Article Figures & Data
Figures
- FIG. 1.
(A) Immunofluorescent foci of ROka and ROka63D in MeWo, U2OS, and Saos-2 cells. Cells infected with ROka or ROka63D were stained with anti-VZV gE mouse monoclonal antibody, followed by fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G antibody. (B) Growth of ROka and ROka63D in U2OS and Saos-2 cells. U2OS and Saos-2 cells were inoculated with VZV-infected MeWo cells. Cells were harvested on days 1 to 6 after infection, and virus titers were determined by plating on MeWo cells. The virus titer on day zero represents the titer of the input inoculum.
- FIG. 2.
Effect of IFN-α (A) and IFN-γ (B) on plaque formation by ROka and VZV deletion mutants in MeWo cells. MeWo cells were untreated or pretreated with IFN-α (1,000 IU/ml) or IFN-γ (1,000 IU/ml) for 18 h and infected with ROka, ROka63D, ROka61D, or ROka67D. Plaques were counted 6 days (for ROka) or 10 days (for deletion mutants) after infection. The efficacy of IFN treatment was measured by the ratio of the number of plaques in IFN-untreated wells to the number of plaques in IFN-treated wells. The results from three experiments were analyzed by one-way analysis of variance, and the Welch modified t test was used to calculate P values. The error bars represent standard deviations.
- FIG. 3.
VZV IE63 expressed in trans overcomes the effect of IFN-α in ROka63D-infected cells. MeWo cells were infected with control adenovirus (Adblank) or adenovirus expressing VZV IE63 protein (Ad63) for 5 h and treated with IFN-α (1,000 IU/ml) for 18 h. The cells were then infected with serial dilutions of ROka63D, and 10 days after infection, the monolayers were stained with crystal violet.
- FIG. 4.
Effects of IFN-α on replication of ROka and ROka63D in U2OS and MeWo cells. Monolayers of U2OS and MeWo cells were untreated or pretreated with IFN-α (1,000 IU/ml) for 18 h. The cells were then infected with ROka or ROka63D. Four days after infection, the cells were harvested and the titer of each virus was determined on MeWo cells. The efficacy of IFN-α treatment was measured as described in the legend to Fig. 3. The error bars represent standard deviations.
- FIG. 5.
Immunofluorescence staining with antibodies to VZV gE and IE62 of VZV-infected, IFN-α-untreated, or IFN-α-pretreated MeWo cells. MeWo cells treated with human IFN-α (1,000 IU/ml) or untreated were infected with ROka, ROka63D, ROka61D, or ROka67D. On the indicated days after infection, the cells were fixed and stained with antibodies to VZV gE (A) or VZV IE62 (B) or with DAPI (A, bottom). The data are representative of one of two independent experiments.
- FIG. 6.
Immunoblots of VZV IE, putative early, and late proteins in MeWo cells untreated (−) or pretreated (+) with IFN-α. (A) MeWo cells untreated or pretreated with IFN-α (1,000 IU/ml) were infected with ROka, ROka63D, or ROka61D. Cell lysates were prepared 2 days after infection and immunoblotted with antibodies to VZV IE62, TK, gE, IE63, ORF61 protein, or cellular β-actin. (B) The VZV protein bands were quantified using a densitometer and normalized to the corresponding β-actin band. The reduction (n-fold) in protein expression due to IFN-α treatment was determined as the ratio of normalized densitometry values from IFN-α-untreated cells to that from IFN-α-treated cells. The data are representative of one of two independent experiments.
- FIG. 7.
Northern blots of VZV IE, putative early, and late transcripts in MeWo cells in the presence (+) or absence (−) of IFN-α. (A) MeWo cells untreated or pretreated with human IFN-α (1,000 IU/ml) were infected with ROka, ROka63D, or ROka61D. Total RNA was isolated 2 days after infection, and Northern blots were performed using radiolabeled DNA probes specific for VZV IE62, TK, gE, and cellular β-actin. (B) Bands were quantified using a densitometer, and each band was normalized to the area of the corresponding β-actin band. Reduction (n-fold) in mRNA expression due to IFN-α treatment was calculated as the ratio of densitometry values from IFN-α-untreated cells to that from IFN-α-treated cells. The data are representative of one of two independent experiments.
- FIG. 8.
Immunoblot of eIF-2α phosphorylation in ROka, ROka63D, or ROka61D-infected MeWo cells. Mewo cells were infected with ROka, ROka63D, or ROka61D; cell lysates were prepared 2 days after infection; and immunoblots were performed using antibodies to phosphorylated or total eIF-2α, VZV ORF4, or gE. Lysates from thapsigargin (Tgn)-treated MeWo cells were used as a positive control, and lysates from uninfected or dimethyl sulfoxide (DMSO)-treated cells were negative controls for eIF-2α-P. The data are representative of one of three independent experiments.
- FIG. 9.
Immunoblot of phosphorylated and total eIF-2α in Cos cells expressing VZV ORF63. Cos cells were transfected with the indicated plasmids, and 2 days later, cell lysates were prepared and immunoblotted with antibodies to phosphorylated or total eIF-2α. The numbers below the bands indicate signal intensities relative to that of untransfected cells. The data are representative of one of three independent experiments.
