Fig 1.
Infectivity, cell-cell fusion, processing and subunit association of Env variants.
(A). The ability of the indicated HIV-1BG505 Env variants to mediate cell-cell fusion was evaluated as described in Materials and Methods. The values shown are normalized to that obtained for wt Env. (B, C). Cell lysates and supernatants from 35S-labeled cells transiently expressing the indicated HIV-1BG505 Env variants were precipitated with a mixture of sera from HIV-1-infected individuals. The precipitated proteins were loaded onto SDS-polyacrylamide gels and analyzed by autoradiography and densitometry to calculate their processing (B) and association (C) indices, as described in Materials and Methods. Means and SEM derived from at least three independent experiments are shown. The results were compared with a paired t-test, and the degree of significance indicated: ***—p < 0.001, **—p < 0.01, *—p < 0.05 and ns—not significant.
Table 1.
Phenotypes of HIV-1BG505 ΔCT mutants.
Fig 2.
Sensitivity of Env variants to inhibition by sCD4, VRC01 and exposure to cold.
Normalized amounts of recombinant luciferase-expressing HIV-1 pseudotyped with the wt and S375W HIV-1BG505 Envs were incubated at 37°C with increasing concentrations (0–200 nM) of sCD4 (A) or VRC01 (B) for 1 hr prior to addition of Cf2Th-CD4/CCR5 cells. In (C), recombinant HIV-1 bearing wt or S375W Envs was incubated on ice for different amounts of time [89]. At the indicated time points, aliquots were removed and frozen at -80°C. After completion of the longest incubation, all samples were thawed and infectivity on Cf2Th-CD4/CCR5 cells was measured. Data are representative of results obtained from at least three independent experiments, performed in quadruplicate.
Fig 3.
Effect of a reducing agent on the ability of Env variants to mediate infection and cell-cell fusion.
(A). Normalized amounts of recombinant luciferase-expressing HIV-1 pseudotyped with the wt and mutant HIV-1BG505 ΔCT Envs were spin-inoculated onto Cf2Th-CD4/CCR5 cells before adding increasing concentrations of DTT (0–5 mM). (B). The ability of the indicated Env variants to mediate cell-cell fusion in the presence of increasing concentrations of DTT was evaluated. The data shown are from a single experiment and are representative of results from at least three independent experiments, performed in quadruplicate.
Fig 4.
Effect of soluble CD4 on MAb binding to HIV-1BG505 Env variants.
The binding of 17b (A), A32 (B) and PGT151 (C) MAbs to the indicated HIV-1BG505 ΔCT Env variants expressed on the cell surface, in the absence and presence of 80 nM sCD4, is shown. The cell-surface expression of the four Env mutants, as determined by the binding of the 2G12 antibody, was comparable and varied no more than 20% from the level of the wt Env. Each signal was normalized for the relative level of Env expression, using the binding of the 2G12 antibody. Means and SEM derived from at least five independent experiments performed in quadruplicate are shown. Statistical significance is indicated, as described in the Fig 1 legend.
Table 2.
Characterization of ligand binding to selected HIV-1BG505 variants by cell-based ELISA.a
Fig 5.
Binding of MAbs and sera from HIV-1-infected individuals to Env variants.
The binding of VRC01 (A), b12 (B), CD4-Ig (C), 4e10 (D), F240 (E), 7b2 (F), 2.2B (G) or sera from HIV-1-infected individuals (patient sera, PS#1) (H) and PS#2 (I) to HIV-1BG505 ΔCT Env variants expressed on the cell surface is shown. Means and SEM derived from at least five independent experiments performed in quadruplicate are shown. Statistical significance is indicated, as described in the Fig 1 legend.
Fig 6.
MAb binding to HIV-1BG505 Env variants on the cell surface.
The binding of PGT121 (A) and PG9 (B and C) to HIV-1BG505 Env ΔCT variants expressed on the cell surface was measured by cell-based ELISA. The data in (C) were generated in the presence of 80 nM sCD4. The means and SEM derived from at least three independent experiments performed in quadruplicate are shown. Statistical significance was assessed using a paired t-test, and is indicated as described in the legend to Fig 1 of the main text.
Fig 7.
Anti-V3 MAb binding to HIV-1BG505 Env variants on the cell surface.
The binding of the indicated MAbs to HIV-1BG505 Env ΔCT variants expressed on the cell surface was measured by cell-based ELISA. The means and SEM derived from at least three independent experiments performed in quadruplicate are shown. Statistical significance was assessed using a paired t-test, and is indicated as described in the legend to Fig 1 of the main text.
Fig 8.
Ligand binding to HIV-1BG505 Env variants.
The binding of each of the indicated ligands to the HIV-1BG505 ΔCT Env variants expressed on the cell surface was measured by the cell-based ELISA, and is plotted relative to the binding of the 2G12 antibody. In some cases, ligand binding was measured in the presence of 80 nM sCD4. The ligands are arranged according to the relative binding to wt Env in the absence of sCD4.
Fig 9.
Ligand binding to HIV-1BG505 full-length wild-type and I559P Env variants.
The binding of wild-type (wt) and I559P HIV-1BG505 Env full-length (gp160) variants expressed on the cell surface was measured by cell-based ELISA, and is plotted relative to the binding of the 2G12 antibody. The ligands are arranged according to the relative binding to wt Env. The means and SEM derived from four independent experiments performed in quadruplicate are shown. Statistical significance was assessed using a paired t-test, and the degree of significance is indicated: ***—p < 0.001, **—p < 0.01, *—p < 0.05 and ns—not significant.
Fig 10.
Insights into the I559P phenotypes from the structure of the soluble gp140 SOSIP.664 Env trimer.
(A). The ribbon structure (PDB 4TVP) of one protomer of the HIV-1BG505 soluble gp140 SOSIP.664 Env trimer [38] is shown, oriented with gp41 at the top of the figure. The gp41 subunit is colored green, the gp120 outer domain is colored blue, and the gp120 inner domain and N- and C-termini are colored orange. The approximate location of isoleucine 559 within the disordered gp41 segment (residues 548–568) (dotted line) is indicated by an asterisk. The gp41 α7 helix forms a coiled coil at the trimer axis. The SOS disulfide bond linking gp120 residue 501 and gp41 residue 605 is labeled. The F240 Cluster I gp41 epitope is highlighted in magenta, and is buried near the trimer axis in the soluble gp140 SOSIP.664 structure. The gp120 core is in the CD4-bound conformation, except for the V1/V2 stem, which is not shown. The β-sandwich of the gp120 inner domain, which is involved in the non-covalent association of gp120 and gp41 [90], loosely interacts with the gp41 fusion peptide in this structure. (B). The soluble SOSIP.664 Env trimer structure [38] was fitted as a rigid body into the tomogram of the HIV-1 virion Env trimer (grey) (EMD 5019) [40], using UCSF Chimera software [91–93]. The soluble gp140 SOSIP.664 ribbon structure is colored as in A and, in addition, the structure of the gp120 V1/V2 and V3 variable regions is shown in red. In the upper panel, the Env spike is oriented such that the viral membrane is at the top of the image. The gp41 disordered region (residues 548–568), including isoleucine 559, and the α7 helix of the soluble gp140 SOSIP.664 structure are out of the virion Env density, as is the α0 helix of the gp120 inner domain. The lower panel shows a view from the perspective of the target cell. Note the density in the gp120 arms of the virion Env tomograms that is not explained by the soluble gp140 SOSIP.664 structure. The approximate angle of approach used by the CD4-binding site (CD4BS) antibodies to engage the HIV-1 Env trimer is indicated by the red arrow. Because these antibodies must access their epitopes by bypassing the adjacent gp120 protomer, their binding is potentially sensitive to changes in the orientation of the gp120 subunits in the quaternary Env trimer structure [42,81].