beads (Pierce Inc). Lysates were further washed with cold PBS and were resolved in 12% SDS-PAGE under denaturing conditions. Equal amounts of immunoprecipitated materials were subjected to 12% SDS-PAGE under denaturing conditions [32] , transferred onto PVDF membranes and subsequently Western blot assay done with anti-human CD59 antibody (at 1: 1000 dilution) to assess the ability of Env association with CD59enriched compartment. As shown in Figure 1A , due to L30E mutation in Gag, Env failed to recruit CD59 in contrast to pNL4.3 wild type. Our data indicate that the mutation in Gag MA region (L30E) restricts the interaction between Gag and Env resulting in down modulation of envelope trafficking to GPI-anchored membranes in primary CD4 + T cells such as CD59. Moreover, in order to assess the effect of L30E mutation in p17 gag on HIV-1 envelope incorporation on virions in primary CD4 + T cells, cell-free virus pellets were obtained by centrifugation as described previously [13] . Equal amounts of virus particles (p24) were resolved in SDS-PAGE under denaturing conditions followed by Western blotting using monoclonal antibodies to p24 (183-H12-5C) and gp41 (1:1 of 2F5 and 4E10). As shown in Figure  1B , L30E substitution in p17 Gag was found to drastically affect envelope incorporation onto virus particles in CD4 + T cells as expected.