates of two to six experiments. (B) RFP reporter expression is CRM1 dependent. SIRC and HeLa cells were transfected as for panel A, with the addition of LMB and expression plasmids encoding either short NLS/NES sequences or the dominant negative nucleoporin ΔCAN/Nup214. (C) Primary T cells were nucleofected with the expression constructs described for panel A and analyzed 24 h later. Shown are arithmetic means ± SEM of results from duplicates of two to four independent experiments. (D) Primary T cells were nucleofected with the nearly full-length HIV-1 reporter construct pNL4-3 GFP (early gene expression) or pNL4-3 Gag-GFP (late gene expression). The MFI of GFP expression in viable cells was determined 24 h later by flow cytometry, and MFIlate/MFIearly ratios are depicted. Results for T cells from individual donors or animals (open circles) and the arithmetic means ± SEM (filled triangles) are depicted.