Novel recombinant engineered gp41 N-terminal heptad repeat trimers and their potential as anti-HIV-1 therapeutics or microbicides

Abstract

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

FIGURE 1.

The HIV-1 gp41 functional domains and N-peptides. A, schematic view of gp41 functional domains. The residue number corresponds to its position in HIV-1_HXB2 gp160. FP, fusion peptide; TR, tryptophan-rich region; TM, transmembrane domain; CP, cytoplasmic domain. B, interactions between the NHR and CHR of gp41 and between N- and C-peptides. The dashed lines between NHR and CHR indicate the interaction between the residues located at the e and g positions in the NHR and the a and d positions in the CHR. C, the sequences of N36Fd and N28Fd. The NHR and CHR sequences are highlighted in blue and orange, respectively. The pocket-forming sequence (aa 565–581) in the NHR and pocket-binding domain (aa 628–635) in the CHR are colored in red and green, respectively. The Fd sequence is colored in purple.

FIGURE 2.

SDS-PAGE analysis of N36Fd (A) and N28Fd (B). The samples were boiled for 5 min or kept at room temperature in the presence of 2% SDS in 1× SDS sample buffer (Novagen) prior to loading. Arrows indicate the positions of monomers and trimers. M = protein marker.

FIGURE 3.

Sedimentation velocity analysis of N28Fd and the N28Fd/C34 mixture (A), and N36Fd and the N36Fd/C34 mixture (B). The buffer was 100 mm CH₃COONa/CH₃COOH buffer (pH 6.0) for N28Fd and N36Fd alone, and PBS (pH 7.4) for the mixtures. The sedimentation coefficient (s) and molecular mass (kilodaltons) of each peak are indicted.

FIGURE 4.

Secondary structure of the peptides N36 and N36Fd (A), and N28 and N28Fd (B), as determined by CD spectroscopy. The spectra of (N36Fd)-Fd (A) or (N28Fd)-Fd (B) were calculated by subtracting the spectra of Fd peptide (A and B) from those of N36Fd (A) or N28Fd (B), respectively. The final concentration of each peptide in water was 10 μm.

FIGURE 5.

CD spectroscopic analysis of the helical bundle formed between C34 and N36 or N36Fd (A), and between C34 and N28 or N28Fd (B). The spectra of (N36Fd+C34)-Fd (A) or (N28Fd+C34)-Fd (B) were calculated by subtracting the spectra of Fd peptide from those of N36Fd+C34 or N28Fd+C34, respectively. The final concentration of each peptide in PBS was 10 μm.

FIGURE 6.

Inhibition of HIV-1-mediated cell-cell fusion by N-peptides and N-peptide trimers. The C-peptides T20, T1249, and T1144, and Fd peptide were included as controls. The IC₅₀ value of each peptide is shown in the figure.

FIGURE 7.

Inhibitory activities of N-peptide trimers on infection by HIV-1 strains IIIB (A), Bal (B), and 93IN101 (C). The inhibitory activities of N36Fd and N28Fd against infection by the HIV-1 strains IIIB (subtype B, X4) in MT cells, Bal (subtype B, R5) in TZM-bl cells and 93IN101 (subtype C, R5) in peripheral blood mononuclear cells were determined by ELISA or luciferase assay as described under “Experimental Procedures.” The IC₅₀ value of each peptide is shown in the figure.

FIGURE 8.

Proteinase K sensitivity of N28Fd. After digestion by proteinase K for different times, the residual amount of N28Fd or T20 was detected by a direct ELISA (top panel), and the remaining antiviral potency of N28Fd or T20 was tested by inhibition of HIV-1_IIIB assay (bottom panel).