Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions

Abstract

The HIV-1 envelope (Env) mediates viral entry into host cells. To enable the direct imaging of conformational dynamics within Env, we introduced fluorophores into variable regions of the glycoprotein gp120 subunit and measured single-molecule fluorescence resonance energy transfer within the context of native trimers on the surface of HIV-1 virions. Our observations revealed unliganded HIV-1 Env to be intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. The distinct properties of neutralization-sensitive and neutralization-resistant HIV-1 isolates support a dynamics-based mechanism of immune evasion and ligand recognition.

Fig. 1. Single-molecule FRET imaging indicates conformational dynamics of HIV-1 Env

(A) Experimental design. Native HIV-1 virions containing a single dually labeled Env molecule among native trimers were immobilized on the surface of passivated quartz microscope slides and imaged via TIRF microscopy. (B) Approximate position of the organic dyes in the variable loops V1 (green; Cy3B or Cy3) and V4 or V5 (red; Cy5(4S)COT or Alexa Fluor 647) of gp120 of HIV-1 Env. The Env structure, with the ribbon structure of the gp120 trimer, is adapted from PDB accession code 4NCO (–8, 11, 45). (C) Ribbon structures of the gp120 inner domain (cyan), outer domain (blue), and V1/V2 region (purple) are highlighted.

Fig. 2. Ligands remodel the conformational landscape of HIV-1 Env

(A) (Top left) Representative fluorescence (Cy3B, donor, green; Cy5(4S) COT, acceptor, red) and (bottom left) FRET (blue) trajectories obtained from a single HIV-1_NL4-3 Env on the surface of an intact virion. Idealization of FRET trajectories (red) was achieved by fitting each trace to a three-state Markov model (28). (Center) FRET trajectories from individual unliganded HIV-1_NL4-3 Env proteins were compiled into a population FRET histogram and fit to the sum of three Gaussian distributions (red) with means 0.1, 0.3, and 0.6 (black). (Right) TDPs displaying the relative frequencies of observed transitions were generated from the idealization of individual FRET trajectories. (B) (Left) Fluorescence and FRET trajectories for the unliganded HIV-1_JR-FL Env. (Center) FRET histogram fit to the sum of three Gaussian distributions with means 0.1, 0.3, and 0.65, and TDP. (C and D) Fluorescence and FRET trajectories, FRET histograms, and TDPs for (C) HIV-1_NL4-3 Env and (D) HIV-1_JR-FL Env in the presence of sCD4_D1D2 (5 μM). (E and F) The same data for (E) HIV-1_NL4-3 Env and (F) HIV-1_JR-FL Env in the presence of sCD4_D1D2 (5 μM) and 17b Fab (2 μM). The number of FRET trajectories and the number of FRET transitions are indicated on the histograms and TDPs, respectively. Histograms represent the means ± SEM determined from three independent populations of smFRET traces.

Fig. 3. Conformational masking of the co-receptor–binding site

(A and B) Population FRET histograms each composed of 100 to 150 smFRET trajectories for (A) HIV-1_NL4-3 Env and (B) HIV-1_JR-FL Env in presence of 17b Fab (2 μM) acquired after 0, 30, 60, and 90 min of incubation. All histograms are displayed as in Fig. 2. (C) Quantification of the occupancy in the intermediate-FRET state for (blue) HIV-1_NL4-3 Env and (red) HIV-1_JR-FL Env. (D) Corresponding neutralization of HIV-1_NL4-3 Env and HIV-1_JR-FL infectivity by 17b Fab. The data are presented as the means ± SEM determined from three independent measurements.

Fig. 4. Ground-state stabilization by broady neutralizing antibodies and an inhibitor

Population FRET histograms for (A) unliganded HIV-1_NL4-3 Env and in the presence of broadly neutralizing antibodies (B) VRC01, (C) PG16, (D) PGT145, (E) 2G12 (all at 0.7 μM), and (F) the small molecule BMS-626529 (100 μM).The same data for (G) unliganded HIV-1_JR-FL Env and in the presence of (H) VRC01, (I) PGT128, (J) 2G12, and (K) BMS-626529. Histograms are displayed as in Fig. 2.