FIG 3.

VZV significantly modulates cell surface NKG2D ligand expression over time. ARPE-19 cells were infected with VZV or mock infected at a 1:10 ratio and harvested at various times postinfection. (A) The percentage of total cells infected over time was monitored by flow cytometry staining for VZV antigen, gE:gI, at each time point, for three independent experiments. The mean percentage of VZV antigen-positive cells is recorded above each time point. (B and C) Mock- or VZV-infected cells were harvested at 24, 48, 72, and 96 hpi and stained with antibodies against MICA, ULBP2, ULBP3, and MHC-I for flow cytometry analysis. (B) One representative histogram at 96 hpi is shown, comparing mock (continuous black lines) and VZV (antigen-positive [dotted black lines]) infections for cell surface NKG2D ligand or MHC-I expression. Isotype control antibody staining is indicated by the gray-filled histogram. (C) The relative MFI fold change over mock infection in surface NKG2D ligand or MHC-I expression (less respective isotypes) over time is presented as the mean ± SEM of data from biological replicates (n = 3). Statistical significance was established by two-tailed paired Student's t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Mock- or VZV-infected protein lysates were harvested at 72 and 96 hpi for immunoblotting with antibodies against MICA, ULBP2, and ULBP3. Corresponding probing for a housekeeping protein (actin) is shown beneath. One representative blot of three independent experiments is shown.