Abstract
T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.
Highlights
HIV6 enters into a target cell in three steps: (i) human immunodeficiency virus (HIV) envelope glycoprotein (Env) surface subunit gp120 binds to CD4, the
They demonstrated that C34 could interact with N36, a peptide derived from the HIV gp41 N-terminal heptad repeat (NHR) region, to form a stable sixhelix bundle (6-HB), representing the gp41 core structure [9]
X-ray crystallographic studies have shown that the 6-HB core consists of three N36 molecules that form a trimeric core and three C34 peptides that bind to the grooves of N36 trimer in an anti-parallel manner [10]. These findings suggest that the binding of gp120 to CD4 and a coreceptor, CXCR4 or CCR5, triggers the conformational changes of gp41, which include the interaction between the gp41 C-terminal heptad repeat (CHR) and NHR to form a stable 6-HB core, bringing the viral and target cell membranes into close proximity. This viral fusion model has been employed for explaining the mechanism by which the CHR-peptides C34 and T20 inhibit HIV fusion, i.e. they interact with the viral NHR to form heterologous 6-HB and block the formation of the homologous 6-HB core between the viral NHR and CHR, thereby inhibiting HIV fusion
Summary
Reagents—MT-2, TZM-bl, and HIV-1IIIB-infected H9 (H9/ HIV-1IIIB) cells as well as T20-resistant HIV-1 strains were obtained from the NIH AIDS Research and Reference Reagent Program. 50 l of H9/HIV-1IIIB cells (2 ϫ 105/ ml), which were pre-labeled with Calcein AM, a fluorescent dye (Molecular Probes, Eugene, Oregon), were incubated with a 50-l peptide at a graded concentration at 37 °C for 30 min, followed by the addition of 100 l of MT-2 cells (1 ϫ 106/ml) in a 96-well plate. The mixture was added to the NC-1-coated plate, followed by incubation at room temperature for 30 min and washing with washing buffer (PBS containing 0.1% Tween 20) 6 times. Concentration in triplicate was incubated with an equal volume of an HIV-1 isolate at 0.01 multiplicity of infection at 37 °C for 30 min, followed by addition of the mixture to 100 l of TZM-bl cells (1 ϫ 105/ml) that were pre-cultured in a 96-well plate at 37 °C overnight. Measurement of Inhibitory Activity of Peptides on Infection of TZM-bl Cells by T20-resistant HIV-1 Strains—To compare the in vitro efficacy of C36 and T20 on infection by T20-resistant HIV-1 strains in TZM-bl cells [20], 50 l of a peptide at graded
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