FIG 4.

GBF1-independent recruitment of PI4KIIIβ by HRV2 3A does not rely on ACBD3. (A) Sensitivity of HRV2 and HRV14 to depletion of ACBD3 or PI4KIIIβ by siRNA treatment. HeLa R19 cells (2,000 cells) were reverse transfected with 2 pmol siRNA and 0.1 μl Lipofectamine 2000 mixed in 20 μl Opti-MEM in a 96-well format. Two days posttransfection, cells were infected with virus for 30 min, after which the medium was replaced with fresh medium. Samples were either immediately frozen to determine virus input levels (indicated with a dashed line) or incubated for 24 h. Significant differences compared to the results of treatment with Scramble siRNA were determined with a Student's t test (** = P < 0.01; *** = P < 0.001). (B) Western blot analysis of cell lysates confirming efficient knockdown by siRNA treatments. (C) HRV2 3A recruits PI4KIIIβ in ACBD3-depleted cells. HeLa R19 cells were reverse transfected with Scramble siRNA or siRNA against ACBD3 as described for panel A. After 48 h, cells were transfected with EGFP-tagged HRV2 3A. At 24 h after DNA transfection, cells were fixed and stained with antibodies against endogenous ACBD3 or PI4KIIIβ. (D) Interaction of HRV 3A proteins with PI4KIIIβ in the mammalian two-hybrid system. Bars show the means of the results from three samples with SD.