Fig 2.

Overexpression of 5ptaseIV prevents RSV Gag from localizing to the PM. (A) QT6 cells were transfected with RSV pGag-GFP alone (top row) or cotransfected with Myc-tagged 5ptaseIV (middle row) or Myc-tagged 5ptaseIVΔ1 (bottom row). Graphs showing the percentage of cells with cytoplasmic (C) or PM-associated (M) Gag are shown at the right of the images. Fisher's exact test (GraphPad Prism) was used to determine whether there were statistically significant differences in Gag localization for untreated, 5ptaseIV-, and 5ptaseIVΔ1-treated cells and for the Gag variants compared to the wild type. (B) QT6 cells expressing Gag-GFP alone (mock) or Gag-GFP plus 5ptaseIV or 5ptaseIVΔ1 were metabolically labeled with [³⁵S]methionine-cysteine, the medium was collected after a 2.5-h labeling period, and the cells were lysed. After immunoprecipitation of cell lysates and supernatants with anti-RSV antiserum (a gift from John Wills and Rebecca Craven, Penn State College of Medicine) (32), the amount of Gag-GFP in the lysates and media was quantified by PhosphorImager analysis (35–37). Budding efficiency, the amount of Gag-GFP in the medium divided by the sum of the amounts in the medium and lysate [M/(M+L)], was calculated. Budding efficiency of Gag-GFP under steady-state conditions was set at 100%. Data from three independent experiments were averaged, with the bars representing the mean ± the standard error of the mean. Statistical analysis was performed using two-tailed Student's t test, with a single asterisk signifying a p value of <0.005 and two asterisks signifying a p value of <0.0005. Autoradiography images from a representative experiment are shown to the right of the graph.