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. 2005 Jan;79(1):364–379. doi: 10.1128/JVI.79.1.364-379.2005

FIG. 4.

FIG. 4.

Hybrid AV2:5 ITR viral genomes have reduced capacity to form circular intermediates. HeLa cells were infected at a multiplicity of infection of 500 DNase-resistant particles/cell with AV2:2/2eGFP, AV5:5/2eGFP, or AV2:5/2eGFP virus. The low-molecular-weight Hirt DNA was extracted at 1 day postinfection, and 1/10th of the DNA was used to transform E. coli Sure cells. (A) Total rescued ampicillin-resistant CFU and head-to-tail circular intermediates with double-D ITR junctions are indicated. Results indicate the mean (± standard error of the mean) for triplicate assays for each experimental point. (B) The predicted structure of a head-to-tail circularized hybrid ITR genome is given with restriction enzyme sites and sequence primers used for molecular characterization. (C) Restriction enzyme analysis of 24 circular intermediates rescued following AV2:5/2eGFP infection. Isolated plasmids p12, p14, p17, p18, and p21 (marked by arrows) were the only five clones that demonstrated an intact 800-bp EGFP SacII fragment and a ≈200-bp ITR SphI fragment that matched the predicted double-D circular intermediate structure seen with the AV2:2 and AV5:5 genomes. These clones were further analyzed by sequencing of the ITR junction with primers EL1080 and EL1081. (D) Sequence results of the five clones containing a double-D ITR junction. The upper line depicts consistent sequences found in all clones and includes an intact D′ sequence from the AAV-5 and AAV-2 ITRs. The boldface partial sequence is the area flanking the ITRs where the SphI site (boxed) resides. Below is the remaining sequence between the two D sequences for each of the individual clones, as marked. The sequence homology to AAV-5 ITR is indicated in black, and homology to AAV-2 ITR is indicated in red. The various palindromic segments of the ITR are indicated by D, A′ B, B′, C, C′, and A, according to the standard nomenclature. The D′ and A segments of the AAV-2 ITR and the A′ and D segments of AAV-5 are marked with underlining. Segments with deletions are marked by an asterisk.