Figure 2

The use of multi-colour LeGO vectors facilitates simultaneous analysis of different RT variants in single assays. (A) Fluorescence microscopy demonstrates that vectors encoding Cerulean (packaged using a wild-type RT gene) or eGFP (packaged using the quadruple RT mutant [D67N/K70R/T215F/K219Q = NRFQ]) efficiently transduce SC1 cells (left panel), but only the vector packaged with the mutant RT facilitates efficient transduction in the presence of AZT (right panel). (B) AZT-sensitivity of the two RT variants in a dose-dependent manner was determined by FACS analysis. (C) Quantification of the FACS data from two independent experiments performed in duplicate. (D) Relative transduction efficiencies of four different lentiviral pseudotypes with the indicated Pol proteins (wild-type [wt], double mutants [D67N/K70R = NR, T215F/K219Q = FQ] or quadruple mutant (NRFQ) marked by tdTomato (red), Cerulean (blue), eGFP (green), or Venus (yellow), respectively. SC1 cells were transduced simultaneously with equivalent viral titres with or without AZT and analyzed 96 h post transduction. Transduction frequency ranged from 20 to 70 %. Shown are the mean values from two independent experiments. (E) AZT per se does not alter the detection sensitivity for any of the four fluorescent proteins tested. Human 293T cells were infected with lentiviral pseudotypes packaged with the quadruple RT mutant and containing a vector encoding the indicated fluorescent protein [eGFP, tdTomato (tdTom), Cerulean (Cer), or Venus]. AZT was added at the indicated concentration 24 h post transduction and analysed 96 h later. Shown are the mean values of four independent experiments. (F) The comparability of different fluorescent vectors was confirmed by assaying the sensitivity of Pol wt or the quadruple RT mutant (Pol NRFQ) to various concentrations of AZT with vectors encoding the indicated markers after transduction of human 293T cells and analysis 96 h post transduction. Transduction frequency ranged from 20 to 55 %. Shown are the mean values of a minimum of three experiments for each vector. P-values that were significantly different between all other data groups, or only for those connected by lines, are denoted with *, for values < 0.05 or **, for values < 0.01.