Recognition and Blocking of HIV-1 gp41 Pre-transmembrane Sequence by Monoclonal 4E10 Antibody in a Raft-like Membrane Environment

José L Nieva,Antonio Cruz,Renate Kunert,Nerea Huarte,Jesús Pérez-Gil,Maier Lorizate

doi:10.1074/jbc.m605998200

Abstract

The conserved (664)DKWASLWNWFNITNWLWYIK(683) (preTM) sequence preceding the transmembrane anchor of human immunodeficiency virus (HIV-1) gp41 glycoprotein subunit is accessible to the broadly neutralizing 4E10 antibody and, therefore, constitutes a potential target for vaccine design. Recently reported structural data are compatible with preTM insertion into the viral external membrane monolayer in the gp41 pre-fusion state (Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J. D., Grisé, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847-852). Here we demonstrate that the broadly neutralizing 4E10 antibody is able to specifically block the membrane-restructuring activity of a peptide mimic inserted into membranes. Recognition and restructuring blocking occurred in the presence of cholesterol, whereas transmembrane versions as those promoted in 1-palmitoyl-2-oleoylphosphatidylcholine:sphingomyelin mixtures could not be effectively arrested. Spectrofluorimetric assays using rhodamine-labeled peptides revealed that recognition correlated better with pore-formation blocking than with membrane-fusion inhibition. The capacity of the antibody to recognize preTM peptides in a raft-like environment was further corroborated employing planar-supported lipid layers and fluorescence microscopy. These data support that membrane-bound epitope recognition by 4E10 results in clustering reorganization of preTM at the membrane interface. We propose that this process might interfere with the formation of fusion-competent complexes at the low spike densities existing in the HIV-1 membrane. This work comprises the first experimental report on a lipid-modulated antibody capacity to bind a membrane-bound epitope sequence and arrest its restructuring activity.

Highlights

Three broadly neutralizing anti-HIV-14 monoclonal antibodies, 2F5, 4E10, and Z13, have been identified to react with the membrane-integral gp41 Env subunit that promotes viral-cell fusion
HIV-1 acquires its membrane by budding from laterally segregated specific cell membrane platforms or “rafts” enriched in Chol and SPM (18 – 20)
Previous work by our group had shown that aromatic-rich gp41 preTM might exist as a membrane interface-residing sequence whose aggregation state and topology were modulated by those lipids [3]

Summary

Introduction

Three broadly neutralizing anti-HIV-14 monoclonal antibodies (mAbs), 2F5, 4E10, and Z13, have been identified to react with the membrane-integral gp Env subunit that promotes viral-cell fusion (for a recent review, see Ref. 1) These three antibodies recognize epitopes within the conserved aromatic-rich domain that precede the gp transmembrane anchor [2,3,4,5,6,7]. The epitope core 671NWF(D/N)IT676 recognized by mAb4E10 locates at the center of the gp41 664DKWASLWNWFNITNWLWYIK683 sequence This sequence has been defined as a distinct domain according to its interfacial hydrophobicity (designated as pre-transmembrane (preTM)) [2,3,4] and attending to its implication in fusion and neutralization processes (designated as a membrane-proximal external region) [1, 5,6,7]. These observations might be important for the design of immunogens aimed at recovering 4E10-like protective responses

Methods

Results

Conclusion