Biochemical characterization of VLPs containing gp41 variants. (A) Incorporation of gp41 derivatives into VLPs. VLPs were produced as described above and analyzed in 10 to 50% sucrose gradients. 20 fractions were collected and loaded on a reducing SDS gel, respectively. gp41 variants were detected by anti-HA antibody and Pr55Gag by anti-p24 MAb 13/5 in subsequent Western Blot analysis. Co-banding of Gag (only shown for basal construct gp41CTM) and gp41 variants in fractions 11–15 indicates incorporation into virus-like particles. Higher molecular weight bands for GCN4- and H3-gp41CTM indicate stable dimeric and trimeric conformations. VLPs produced by transfection of Gag without gp41 were negative for anti-HA-antibody staining (data not shown). (B) Quantification of gp41 derivatives in VLPs. 0.25 μg of indicated lysed pseudotyped VLPs were loaded onto a slot blot, and stained with 4E10 and anti-human-HRP antibody. (C) Functional preservation of bnMAb epitopes on VLPs. VLP morphology and epitope preservation were verified by immuno-gold labeling and electron microscopy. VLPs were purified and incubated with human bnMAb 4E10 and gold-labelled anti-human antibody. VLPs pseudotyped with gp41CTM, GCN4-gp41CTM and H3-gp41CTM showed specific staining with 4E10. VLPs without pseudotyping exhibited no labeling (right).