Fig. 5

Inability of protease or low pH treatments alone to trigger fusion with the plasma membrane of entry-resistant cells. HSV-1 KOS was bound to B78 cells (MOI of 10) for 2 h at 4 °C. a Confluent cell monolayers were chilled at 4 °C and washed with ice-cold PBS. HSV-1 strain KOS in bicarbonate-free culture medium supplemented with 20 mM Hepes and 0.2 % BSA was added at an MOI of 10 for 2 h at 4 °C. Monolayers were rinsed with ice-cold PBS, and then PBS containing different concentrations of trypsin was added to cells for 25 min at 4 °C. Trypsinization was halted by addition of soybean trypsin inhibitor (8000-16,000 BAEE units; GIBCO-BRL) and 5 % fetal bovine serum for 15 min at room temperature. Complete cell culture medium was added, and cultures were incubated for 7 h at 37 °C. b Serum-free, bicarbonate-free culture medium with 0.2 % BSA and 5 mM (each) HEPES (Life Technologies), 2-(N-morpholino)ethanesulfonic acid (MES; Sigma), and sodium succinate (Sigma) was adjusted with HCl to achieve pHs ranging from 7.0 to 4.5 [69]. Confluent cell monolayers were chilled at 4 °C and washed with ice-cold PBS. HSV-1 strain KOS was added at an MOI of 10 for 2 h at 4 °C. Cells were washed with warm PBS. Warmed media adjusted to different pHs were added. Samples were incubated at 37 °C for 10 min. A pre-titrated amount of NaOH was added to return each sample to pH 7.4. Complete cell culture medium was added, and cultures were incubated for 7 h at 37 °C. Each value is the mean of quadruplicate determinations with standard deviation. Representative experiments of at least three independent experiments are shown