Figure 6. Progression of dsDNA synthesis by HIV-1 RT on circular ssDNA templates in the presence of NCp7, Vpr and IN.

The polymerase reaction was carried out in a RT buffer containing 50 mM Tris-acetate pH 7.8, 50 mM sodium acetate, 6 mM magnesium acetate and 0.5 mM DTT at 37°C for 40 min. (see (Hameau et al., 2001) and (Mirambeau et al., 2007) for more details). Upper left panel: comparison by agarose gel electrophoresis of DNA synthesis by RT in the absence (labeled RT) and presence of NCp7 (A), NCp7 and Vpr (B), NCp7, Vpr and IN (C). Concentrations of primed-ssDNA circles, RT and NCp7 were, respectively, 5 nM, 100nM, 3.4 μM. In B and C, the Vpr concentration was 2 μM. In C, the IN concentration was 0.5 μM. ssDNA and proteins were premixed for 4 min. at 37°C, followed by incubation with RT for 2 min. before addition of dNTPs (100 μM each) to start the reaction. DNA products were heated to 70°C for 10 min. in the presence of 1% (w/v) SDS and 20 mM EDTA before gel electrophoresis on a 1% agarose gel in 0.5x TBE. Upper right 4 panels: TEM images of ssDNA control followed by dsDNA produced by RT alone (at either 2 or 40 min.) or with Vpr (same concentrations as in B) analyzed after 40 min. at 37°C. Lower panels, a-c) RT polymerization observed by TEM at 40 min. under polymerization conditions described for the agarose gel analysis described above. 5 μl-aliquots of the polymerization assay were diluted 5-fold in the reaction buffer without DTT and next deposited onto the EM grid. The scale bars correspond to 250 nm.