Figure 6
From: In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis
LAINefP72A/P75A replicates in A3.01 T cells and is functional for CD4 downregulation. pLAI, pLAINeffs and pLAINefP72A/P75A proviral clones were transfected into 293T cells and virus harvested from the media. (A) Nef (α-Nef) and p24gag (α-p24) proteins were detected by Western blot analysis of 293T producer cell lysates. Control is non-transfected 293T cells. GAPDH (α-GAPDH) is a protein loading control. (B) A3.01 cells were infected with LAI and LAINefP72A/P75A at multiplicity of infection of 0.05 and viral production followed for 20 days with ELISA for p24gag. (C) LAI, LAINeffs and pLAINefP72A/P75A were titered using HeLa-MAGI indicator cells [82] and p24gag quantitated by ELISA. Infectivities were normalized to LAI (100%). (D) Nefs encoded by LAINefP72A/P75A and LAI were expressed in CEM cells following transduction with retroviral vectors (LXSN). CEM cells expressing LAI Nef and LAI NefP72A/P75A were analyzed by flow cytometry for cell surface CD4 and MHC Class I expression. LXSN is the negative control. Percentage of cells in each quadrant out of total cells indicated.