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J. Biol. Chem., Vol. 281, Issue 51, 39598-39606, December 22, 2006

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

Maier Lorizate¹, Antonio Cruz², Nerea Huarte, Renate Kunert^¶, Jesús Pérez-Gil², and José L. Nieva³

From the Biophysics Unit (Consejo Superior de Investigaciones Científicas-UPV/EHU) and Biochemistry Department, University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain, ^¶Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria, and Department of Biochemistry and Molecular Biology I, Faculty of Biology, Universidad Complutense, 28040 Madrid, Spain

The conserved ⁶⁶⁴DKWASLWNWFNITNWLWYIK⁶⁸³ (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.

Received for publication, June 22, 2006 , and in revised form, October 6, 2006.

^* This study was supported in part by the Spanish MCyT Grants BFU2005-06095/BMC (to J. L. N.) and BIO2006-03130 (to J. P.-G.) and by University of the Basque Country Grant 042.310-13552. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a pre-doctoral fellowship of the Basque Government.

² Supported by Community of Madrid Grant CAM-S-505-MAT-283 and FP6 of European Union Grant MEST-CT-2004-007931.

³ To whom correspondence should be addressed: Biophysics Unit (CSIC-UPV/EHU), University of the Basque Country, P. O. Box 644, 48080 Bilbao, Spain. Tel.: 34-94-6013353; Fax: 34-94--6013360; E-mail: gbpniesj{at}lg.ehu.es.

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