The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation. A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24CA-normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of dNTPs and permeabilizing agent melittin. Samples without dNTPs were used as a control. DNA was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.