Dependence of ANG path entry on intracellular low pH and endocytosis. (A) Effect of alteration of intracellular pH on ANG path entry. CHO cells expressing either nectin-1 or nectin-2 were treated with the indicated concentrations of ammonium chloride (NH4Cl) for 30 min. HSV-1 strain ANG path was added (MOI of 10) for 6 h in the continued presence of NH4Cl. Cells contain the lacZ gene under the control of an HSV-inducible promoter. Entry was measured as the % of beta-galactosidase activity relative to that obtained in the absence of agent. Data shown are means of quadruplicate determinations with standard deviation. (B) Effect of inhibition of endocytic uptake on ANG path infection. ANG path was added to CHO-nectin-1 or CHO-nectin-2 cells (100 PFU/well) in control medium (A) or hypertonic medium containing 0.3 M sucrose. At 30 min post-infection, medium was removed, extracellular virus was acid-inactivated, and plates were incubated for 24 h. Syncytia were detected by immunoperoxidase staining and quantified. Shown are representative data from at least three independent experiments.