Nef expression increases p-Erk1/2 induction in infected and transduced primary T cells. (A) Representative micrographs of uninfected (mock) primary human T lymphocytes or after infection with the indicated HIV-1 IRES.GFP reporter viruses (infected cells in green) in conjugates with SEE-pulsed Raji B cells (in blue) after staining for p-Erk1/2 (in red). Arrows indicate infected cells in B-cell/T-cell conjugates. Note the higher level of p-Erk1/2 in the HIV-1 WT–infected cell in conjugate with a B cell compared with the ΔNef–infected cell and uninfected cells. Scale bar indicates 10 μm. (B) As in panel A, the frequencies of primary human T lymphocytes from 2 different donors with elevated p-Erk1/2 levels were analyzed. (C) Total per-cell levels of p-Erk1/2 in primary human T lymphocytes infected with WT or ΔNef HIV-1. Values for individual cells are presented, with the arithmetic mean indicated by the black line relative to uninfected control cells in conjugates. Asterisks indicate statistical significance by Student t test analysis. ***P ≤ .001; **P < .005; *P < .05. (D) Primary CD4+ T lymphocytes were lentivirally transduced for expression of GFP or Nef.GFP, stimulated with anti–CD3 Ab for the indicated times 48 hours after transduction, and cell lysates were analyzed by Western blotting for the detection of total (Erk1/2), active (p-Erk1/2) Erk, or active Lck (p-Lck394). Expression of GFP and Nef.GFP was analyzed with an anti-GFP Ab. (E) Quantification of p-Erk1/2 levels in the Western blot shown in panel D. The intensities of the bands were analyzed using Quantity One Version 4.6.5 software and are plotted as the ratio of p-Erk1/2 to total Erk1/2 levels. The value for the GFP control at t0 was arbitrarily set to 1, and all other values are plotted relative to this.
