Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

Abstract

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.

FIGURE 1.

Schematic of CCR5 and ECL2 peptide constructs used in this study. A, the region of CCR5 corresponding to ECL2 is shown in red, and the helical N terminus is labeled and shown as a cylinder. B, amino acid sequences of the ECL2 peptide constructs predicted to reside in the extracellular region of ECL2. C, C-terminal ECL2 peptide sequences that contain additional residues predicted to reside in TM5.

FIGURE 2.

Relative IC₅₀ values of the extracellular ECL2 peptides (Fig. 1B) for inhibition of viral entry of R5, X4, and dual tropic HIV-1 strains. Numerical values are provided in Table 1, and representative inhibition curves are shown in supplemental Fig. S1. The legend for the HIV-1 strains used is shown in the inset in each graph. A dramatic reduction in activity can be seen readily for peptides 2N, 3, and 4.

FIGURE 3.

Histograms showing relative IC₅₀ values for 2C-TM5 peptides in neutralization (A) and cell-cell fusion (B) assays. The legend for the HIV-1 strains used is shown in the inset in A and B. Numerical values are provided in supplemental Tables S2 and S3.

FIGURE 4.

STD NMR ¹H spectra of peptide 2C in the presence of soluble gp120s. A, difference (DIFF) and reference (REF) spectra for 2C in the presence of R5 strain YU2. Expansions of the aromatic regions of 2C in complex with YU2 and X4 strain HXB2 are shown in B and C, respectively. Reference and difference spectra are colored black and red, respectively. Proton signals showing enhancements and therefore involved in gp120 binding are labeled. Weak enhancements from the β- and γ-protons of Gln and the β-protons of several of the aromatic amino acids appear as an envelope in the full spectra and are labeled collectively as Q^β/γ and Ar^β. Spectra were normalized to the Trp ϵ3/ζ2 signal. D, amino acid sequence of 2C with residues involved in binding displayed as large red letters.

FIGURE 5.

Two-dimensional ¹H-¹³C HSQC STD NMR spectra for 2C in the presence of soluble gp120 from YU2 (A) and HXB2 (B) HIV-1 strains. Cross-peaks are labeled according to Fig. 4C. Histograms showing the relative peak intensities (normalized to H^δ of Phe-189) for individual aromatic side chain protons involved in binding to gp120s from YU2 (C) and HXB2 (D) strains. Peak intensities were obtained by integrating peak volumes using the program PIPP (35) and correspond to cross-peaks within individual spectra. Peaks that remained overlapping in the two-dimensional spectra are labeled as pairs in A and B; and the average of their combined intensities is plotted in C and D and denoted with asterisks. Cross-peaks for His-181 and Phe-182 are labeled red in C and D to point out the changes in intensity when binding YU2 and HXB2. All spectra were recorded on solutions containing unlabeled, synthetic peptides.

FIGURE 6.

NOESY spectra of ECL2 peptide 2C. A, NOESY spectrum of free 2C. B, transferred NOESY spectrum of 2C in the presence of YU2 gp120. C, expansion of and assignments for cross-peaks in the NH-C^αH/C^βH region of the spectrum for 2C in complex with gp120. In spectra for the complex, only intraresidue (black labels) and sequential (i to (i+1)) (red labels) correlations were observed; no long range correlations were detected. Spectra were recorded on samples prepared under identical conditions (with concentrations of 800 μm peptide for free 2C, and 800 μm peptide/15 μm gp120 for the complex) and with identical acquisition parameters at 600 MHz; τ_m = 150 ms.