FIG. 7.

Time course of initiation of plus-strand DNA synthesis. The assay for initiation of HIV-1 or HIV-2 plus-strand DNA synthesis was performed as described in Materials and Methods. Aliquots were removed at the indicated times, and samples were run in 8% sequencing gels. The amount of ³²P-labeled 20-nt DNA formed was calculated by comparing the volume of the 20-nt band at each time point with the value for the volume of the 35-nt RNA-DNA extension product synthesized with T4 DNA polymerase, which was set at 100%. (A) HIV-1 substrate. Reactions were carried out with the following amounts of enzyme: 1 pmol of HIV-1 RT (circles) and 2 pmol of HIV-2 440 RT (squares). (B) HIV-2 substrate. Reactions were carried out with 1 pmol of HIV-1 (circles) or 2 pmol of the three HIV-2 RTs: 440 (squares), 470 (triangles), and 484 (inverted triangles). (C) HIV-1 and HIV-2 substrates. The reaction mixtures contained a final monocation concentration of 5 mM instead of 58 mM, and reactions were carried out with the HIV-1 substrate and 1 pmol of HIV-1 RT (circles) and with the HIV-2 substrate and 1 pmol of HIV-2 440 RT (squares). (D) Schematic diagram of the initiation assay. The procedure was the same as that used for the experiment in Fig. 6, except that in this assay, both primer extension and primer removal are catalyzed by RT. The representation of the nucleic acid reactants is the same as in Fig. 6C. The vertical arrow in step 2 indicates the site of RNase H cleavage.