Figure 2

M48U1 induces gp120 shedding. A) “Trimer VLPs” (E168K+N189A mutant) expressing trimeric Env of the subtype B virus JR-FL were treated with graded doses of M48U1 or sCD4. Shedding was then assessed using BN-PAGE-Western blot analysis of VLP Env. Below the gel, the density of trimeric gp41 stumps (indicated by an arrow on the gel) is shown, as determined using ImageJ densitometry software (NIH, Bethesda, USA, http://imagej.nih.gov/ij/). Second, we coated the same trimer VLPs of JR-FL B) or the subtype B transmitted/founder viruses REJO and WITO (C) on an ELISA plate at 20x the concentration present in transfection supernatants, treated then with graded doses of M48U1 or sCD4, as indicated and then measured ELISA binding by mAb 7B2 that reacts with a cryptic epitope of gp41 that only becomes exposed upon the loss of gp120 from native trimers (i.e. gp120 shedding). D) Finally, virus particles were isolated with magnetic CD44 microbeads from PBMC culture supernatant at 0 h, 24 h and 72 h after M48U1 exposure. Gp120 was then quantified in virion and virion-free fractions using a D7324-based gp120-ELISA. Shedding was expressed as a ratio of free gp120 in the supernatant and intact Env in the virion fraction and compared to the untreated control cultures (i.e., medium). Values are the means +/− SEM of two independent measurements. E) Env incorporation in the virion fraction was determined by quantifying both the gp120 and p24 content in ELISA and plotting the gp120/p24 ratio.