Nuclear protein composition and metabolism of HeLa cells after infection with herpes simplex virus.

Cells. HeLa cells (American Type Culture Collection, Rockville, Md.) were grown in 80 ml culture medium in Roux bottles. Cells were grown in Eagle's medium (7) which contained Hanks' balanced salt solution (1 1), 10% bovine serum, 100 @zg/mlstreptomycin sulfate, and 100 units/ml penicillin G. Bottles were incubated at 36°in an humidified atmosphere of 5% CO2 , and the cells were passed by trypsinization every 4 days. Cells were growing logarithmically when used in all experiments. Virus. The MP strain of herpes simplex was used (12). High virus titers were produced by washing 1 ml virus over HeLa monolayers in l2-oz bottles. After 1 hr at 36°,10 ml growth medium were added and the incubation was continued. The CO2 content of the atmosphere was adjusted to maintain the cells at pH 6.6, the pH of maximal virus production (28). After 18 hr the cultures were frozen and thawed 3 times to release cell-bound virus. Virus titers were measured by plaque counts on HeLa cell monolayers (10). Titers were usually 2 to 5 x 106 plaque-forming units/mi. In all experiments cells were infected with 2 to 5 plaque-forming units/cell. Control bottles were sham-infected with equivalent volumes of warm growth medium. All bottles were then incubated for 1 hr at 36° . Monolayers were washed twice with 10 ml of warm Eagle's medium containing 1% bovine serum and 0.3% pooled human @-globulln(Merck Sharp and Dohme, West Point,Pa.). Growth medium, 80 ml, was then added and the incubation was continued. Cell-associated virus was measured by removing the growth medium, washing the cells twice in fresh growth medium, and freeze-thawing the cells 3 times in 20 ml growth medium. The solution was then assayed for virus content as above (10).