FIG 8.

Binding of 10E8-3R to native Env trimers on virions. (A) HIV-1 virions displaying Comb-mut Env were incubated with Fabs 10E8-WT or 10E8-3R, followed by treatment with mild detergent and the resolution of Env-Fab complexes using BN-PAGE Western blotting. Binding of an antibody to the Env trimer causes it to run more slowly on the gel and the cognate band to shift upwards on the blot. Estimation of the migration of a trimer with one, two, or three Fabs bound (left) is based on gel mobility shifts by control Fabs PGT126 (3 Fabs/trimer), PGT151 (2 Fabs), and PG9 (1 Fab). (B) BN-PAGE gel mobility shift analysis was performed as described above for panel A, except that after Fabs and virus were incubated together for 30 min, the virus was pelleted, and unbound Fab was removed. Thus, the gel mobility shift reflects only binding to the trimer while it was incorporated into the virion membrane. (C) Intensities of bands on BN-PAGE Western blots were quantified by using ImageJ software, and the percentage of the trimer bound by antibody was calculated by comparing the intensities of the unliganded trimer band in the presence and absence of Fab. The results shown are the averages of data from four (standard assay) and two (washout) independent BN-PAGE Western blot assays.