Asymmetric Deactivation of HIV-1 gp41 following Fusion Inhibitor Binding
Figure 6
Overlap of the C37- and 5-Helix-sensitive intermediate states.
(A) C37W628A inhibitory activity against HIV-1 by standard assay (squares) or in a 5-Helix-washout assay after Env was first trapped in the 5-HelixL556A/V570A-bound state (circles). In the standard assay, C37W628A was coincubated with virus and cells at the beginning of infection. In the 5-Helix-washout assay, target cells preincubated with HIV-1 and 5-HelixL556A/V570A were washed with media containing C37W628A. (B) 5-HelixL556A/V570A inhibitory activity against HIV-1 by standard assay (squares) or in a C37-washout assay after Env was first trapped in the C37W628A-bound state (circles). (C) Antiviral activity of C37N656D in the presence of 30 nM 5-HelixV549E. The individual IC50 values for C37N656D and 5-HelixV549E were determined to be 130±10 nM and 54±2 nM, respectively. The solid line denotes the titration expected if C37 and 5-Helix target separate intermediate states (additive inhibition). The dotted line denotes the titration expected if C37 and 5-Helix could bind simultaneously to the same intermediate state (synergistic inhibition). These different binding scenarios are depicted schematically in Figure S7, and their quantitative evaluations are presented in Text S1. (D) Concentration dependence to synergistic inhibitory activity. Combination indices were calculated following the method of Chou and Talalay [49] for inhibition experiments performed with C37N656D and either 10 nM (circles) or 30 nM (squares) 5-HelixV549E. A diminishing combination index below unity indicates increasing synergistic activity.