Identification of V3 Loop-binding Proteins as Potential Receptors Implicated in the Binding of HIV Particles to CD4+Cells

doi:10.1074/jbc.273.34.21988

Identification of V3 Loop-binding Proteins as Potential Receptors Implicated in the Binding of HIV Particles to CD4⁺Cells*

From the ^‡Unité de Virologie et Immunologie Cellulaire, ERS 572 CNRS, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15 and ^§Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 15 rue Descartes, 67084 Strasbourg Cedex, France

Abstract

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4⁺ cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kψ(CH₂N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kψ(CH₂N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kψ(CH₂N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4⁺ cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

Footnotes

↵* This work was supported by grants from Institut Pasteur, Paris, CNRS, Agence Nationale de la Recherche sur le SIDA, and SIDACTION; by a grant from Agence Nationale de la Recherche sur le SIDA (to C. C.); by scholarships from SIDACTION (to N. B. and N. S.); by postdoctoral fellowships from Ministerio de Educacion y Ciencia (Spain) and Ministère des Affaires Etrangères (France) (to J. B.); and by a grant from Fondation Mondiale pour la Recherche et Prevention SIDA (France) (to E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed. Tel.: 33-1-4568-8776; Fax: 33-1-4061-3012; E-mail:arahovan{at}pasteur.fr.
↵1 The abbreviations used are: HIV, human immunodeficiency virus; TASP, template-assembled synthetic peptide; PHAP I and PHAP II, putative HLA class II-associated proteins I and II; gp120, the external envelope glycoprotein of HIV-1; V3 loop, the hypervariable region of about 36 amino acids in gp120; V3-BPs, V3 loop-binding proteins; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorting; AZT, azidothymidine; MMLV, Moloney murine leukemia virus; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; LTR, long terminal repeat; Fmoc,N-(9-fluorenyl)methoxycarbonyl.
↵2 A. G. Hovanessian and F. Puvion-Dutilleul, manuscript in preparation.
- Received March 23, 1998.
- Revision received June 4, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.