Figure 4.

MPER antibodies induce gp120 shedding from virions and HIV envelope–expressing cells. (A) To analyze gp120 shedding by size-exclusion chromatography, JR-FL pseudovirus was treated with 25 µg/ml of CD4-IgG2, b12, 2G12, 2F5, 4E10, or total human IgG (huIgG) for 20–25 h. Samples were then separated on a Sephacryl S-1000 column, and collected fractions were analyzed for virion content and viral infectivity (relative light units [RLU] luciferase reporter production; relative light units are depicted in units of 3 × 10³). One of three independent experiments is shown. (B) Shedding of gp120 from envelope-expressing cells was detected by Western blot analysis. 293-T cells expressing ΔCT JR-FL gp160 were treated with 10 µg/ml CD4-IgG2, 50 µg/ml 2G12, 50 µg/ml 2F5, or 50 µg/ml human IgG as control for the indicated time periods to allow for shedding. After cell lysis, total protein was separated by SDS-PAGE and analyzed by Western blotting for gp120 content. gp120 content was quantified by densitometric analysis, and shedding induced by neutralizing mAbs was expressed in relation to human IgG control. One of three independent experiments is shown. (C) 293-T cells that were mock transfected or transfected with ΔCT JR-FL gp160 were treated with 10 µg/ml CD4-IgG2, 50 µg/ml 2F5, or 50 µg/ml human IgG as control for 20 h to allow for shedding. Cell surface–associated gp120 was detected by flow cytometry upon staining with biotinylated 2G12 and streptavidin-APC. One of three independent experiments is shown. (D) Induction of gp120 shedding from PBMC-derived, replication-competent HIV isolates was analyzed using PBMC-derived viruses JR-FL, DH123, and NL4-3. Coreceptor usage of viruses is indicated. Viruses were incubated with 10 µg/ml CD4-IgG2, 100 µg/ml 2F5, and 100 µg/ml 4E10 or left untreated for 24 h (mock control). The extent of gp120 shedding in mAb-treated samples was analyzed as described in Fig. 3 B. Data are expressed in relation to the mock control. Mean and SEM of three independent experiments are depicted.