Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics

Abstract

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.

Fig 1.

Coreceptor use of NLHX and NLHX V3 chimeric Envs. Coreceptor use of Envs determined in a cell/cell fusion luciferase reporter assay, using QT6 effector cells expressing Env and QT6 target cells expressing indicated receptors (control; pcDNA3.1-transfected cells) (A), and by infection using luciferase reporter pseudotype viruses to infect U87 cells expressing indicated receptors (B). Results, presented from representative experiments, are expressed in relative light units (RLU) ± SD, determined on replicate wells.

Fig 2.

T-20 sensitivity of NLHX, NLHX^SF162-V3, NLHX^ADA-V3B, YU2, and YU2 K421D Envs. Relative T-20 sensitivities of Envs determined in a cell/cell fusion luciferase reporter assay, using QT6 effector cells expressing Env and QT6 target cells expressing CD4 and indicated coreceptors (A), and by infection using luciferase reporter pseudotype viruses to infect U87/CD4 cells expressing indicated coreceptors (B). Results are from a representative experiment ± SD determined on replicate wells.

Fig 3.

TAK-779 sensitivity of NLHX^SF162-V3 and NLHX^ADA-V3B Envs determined in a cell/cell fusion luciferase reporter assay, using QT6 effector cells expressing Env and QT6 target cells expressing CD4 and CCR5 (A), and by infection using luciferase reporter pseudotype viruses to infect U87/CD4/CCR5 cells (B). Results are representative of at least three experiments ± SD determined on replicate wells.

Fig 4.

Receptor binding efficiencies of NLHX, NLHX^SF162-V3, and NLHX^ADA-V3B Envs determined by immunostaining and flow cytometry analysis of gp120 binding to the surface of NP2/CD4 cells and T-REx/CCR5 cells. % gated, percentage of cells immunostained for bound Env. Dotted line, assay background. Results are representative of at least four experiments.

Fig 5.

Fusion kinetics of NLHX^SF162-V3 and NLHX^ADA-V3B Envs determined in a dye transfer content mixing assay using QT6 effector cells expressing Env and labeled with calcein AM and 3T3/CD4/CCR5 target cells labeled with CMTMR. Results represent the average fusion observed in five fields photographed at ×10 magnification. T-distribution tests showed the rate differences to be statistically significant (P < 0.05).

Fig 6.

Impact of CCR5 expression on the T-20 sensitivity of NLHX^SF162-V3 (A) and BaL (B) luciferase reporter pseudotype virus infection. (Insets) Absolute infection levels. RLU, relative light units.