FIGURE 4.

PACS-2 is required for Nef to assemble the SFK—ZAP-70/Syk—PI3K cascade. A, H9 CD4⁺ cells were nucleofected (Amaxa) with a control (scr) siRNA or siRNAs specific for PACS-1 or PACS-2 on day 1 and again on day 3. On day 5, cells were infected with either VV:WT or VV:Nef/f (m.o.i. = 10, 8 h). Nef was immunoprecipitated (IP), and co-immunoprecipitating Tyr(P)²⁹²Zap70 was detected by Western blot (WB). The amount of Nef-associated PI3K activity was quantified as described under “Experimental Procedures.” Bottom, Western blot showing expression of PACS-1, PACS-2, and actin. Error bars represent the mean ± S.D. of three independent experiments. B, H9 cells were transfected on day 1 with plasmids expressing p85α/f, p85αR₃₅₈A/f, p85αR₆₄₉A/f, or p85αRARA/f. On day 3, the cells were infected with VV:WT (m.o.i. = 10, 8 h) or co-infected with VV:Nef (untagged, m.o.i. = 10, 8 h) and VV:Hck (m.o.i. = 2, 8 h). Cells were harvested, and FLAG-tagged p85α was immunoprecipitated with mAb M2 and co-precipitating Tyr(P)²⁹²ZAP-70 detected by Western blot. Data are representative of two independent experiments. C, H9 cells were transfected with siRNAs as described in A. On day 5, the cells were co-infected with VV:WT or VV:Nef/f (m.o.i. = 10, 8 h) and VV:Hck (m.o.i. = 2, 8 h). Nef was immunoprecipitated, and co-immunoprecipitating Hck was detected by Western blot. Data are representative of three independent experiments. D, parallel plates of H9 CD4⁺ cells were infected or not with VV:Nef/f (m.o.i. = 6 each) and VV:PACS-2ha (m.o.i. = 5 each) and with decreasing amounts of VV:WT (m.o.i. in lane 2 = 14, lane 3 = 3, lane 4 = 2, and lane 5 = 1) or VV:Hck (m.o.i. in lane 4 = 1, lane 5 = 2, lane 6 = 3; total m.o.i. = 14, 18 h) and harvested as indicated under “Experimental Procedures.” Nef/f was immunoprecipitated with mAb M2, and co-precipitating Hck and PACS-2ha were detected by Western blot. Error bars represent mean and S.D. from three independent experiments.