Effects of different IN mutants on HIV-1 reverse transcription and DNA nuclear import. Dividing C8166 T cells were infected with equal amounts of different HIV-1 IN mutant viruses. A) At 12 hours post-infection, 1 × 106 cells were lysed and the total viral DNA was detected by PCR using HIV-1 LTR-Gag primers and Southern blot. B) Levels of HIV-1 late reverse transcription products detected in panel A were quantified by laser densitometry and viral DNA level of the wt virus was arbitrarily set as 100%. Means and standard deviations from two independent experiments are presented. C) At 24 hours post-infection, 2 × 106 cells were fractionated into cytoplasmic and nuclear fractions as described in Materials and Methods. The amount of viral DNA in cytoplasmic and nuclear fractions were analyzed by PCR using HIV-1 LTR-Gag primers and Southern blot (upper panel, N. nuclear fraction; C. cytoplasmic fraction). Purity and DNA content of each subcellular fraction were monitored by PCR detection of human globin DNA and visualized by specific Southern blot (lower panel). D). The percentage of nucleus-associated viral DNA relative to the total amount of viral DNA for each mutant was also quantified by laser densitometry. Means and standard deviations from two independent experiments are shown.