Figure 3

LAINef fs Δ - 1 and LAINef fs Δ - 13 encode Nefs that are specifically defective for downregulating surface expression of CD4. (A) Nefs encoded by LAINeffs∆-1, LAINeffs∆-13 and LAI were expressed in CEM cells following transduction with retroviral vectors (LXSN). Upper Panel, A Western blot demonstrates LAI Neffs∆-1 and LAI Neffs∆-13 were expressed at comparable levels as wild type (α-Nef). LXSN and LXSNNeffs served as negative controls. GAPDH is a protein loading control (α-GAPDH). Lower Panel, CEM cells expressing LAI Nef, LAI Neffs, LAI Neffs∆-1 and LAI Neffs∆-13 were analyzed by flow cytometry for cell surface CD4 and MHC Class I (MHCI) expression. LXSNLAINef was the positive control. LXSN and LXSNLAINeffs were negative controls. Percentage of cells in each quadrant out of total cells is indicated. (B) Nefs encoded by LAI, LAINeffs∆-1 and LAINeffs∆-13 were expressed in transfected 293T cells. Control, 293T cells transfected with empty vector. Upper Panel, Lysates from transfected cells were analyzed by Western blot (α-Nef). Lower Panel, Total p21 activated protein kinase-2 (PAK2) in lysates of transfected cells lysates were immunoprecipitated with anti-PAK2 antiserum (α-PAK2) and analyzed by the in vitro kinase assay (IVKA). Control cells had no activated PAK2. Arrow, autophosphorylated PAK2. (C) pLAI, pLAINeffs, pLAINeffs∆-1 and pLAINeffs∆-13 proviral clones were transfected into 293T cells and virus harvested from the media. LAI, LAINeffs, LAINeffs∆-1 and LAINeffs∆-13 were titered using HeLa-MAGI indicator cells [82] and p24^gag contents were quantified by ELISA. Infectivities (blue cells per ng p24^gag) from six determinations of each virus were normalized relative to LAI (100%). Significant comparisons are indicated by lines and arrows above respective bars (*, p < 0.05). (D) A3.01 cells were infected with LAI, LAINeffs∆-1 and LAINeffs∆-13 at multiplicity of infection of 0.05 and viral production followed for 20 days with ELISA for p24^gag.