Frequency of inter- and intrasubtype HIV-1 recombination in an in vitro, single cycle, and multiple cycle assay systems. Panel A indicates the nucleotide genetic distances that separate the env genes in each pair of subtype A and D primary HIV-1 isolates employed in this study. The recombination frequencies of each pair in panel B were calculated in three systems. For in vitro, the synthesis of minus strand DNA on the donor RNA template was catalyzed by RT. Products were PCR amplified, cloned, and blue/white colonies were screened to calculate recombination frequencies. For the single-cycle system, recombination occurred in a cell infected with a heterozygous virus particle. Recombinants were identified by PCR and by the same blue/white colony screening described in Figure 1 and in the Materials and Methods. Finally, the recombination frequency in the multiple cycle system was measured by quantitative PCR using isolate- or subtype-specific primers (see Materials and Methods and Figure 4). The sequence identity between each HIV-1 pair is shown as line graph with the scale on right of panel B. Panel C shows a plot of recombination frequency in the single cycle (filled circle) or in vitro (open circle) systems versus sequence identity. The r value represents the Pearson product moment correlation for in vitro (open circle) and single cycle (filled circle) assays.