Figure 3.

All HIV Env variants are expressed at comparable levels in infected BHK-21 cells. BHK-21 cells were infected with VSV-GP, VSV-GP-gp140:G*, VSV-GP-gp160*, VSV-GP-gp140:G-linker, VSV-GP-gp140:G, VSV-GP-Δ147, VSV-GP-TM1 and VSV-GP-TM2 with a MOI of 0.1. Twenty-four hours after infection, cell lysates were prepared for western blotting (A) or cells were trypsinized and analyzed by flow cytometry (B). As a negative control, cell lysates from non-infected BHK-21 cells (Mock) were used. (A) The non-cleaved HIV Env precursor protein (gp160) and the cleaved gp41 protein were detected with an anti-HIV-1 gp41 monoclonal antibody (4E10, upper two blots). The non-cleaved Env precursor and the cleaved gp120 subunit were detected on a separate membrane using an anti-HIV-1 gp120 monoclonal antibody (16H3, third blot). As a loading control, an anti-actin antibody was used (lower blot). (B) Cells were stained with HIV bnAbs against the V1V2 loop of gp120 (PG9, PG16), the V3 loop of gp120 (PGT121), the CD4 binding site (b12, VRC01, 3BNC117, CH106) or the MPER of gp41 (4E10) and fluorescently-labeled anti-human secondary antibodies. As a loading control, the LCMV-GP-specific WEN4 was used. The ratio of the geometric mean fluorescence of every single HIV-specific antibody relative to the geometric mean fluorescence of the LCMV-specific WEN4 was calculated. The graph shows the mean ratio of duplicate samples for each antibody and virus.