Article Figures & Data
Figures
- Fig 1
PRV infection leads to a US3-dependent suppression in S3 cofilin phosphorylation. (A) ST cells were mock inoculated or inoculated (MOI of 10) with WT PRV, US3null PRV, or US3rescue PRV. At 6 hpi, total cell lysates were subjected to Western blotting to detect phospho-S3 cofilin, total cofilin, US3, and gE. (B) Relative cofilin phosphorylation levels based on the phospho-S3 cofilin/cofilin ratio (with mock infection set to 1) are represented as means + standard errors of the means of data from three independent experiments, with ** indicating P values of <0.01 and *** indicating P values of <0.001. (C) ST cells were inoculated with WT PRV (MOI of 10) and lysed at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, or 24 h postinfection. Total cell lysates were subjected to Western blotting to detect phospho-S3 cofilin, total cofilin, and US3. (D and E) ST cells were transfected with US3 or with a control plasmid encoding DsRed (23) and stained for US3 and phospho-S3 cofilin. Panel D shows quantification of fluorescein isothiocyanate (FITC) (p-S3-cof) pixel intensities of 8 randomly chosen US3- or control plasmid-transfected cells, which were determined using ImageJ. Data shown represent means + standard errors of the means, with * indicating P values of <0.05.
- Fig 2
The kinase activity of US3 is required to suppress phosphorylation of cofilin. (A) ST cells were mock inoculated or inoculated (MOI of 10) with WT PRV, kinase-inactive D223A US3 PRV, or D223Arescue PRV. At 6 hpi, total cell lysates were subjected to Western blotting to detect phospho-S3 cofilin, total cofilin, US3, and gE. (B) Means + standard errors of relative cofilin phosphorylation levels from three independent experiments, with *** indicating P values of <0.001.
- Fig 3
Overexpression of S3D phosphomimetic cofilin interferes with the ability of US3 to cause cell rounding and cell projections. (A and B) ST cells were transfected with US3 encoding plasmid or cotransfected with plasmids encoding US3 and GFP-tagged WT cofilin, S3A cofilin, or S3D cofilin. At 24 h posttransfection, cells were fixed and stained for US3 and nuclei and analyzed for expression of cofilin (GFP; green) and US3 (red). Panel A shows the percentage of transfected cells displaying actin rearrangements, as assessed by cell rounding and the formation of cell projections (means + standard errors of the means; data from three independent experiments), with * indicating P values of <0.05 and ** indicating P values of <0.01. Small blue dots in panel B represent leftover plasmid DNA-containing transfection reagent in cells and on the cover glass.
- Fig 4
Group I PAKs are involved in US3-mediated suppression of S3 cofilin phosphorylation. (A and B) ST cells treated with or without 33 μM group I PAK inhibitor IPA-3 were either mock inoculated (A) or inoculated with WT PRV (B). At 6 hpi, total cell lysates were subjected to Western blotting to detect phospho-S3 cofilin, total cofilin, and US3. Values were normalized to mock (A) or to PRV and IPA-3 (B). The graphs represent the means + standard errors of the means from three independent experiments, with * indicating P values of <0.05.
