FIG. 5.

Localization of HIV-1 cDNA after infection with each mutant. (A) Quantification of HIV-1 cDNA in the nuclear fraction. 293T cells were infected with each virus (WT, N144Q, PYNP>KL, KKK>AAA, or D116G). At 24 h postinfection, nuclei were isolated from the infected cells as described in Materials and Methods. A DNA sample from each nuclear fraction was subjected to real-time quantitative PCR with the M667-AA55 (R/U5-specific) or the M661-M667 (R/gag-specific) primer pair. The values shown are the copy number (R/U5) of each mutant per 10⁴ nuclei relative to that of the WT, taken as 100%. Means and standard deviations from three independent experiments are shown (n = 5). (B) HeLa cells (4 × 10³) were seeded on silane-coated glass slides. Cells were infected with each IN mutant (PYNP>KL, KKK>AAA, or D116G) or with the WT (∼70 ng of p24). At 6 h postinfection, the cells were fixed with 4% paraformaldehyde and subjected to FISH analysis with an FITC-labeled HIV-1-specific PNA probe corresponding to nt 2878 to 2994 of the pol region of pNL43 (1). The signals were observed with a confocal microscope. Representative medial sections were mounted by using Adobe Photoshop software. The representative cells in each slide are shown with lower (left) and higher (right) magnification scales.