θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

Stephen A Gallo,Wei Wang,Satinder S Rawat,Grace Jung,Alan J Waring,Alexander M Cole,Hong Lu,Xuxia Yan,Norelle L Daly,David J Craik,Shibo Jiang,Robert I Lehrer,Robert Blumenthal

doi:10.1074/jbc.m602422200

Abstract

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Highlights

Retrocyclin-1, a ␪-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry
We examined the fusion of CD4-independent 8x Env [39] with mouse fibroblast 3T3 target cells bearing CD4 and CXCR4, or bearing CXCR4 alone
When Fluorescence Recovery after Photobleaching (FRAP) experiments were performed to assess the effect of retrocyclin-1 on the lateral mobility of CD4 in medium that contained 10% fetal calf serum, we found the lateral mobility of CD4 to be intact (Table 1) and concluded that cross-linked surface barricades on the target membrane did not form under the experimental conditions used in our fusion experiments

Summary

Introduction

Retrocyclin-1, a ␪-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. Because the cross-linked barrier mechanism would not explain such selectivity, we examined the inhibitory effects of retrocyclin-1 on fusion mediated by HIV-1 Env. We found that retrocyclin-1 prevents HIV-1 entry by binding the C-heptad repeat of gp41 in a lectin-independent manner that prevents 6HB formation.

Results

Conclusion