Abstract
The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.
Highlights
From the Departmentsof $Cell Sciences and §§Molecular Genetics,SmithKline Beecham Pharmaceuticals, Kingof Prussia, Pennsylvania 19406, VBecton Dickinson Monoclonal Center Inc.,S a n Jose, California 95231, the IIDepartment of Biochemistry and Molecular Biophysics and §§Howard Hughes Medical InstituteC, ollege of Physicians and Surgeons, Columbia University, New York, New York 10032, and the **Retroviral Immunology Group, Academic Departmentof Genito-Urinary Medicine, University College and Middlesex Schoolof Medicine, London S W l, United Kingdom
Attachment of human immunodeficiency ViNS (HIV)’ to cells is mediated through a high affinity interaction of the viral envelope glycoprotein gp120 with the cell surface recepantibodies which recognize distinctepitopes tor CD4 [1,2,3,4,5,6,7,8,9,10,11].CD4 isa 55-kDa transmembrane glycoprotein associated with these regions in D l
Within D l, a loop (C’C”) that is analogous to CDR2 of IgV, chains has been implicated as an important determinant for high affinity recognition of gp120
Summary
Cross-blockingof CD4 mAbBinding-HPB-ALL cells or peripheral blood mononuclear cells (IO6) were incubated with unlabeled mAb (fivetimes the titre pointf)or 20 min at 37 "C. The antibodies were incubated with soluble CD4 proteins for 30 min followed by the addition of SupTl cells. After a 30-min incubation, the cells were washed, stained with FITC-labeled goat anti-mouse antibody, washed again, fixed, and analyzed by flow cytometry. Following a further 30-min incubation and washing, 50 p1 of 1pg/ml biotin-labeled gp120 mAb (Dupont 9284, Ref. 28) was added. 5 X 10' CEM cells were incubated with or without antibody followedby the addition of FITC-labeled virus (final incubation volume was 200pl) for 30 min at 37 "C, washed, fixed with paraformaldehyde, and analyzed by flow cytometry. Supernatantswere inactivated by heating for 30 min at 56 "C in a 1%Empigen detergent, diluted 100-1000-fold into Tris-buffered saline-0.1% Empigen, and 100 p1 was added to 96-well microtitre plates coated with adsorbed sheep anti-p24 peptide polyclonal antibody D7320(Aalto BioReagents, Ireland).
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