Figure 1
Effect of dNTP concentration on RNA-dependent DNA polymerization activity for lentiviral RT proteins. (A) 5’ 32P-labeled 17-mer primer (P) annealed to 40-mer RNA template. (B) The T/P was extended by 18 purified RT proteins under the condition described in Experimental Procedures at different dNTP concentrations (lanes 1–10: 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 100, nM, 50 nM, 25 nM). HIV-1 strains used were HXB2, NL4-3, 94CY, 92RW, 93IN, 94UG, and 93BR. HIV-2 strains used were Ghana1, ST1, ROD, and ROD10. SIV stains used were Mac239, Mne CL8, Mne 170, Agm155-4, Agm Gri-1, Agm 9063–2, and Agm Tan-1. RT activity used in this assay generated approximately 50% primer extension as determined by 40 bp fully extended product (F) at the highest dNTP concentration (lane 1). Among 18 RT proteins, the reactions with HIV-1 94CY, HIV-2 ROD, and SIVagm 9063–2 are shown in this figure, “*” indicates pause sites produced by kinetic delays of dNTP incorporations at lower dNTP concentrations. (−) no RT control. T: dNTP concentrations found in activated CD4+ T cells, M: dNTP concentrations found in macrophages.