We next assessed if there is any correlation between disruptions of envelope association with CD59+ compartment in primary CD4 + T cells and its association with DRM of the same cell type. The DRM assay was Figure 2 Envelope association with DRM in primary CD4 + T cells. A. CD4 + T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm 3 ) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39] . carried out essentially as described previously [8] . Briefly, primary CD4 + T cells infected with VSV-G pseudotyped pNL4.3 WT and pNL4.3 (L30E) were lyzed with cold 0.5% Triton-X-100 and fractionated through sucrose density gradient centrifugation in an ultracentrifuge (Beckman Coulter Inc) at 1,00,000 × g for 8-12 hours at 4°C. Ten equal fractions were collected and were examined for presence of gp160, CD59 and GM-1 ganglioside by Western blot using human anti-gp41 monoclonal antibody (2F5) [31] and 4E10 [33] , mouse anti-human CD59 (BD Biosciences Inc) and cholera toxin conjugated to horse radish peroxidase (HRP) (Sigma, Inc.) respectively. As shown in Figure 2 , L30E mutation in Gag restricted envelope association with DRM fractions of CD4 + T cells in contrast to the wild type. Our results were further substantiated by the presence of both CD59 and GM1 in DRM fractions under same experimental conditions.