Figure 2

Frequent integration of the IN-CA defective virus into the DSB site. (A) PCR amplification of provirus DNA integrated into the I-SceI site after infection of WT virus (left) or NL-Luc-IN-D64A-E(−)R(−) virus (D64A virus) (right). PMA-treated THP-1/I-SceI cells were used. Each lane shows an independent result that was obtained from cells cultured in a single well of 6-multiwell. For each test group, six wells were independently infected with viruses. M, molecular marker. Arrowheads indicate amplicons of viral DNA integrated in the I-SceI site, which was further confirmed by sequence analysis. (B) Sequence data of D64A provirus DNA that was integrated in the I-SceI site. A representative result is shown at the top. Asterisks indicate the pAC. Dots indicate identical nucleotides to those of the representative sequence. Dashes indicate deleted nucleotides. (C) Experimental protocol for evaluating the frequency of viral integration into the DSB site. I-PpoI-qPCR and EGFP-qPCR analyses were done for quantification of I-PpoI site-specific and total proviral DNA copy numbers, respectively. Representative data of two independent experiments was shown. Error bars, s.d. of triplicate assays. (D) Evaluation of I-SceI site-targeting efficiency. PMA-treated THP-1/I-SceI cells were infected with WT or D64A virus for 2 h, and cells were harvested 48 hpi for the I-SceI-qPCR analysis (see Methods section). To cleave the I-SceI site, cells were infected with the Ad-I-SceI at an MOI of 100 from 1 h post HIV-1 infection. Treatment with RAL and KU55933 was conducted from −2 h to 48 hpi. Effects of RAL and KU55933 were evaluated on 11 samples that were prepared from three independent experiments. Each dot indicates copy numbers of provirus DNA that had integrated in the I-SceI site in 10³ cells, which were infected as a single test sample.