FIGURE 2.

Nef markedly inhibited biochemical readouts of Gα_i2 activation. Nef inhibits chemoattractant-mediated calcium flux in Jurkat cells (A1) and the U937 cell line (A2). Cells were co-transfected with CD8 and Nef, co-expressers were purified by CD8-positive selection (Stem Cell Technologies), and Nef effect in Jurkat cells was evaluated by measuring cell surface CD4 expression. The time course of CXCL12 (10 nm)-initiated calcium flux profiles was obtained using FlexStation III and the recommended assay. Results are representative of four experiments. Agonist (CXCL12 followed by CCL2) dose (10 or 100 nm)-response profiles of intracellular calcium flux in U937 transfectants were analyzed by fluorescence ratiometry in a PTI fluorimeter (33). B1, time course of CXCL12-initiated Ca²⁺ flux in HeLa cells expressing CerFP or Nef-CerFP (red) was monitored by video capture at 30 frames/s of Fluo-4 emission (green) up to 150 s after CXCL12 addition. The arrows denote CerFP or Nef-CerFP cells (expressed at ∼10–15% efficiency around 12–16 h post-transfection) to highlight their difference in Fluo-4 intensity. Fluorescent data were collected from ∼10 ROIs for each field, the calcium flux was measured in >5 fields for each condition in an experiment, and the experiments were repeated three times (n = 50–60). The change in the intensities was analyzed using the Leica software followed by graphing using EXCEL. Ca²⁺ flux profiles of a few (to avoid clutter) representative cells (ROIs) expressing CerFP (left) or Nef-CerFP (right) are shown in B2, with the ordinate showing relative intensity of Fluo-4 emission. CXCL12-initiated Ca²⁺ flux is profiled in purified CEM cells co-transfected with CD8 and WT, null, or other Nef mutants. CEM cells were transfected with CD8, WT, null, or the indicated Nef mutants, and CD8(+) cells were purified prior to measurement of Ca²⁺ flux (as described above) (B3). Nef expression enhanced cAMP levels under basal conditions or after Gα_s activation by isoproterenol (C) or by forskolin stimulation of adenylyl cyclase (D). However, Nef did not further enhance the cAMP levels after isoproterenol treatment with transfectants overexpressing Gα_i3 (E). Jurkat cells were cotransfected with CD8 and Nef or vector (for C–E) and with a Gα_i3 expression plasmid (only for E). Transfected cells were purified by CD8 selection and assayed for cAMP production as described under “Experimental Procedures” (n = 4; error bars represent S.E.; *, p < 0.05).