The potent human immunodeficiency virus type 1 (HIV-1) entry inhibitor HR212 blocks formation of the envelope glycoprotein gp41 six-helix bundle

Abstract

HR212, a recombinant protein composed of the heptad repeat, is a rationally designed human immunodeficiency virus type 1 (HIV-1) fusion inhibitor. This protein can be easily produced by Escherichia coli at a low cost. Previously, studies indicated that HR212 can efficiently inhibit the entry and replication of both laboratory and clinical HIV-1 strains, and this protein is more stable and less sensitive to proteinases than T20. The procedure of HIV-1 entry into the host cells can be divided into three main steps: gp120-CD4 interactions, coreceptor binding, and gp41 six-helix bundle formation and subsequent membrane fusion. The present study demonstrates that HR212 does not block gp120-CD4 binding or interfere with binding to the coreceptors CXCR4 and CCR5. Instead, HR212 efficiently blocks the six-helix bundle formation between peptides derived from the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) region of gp41. Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) indicated that HR212 could form a complex with peptide N36 to block gp41 fusogenic core formation. These results suggest that HR212 inhibits HIV-1 entry by targeting the NHR region of gp41. Therefore, HR212 can potentially be developed as a novel, high-efficiency, specific HIV-1 entry inhibitor.

FIG. 1.

(A) Cell–cell fusion assays examining the inhibitory effects of HR212 on HIV-1-mediated cell–cell fusion between H9/HTLV-III_B and MT-2 cells. (B) Inhibition of gp120 binding to CD4 by HR212. The CD4-based ELISA was carried out in the presence of HR212 (left bar) or the CD4 antibody (RPA-T4) (right bar). (C) Inhibition of the anti-CXCR4 mAb 12G5 binding to CXCR4. Inhibition of HR212 (20 μM, left bar) and the positive control AMD3100 (10 μM, right bar) on the binding of the anti-CXCR4 antibody 12G5 to U373-MAGI-CXCR4CEM cells was determined using a cell-based enzyme-linked immunosorbent assay (ELISA). (D) Inhibition of gp120–CD4 complex binding to CCR5. Inhibition of HR212 (20 μM, left bar) and the CCR5 antagonist maraviroc (1 μM, right bar) on the binding of the gp120–CD4 complex to cells expressing CCR5 was determined using a cell-based ELISA. Each sample was tested in triplicate in two independent experiments, and the data are presented as the means±the standard deviations.

FIG. 2.

HR212 inhibited the gp41 6-HB formation as determined by native polyacrylamide gel electrophoresis (N-PAGE) and ELISA. (A) The N36 or/and C34 peptides were incubated with or without HR212 at 37°C followed by N-PAGE analysis. (B) Dose-dependent inhibition of six-helix bundle (6-HB) formation by HR212 as assessed by N-PAGE. (C) 6-HB formation between N36 and C34-biotin was measured by ELISA. The C34-biotin with or without N36 at the specified concentrations was added to wells precoated with mAb NC-1 (0.8 μg/well). (D) Inhibition of the 6-HB formation by HR212 was analyzed by ELISA. The preincubated (37°C, 30 min) mixtures of the N36 peptide and HR212 in phosphate-buffered saline (PBS) at the specified concentrations were added to C34-biotin. The final concentration of peptides was 10 μM. The percentage of inhibition of 6-HB formation by HR212 was calculated as described in the experimental section.

FIG. 3.

CD spectra illustrating HR212 disruption of the α-helical conformation of the complex N and C peptides. (A) CD spectra for N36, C34, and their complex. (B) CD spectra for HR212 and the mixtures of N36 and C34 with or without HR212.

FIG. 4.

The interaction between HR212 and the N36-FITC and C34-FITC peptides was determined by FN-PAGE. The final concentrations of the peptides and HR212 were 10 μM. The gel was imaged using a Typhoon 9200 image scanner (Amersham, Sweden).