FIG. 7.

N/N.oriT replication in the absence of normal provirus formation. (A) 1D Jurkat cells (1.6 × 10⁸) infected with 8.0 × 10⁸ RT cpm of WT (W), N/N (N), N/N.oriT (T), or mock-treated (m) supernatant for 4 h at 37°C were lysed at the indicated times, and unintegrated nuclear DNA was detected by Southern blotting. Whereas the first four lanes were exposed to X-ray film overnight, the remaining lanes were exposed to film for 3 h. Linear DNA was not detected in this analysis, likely because of its loss during alkaline lysis (3). (B) Inverse PCR. Lanes: 1, genomic DNA isolated from mock-infected cells; 2, 1 ng of plasmid pNL4-3 (1) mixed with 10 μg of mock-infected genomic DNA prior to digestion with HindIII; 3 and 4, genomic DNA isolated at 3 dpi with the indicated viruses; 5, mock-infected DNA mixed with Hirt supernatant from approximately 10⁴ N/N.oriT-infected 1D Jurkat cells prior to HindIII digestion; 6, mock-infected DNA plus 1 ng of pN/N.oriT. Whereas the gel in lane 3 was exposed to film overnight, the gel in lanes 1, 2, and 4 to 6 was exposed to film for 5 h. The migration positions of the 1,102-, 358-, and 261-bp internal PCR products are indicated on the right; migration positions of molecular mass standards (sizes in base pairs are shown) are indicated on the left. (C) Inverse PCR strategy. Solid lines, HIV-1 DNA; open boxes, HIV-1 LTRs; vertical arrows, relevant HindIII sites; dashed lines, cellular DNA; black box, oriT; P, location of PCR primers (not drawn to scale); X, variably sized products indicative of provirus distribution. Whereas the HindIII sites at positions 8,131 and 9,606 in WT HIV-1_NL4-3 yielded a 1,102-bp internal PCR product (38), oriT introduced an additional HindIII site, thereby reducing the size of the WT product to358 bp. The WT and N/N.oriT viruses each yielded a 2-LTR-specific 261-bp product (38).