Stocks of the UL25 deletion mutant (UL25-null) (36 (link)) were generated by using a complementary African green monkey cell line (both gifts from Fred L. Homa, University of Pittsburgh, Pittsburgh, PA). At roughly 75% confluence, monolayers in 175-cm2 flasks were infected with virus stock at a multiplicity of infection (MOI) of 0.1 in 1 ml of phosphate-buffered saline (PBS) without calcium or magnesium per flask for 40 min at room temperature, followed by 5 min at 37°C. The cells were then overlaid with 15 ml virus growth medium (1× minimal essential medium [MEM] Alpha, 1.5% [vol/vol] penicillin-streptomycin; Corning) and incubated at 37°C. At 48 h postinfection, virus-containing medium was clarified by centrifugation at 1,500 rpm for 5 min, and crude cell debris were removed from the supernatant by pelleting at 6,000 rpm for 15 min in a Beckman SW28 rotor. Virus was pelleted for 45 min at 19,000 rpm in a Beckman SW28 rotor. The virus pellets were resuspended in small volumes of PBS without calcium or magnesium, and aliquots of 500 μl were frozen at −80°C. The virus titer was assessed in complementary cells 48 h after infection.
Stocks of the procapsid-producing M100 line (35 (link)) were generated similarly using the complementary F-3 cell line. Stock titers were assessed as described earlier (5 (link)).