We first examined whether a point mutation (L30E) in matrix domain of Gag previously reported to abrogate envelope incorporation, infectivity [23] and DRM association [13] in cell lines affect infectivity and modulate envelope association with CD59-enriched compartment in primary CD4 + T cells which are predominantly the natural targets in vivo during entire course of HIV-1 infection. CD59 marker was selected as it is linked with phosphatidylinositol and segregates into rafts. Primary CD4 + T cells were purified by negative selection from whole blood using RosetteSep® Kit (Stem Cell Technologies Inc.) following manufacturer's protocol. Briefly, CD4 + T Cell Enrichment Cocktail was added at a final concentration of 50 μl/ml of whole blood and incubated at room temperature for 20 min. The mixture was diluted with RPMI/2% Fetal Bovine Serum (GIBCO, Inc), layered onto Ficoll Hypaque (Sigma, Inc) and centrifuged for 20 min at 1200 × g at room temperature. The enriched CD4 + T cells in the interface of density medium and plasma yielded approximately 98% purity; subsequently stimulated with phytohemagglutinin (0.5 ug/ml) and interleukin-2 (10-20 U/ml) before infection with virus stocks. Primary CD4 + T cells were infected with equal infectivity titers of VSV-G pseudotyped pNL4.3 WT and pNL4.3 L30E viruses and the cell lysates made from CD4 + T cells infected with wild type and L30E expressing equal amounts of p24 and gp41 were incubated with anti-gp41 monoclonal antibodies to immuno-precipitate Env. Briefly, CD4 + T cells were lyzed with 1% Triton X-100 in PBS (pH 7.4), and incubated with 1:1 mixture of gp41 monoclonal antibodies 2F5 and 4E10 (1:1000 dilution) for 4 hours at 4°C followed by additional 1 hour incubation with Protein A/G Figure 1 A. L30E Gag confers defective association of Env with CD59-enriched membranes in primary CD4 + T cells. Primary CD4 + T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were lyzed and intracellular p55, gp41 and CD59 contents were measured in total cell lysates (as shown here in upper panels). Cell lysates expressing comparable p55 and gp41 were immunoprecipitated with gp41 antibodies 2F5 and 4E10 as described in the Materials and Methods resolved in 12.5% SDS-PAGE and electrophoretically transferred onto PVDF membrane. The level of CD59 co-immunoprecipitated with envelope was detected by Western blotting using monoclonal anti-CD59 antibody. B. Defective incorporation of HIV-1 envelope proteins onto virus particles due to L30E Gag substitution. Equal amounts of cell free virus particles were resolved in SDS-PAGE and Western blotting done by monoclonal antibodies to gp41 and p24 as described in the text. Note that with L30E gag mutation, envelope incorporation onto virions is severely abrogated.