Figure 1.
Schematic models of the HIV-1 CTT.
A.) Traditional CTT model with one membrane-spanning α-helix and a completely intracytoplasmic localization of the remaining CTT sequence. LLP domains have been placed at their presumed membrane-localized position. B.) Alternative CTT model with multiple MSD segments as proposed by Hollier and Dimmock [15]. This model proposes three membrane-spanning β-sheets and an extracellular localization of the KE.
Figure 2.
Sequence alignment of variant Env CTT.
Sequence alignment of the membrane-spanning domain (MSD) and CTT of the Env proteins used in this study. All Env proteins were full-length gp160, however, only the sequences from K665 to the C-terminus are shown for simplicity. Structural and sequence domains are indicated in boxes above the corresponding sequence. VSV-G epitope tag indicated in bold; SAR1 and 1577 epitope indicated in gray box. KE – Kennedy epitope; LLP – lentivirus lytic peptide, MSD – membrane spanning domain.
Figure 3.
FACS analysis of intact Env-expressing cells demonstrates extracellular exposure of the KE sequence.
Cells transfected with HIV-1 89.6 WT, VSV-G-KE, or VSV-G-LLP2 were analyzed by FACS to determine VSV-G epitope accessibility to anti-VSV-G MAb. A.) Intact, non-permeabilized Env-expressing cells were stained with α-VSV-G (AlexaFluor (AF) 700) and α-actin (AF 488). B.) Intact cells expressing Env were stained for surface exposure of the KE using a native KE antibody, SAR1. C.) Same as (B) except cells were permeabilized prior to staining.
Figure 4.
VSV-G epitope insertions do not disrupt Env association with detergent-resistant membranes.
Env association with detergent resistant membranes was determined by sucrose gradient floatation followed by western blot analysis. Lane 1 represents the top of the gradient and lane 12 the bottom of the gradient. Blots were stained with anti-gp41 MAb Chessie 8 to determine the localization of gp41 in the bands shown.
Figure 5.
Anti-KE MAbs do not bind to intact virions.
The indicated proteins and viral particles were immunoprecipitated using reference MAbs coupled to protein G-coated paramagnetic beads. (Top) Open bars represent % of target antigen precipitated when incubated with solubilized virus, while closed bars represent the % of input p24 precipitated by the corresponding MAb under native (intact virus) conditions. # = p<0.05 for MAbs compared to IgG control with solubilized virus; * = p<0.05 for MAbs with intact virus compared to IgG control. (Bottom) Representative p24 bands immunoprecipitated using the MAbs indicated in top panel with intact virus (Intact) or the bands of the target antigen from each MAb in detergent-disrupted virus (Solublized).
Figure 6.
Comparison of MPER and KE MAb binding using SPR spectroscopy.
MAbs 2F5 (MPER) and SAR1 (KE) were compared for relative binding rates and affinity using SPR spectroscopy to monitor antibody binding to purified intact HIV-1 virions. RU – resonance units.