FIGURE 4.

Effect of MβCD-treatment of HSV-1 on cell attachment and on the kinetics of endocytotic internalization. 5 mM MβCD-treated or mock-treated HSV-1 was added to the indicated cells (MOI) on ice for 1 h. (A) Cells were rinsed twice with cold PBS, and cell-associated virions were quantitated by qPCR. Data are expressed as % of untreated virus that successfully attached. nd, not determined. (B,C) To determine the kinetics of endocytosis of HSV-1, cells were washed and incubated at 37°C, and extracellular virus was inactivated by acid treatment at the indicated times. At 8 h p.i., cells were fixed and fields of ∼2000 cells per sample were evaluated. Total cell number was evaluated by nuclear staining with 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI), and infected cells were detected by immunofluorescence with an anti-HSV polyclonal antibody. Infectivity at 90 min p.i., was set to 100%. Data are means of triplicate determinations with standard deviation. The results shown are representative of at least two independent experiments.