FIG 9.

PP2A depletion is not sufficient to rescue Cts2 vaccinia viral DNA replication. (A) CV1 cells were treated with 50 nM control or PP2A siRNA at 37°C for 72 h. Whole-cell lysates were used for Western blot analyses using anti-BAF or anti-PP2A antibodies. Bands shown were all taken from the same blot with the same exposure times; however, some irrelevant lanes have been removed for clarity. (B) Western blot analysis of BAF phosphorylation in various cell treatments. CV1 cells were transfected with 50 nM control, PP2A, or PP4 siRNA at 37°C for 72 h. Cells were then infected with WT or Cts2 vaccinia virus at an MOI of 5 at 40°C for 24 h. Western blot analyses were performed using the indicated antibodies. (C) Viral DNA replication of CV1 cells transfected with 50 nM control or PP2A siRNA at 37°C for 72 h followed by infection with WT (gray bars) or Cts2 (black bars) virus at an MOI of 5 at 40°C for 24 h. qPCR was performed on purified DNA by using vaccinia virus DNA-specific primers. Viral DNA replication was calculated relative to control siRNA in each infection set (n = 3). Error bars represent standard deviations. (D) Model of BAF phosphoregulation by cellular and viral enzymes. Filled arrows indicate movement of BAF proteins within the cell, while open arrows indicate BAF's (de)phosphorylation by cellular and viral enzymes.