Pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis experiments were carried out as described previously (28). Briefly, about 3.2 × 10⁶ CV1 cells in a 60-mm-diameter dish were infected with the indicated viruses at an MOI of 5 PFU per cell in the presence or absence of 300 μg/ml PAA for 18 h. Cells were collected in cold PBS and pelleted by spinning them at 4,000 rpm for 5 min in a refrigerated tabletop centrifuge. The pellets were resuspended in 150 μl of PBS and mixed with 300 μl of 1.2% agarose, and the mixture was poured onto the bottom of a 10-mm-diameter dish to form an agarose block. The agarose block was digested with 100 μg/ml proteinase K in digestion buffer (10 mM Tris [pH 8.0], 100 mM EDTA, 1% [wt/vol] N-lauroylsarcosine sodium salt [Sarkosyl]) for 20 h at 37°C and washed with storage buffer (10 mM Tris [pH 8.0], 10 mM EDTA). Roughly equally sized agarose plugs were sliced and loaded into the wells of a 0.8% agarose gel, and the wells were sealed with 0.8% low-melting-point agarose. The gel was run in 0.5× TBE buffer (1× TBE buffer contains 89 mM Tris, 89 mM boric acid, and 2 mM EDTA [pH 8.0]) at 6 V/cm for 16 h at 14°C, with an angle of 120° and a pulse time of 45 to 70 s, with a Bio-Rad CHEF-DR II pulsed-field electrophoresis system. After electrophoresis, the gel was soaked in 0.25 M HCl for 45 min to depurinate the DNAs, and the DNAs were further denatured, neutralized, and transferred onto a positively charged nylon membrane as described above. DNAs were UV cross-linked to the membrane and hybridized with the denatured ³²P-labeled BamHI P fragment of the HSV-1 genome as described above. The bound probe was revealed by exposure of the membrane to X-ray film at −80°C in the presence of intensifying screens.