CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor

Abstract

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

FIG. 1.

Recognition of sgp140 trimers from R5, X4, and R5X4 HIV-1 strains by the DP178 peptide. 293T cells were transfected with plasmids expressing sgp140 trimers of different HIV-1 strains and labeled with [³⁵S]methionine. (A) The sgp140 glycoproteins in the radiolabeled supernatant were precipitated by either PS from HIV-1-infected individuals (top) or the synthetic peptide DP178-C9 (bottom). (B) After SDS-PAGE, the proteins were quantified, and the percentages of the sgp140 trimers precipitated by DP178 are shown. In the protein names, -G represents sgp140-GCN4 trimers, and -F represents sgp140-fibritin trimers. The experiments were repeated with similar results.

FIG. 2.

Recognition of chimeric sgp140 trimers by the DP178 peptide. sgp140-GCN4 trimers from the HXBc2 or ADA HIV-1 strain or chimeric sgp140-GCN4 trimers with V1/2 or V3 loops derived from different coreceptor-specific strains were expressed in 293T cells, which were radiolabeled with [³⁵S]methionine. The cell supernatants were precipitated by either PS from HIV-1-infected individuals (top) or DP178-C9 (bottom) at 4°C overnight. The precipitates were analyzed by SDS-PAGE and autoradiography. HX(YU2V3) is the sgp140-GCN4 trimer of the HXBc2 HIV-1 strain with a V3 region derived from the YU2 strain. HX(YU2V1/2) is the sgp140-GCN4 trimer of the HXBc2 HIV-1 strain with the V1/V2 variable loops derived from the YU2 strain. ADA(MNV3/440) is the sgp140-GCN4 trimer of the ADA strain with residue 440 and the V3 region corresponding in sequence to that of the MN strain.

FIG. 3.

Precipitation of gp120 envelope glycoproteins (wild type or with variable loops deleted). The gp120 proteins were precipitated from the supernatants of [³⁵S]methionine-labeled 293T cells by either PS from HIV-1-infected individuals or DP178-Ig. The precipitations were performed in the absence (−) or presence (+) of 2 μg of sCD4/ml at room temperature for 3 h, followed by SDS-PAGE of the precipitated proteins and autoradiography. The experiment was repeated with similar results.

FIG. 4.

Effects of T-20 on antibody binding to HXBc2 gp120. (A and B) HXBc2 gp120 glycoproteins in the supernatant of transiently expressing 293T cells were captured on ELISA plates by a sheep antibody (D7324) against the carboxyl-terminal 15 amino acids of gp120. (A) Bound gp120 glycoproteins were incubated with various concentrations of T-20 peptide and the indicated concentrations of CD4-Ig at room temperature for 1 h. The bound CD4-Ig was detected with an HRP-conjugated anti-Ig secondary antibody and TMB substrates. (B) Bound gp120 glycoproteins were incubated with various concentrations of T-20 peptide in the absence (bottom right) or presence (+sCD4; 2 μg/ml) of sCD4 at room temperature for 1 h, followed by incubation with the indicated concentrations of antibodies at room temperature for another hour. The antibodies bound to gp120 were detected with an HRP-conjugated anti-Ig secondary antibody and TMB substrates. The means and standard deviations of the optical densities at 450 nm (O.D.₄₅₀) are shown. (C) Radiolabeled HXBc2 gp120 was incubated with sCD4, the indicated amounts of antibodies (Ab), and biotinylated DP178-Ig. Avidin-Sepharose was used for precipitation, and the gp120 bound to the beads was analyzed by SDS-PAGE and autoradiography.

FIG. 5.

Effects of T-20 on HXBc2 gp120 binding to CXCR4 PMPLs. CXCR4 PMPLs were incubated with purified HXBc2 gp120 in the absence (black peak) or presence (green and red peaks) of sCD4. Incubation with CXCR4 PMPLs was performed with no added peptide (A), with the addition of 1 μg of the T-20 peptide (B), or with the addition of 10 μg of the C9 peptide (C). The gp120 glycoproteins bound to the CXCR4 PMPLs were detected by FACS analysis, using the C11 antibody followed by a phycoerythrin (PE)-conjugated anti-human Ig secondary antibody.

FIG. 6.

Sequence requirements on the T-20/DP178 peptide for gp120 recognition. (A) Radiolabeled YU2 sgp130(−/GCN4) glycoproteins were incubated with PS from HIV-1-infected individuals, DP178-Ig, or C34-Ig and protein A-Sepharose. In parallel, radiolabeled HXBc2 gp120 glycoproteins, in the absence (−) or presence (+) of sCD4, were incubated with PS, DP178-Ig, or C34-Ig. The precipitated proteins were analyzed by SDS-PAGE and autoradiography. (B) Radiolabeled HXBc2 gp120 was incubated with DP178-Ig and protein A-Sepharose in the presence of the indicated concentrations of T-20 or scrT-20. The precipitated gp120 glycoprotein was analyzed by SDS-PAGE and autoradiography.

FIG. 7.

Effects of T-20 or scrT-20 on infection by HIV-1 with different envelope glycoproteins. Recombinant, luciferase-expressing HIV-1 containing the indicated envelope glycoproteins was incubated with target cells in the presence of various concentrations of either T-20 (A) or scrT-20 (B). For each virus, the luciferase activity in the target cells is normalized to the amount observed for the same virus in the absence of peptide. The results shown represent the means and standard deviations determined from two independent experiments.