Multifaceted Mechanisms of HIV-1 Entry Inhibition by Human α-Defensin

Lusine H Demirkhanyan,Mariana Marin,Sergi Padilla-Parra,Changyou Zhan,Kosuke Miyauchi,Maikha Jean-Baptiste,Gennadiy Novitskiy,Wuyuan Lu,Gregory B Melikyan

doi:10.1074/jbc.m112.375949

Abstract

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.

Highlights

Human neutrophil peptide 1 (HNP-1) inhibits human immunodeficiency virus type 1 (HIV-1) infection by a poorly defined mechanism
We examined the effect of human neutrophil peptides (HNPs)-1 on HIV-1 entry and fusion and found that, surprisingly, this ␣-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers
Defensin Inhibits HIV-1 Env-mediated Fusion—We examined the ability of the human neutrophil peptide 1 (HNP-1, hereafter referred to as defensin) to block HIV-1 fusion, using a direct virus-cell fusion assay based on the cytosolic delivery of the ␤-lactamase (BlaM) incorporated into the pseudovirus core [34, 48]

Summary

Background

Human neutrophil peptide 1 (HNP-1) inhibits HIV-1 infection by a poorly defined mechanism. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum This demonstrates that binding of ␣-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through crosslinking virus and/or host glycoproteins This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. Our experiments revealed the striking ability of HNP-1 to interfere with multiple steps of HIV-1 entry This defensin bound to Env glycoprotein, as well as to CD4 and likely to coreceptors, without inactivating the virus or compromising the cell viability. The lack of correlation between the defensin binding and inhibitory activity suggests that, in addition to the ability to engage multiple targets, the antiviral effect may require defensin oligomerization, which could be disrupted by serum

EXPERIMENTAL PROCEDURES

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DISCUSSION