Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding.

Abstract

SummaryHuman immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and synrytium formation . Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain ofCD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and synrytium formation by a number of HIV isolates are blocked . These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion .C D4 is the high affinity cellular receptor for HIV and is found predominantly on thymus-derived T lymphocytes (1) . The structure predicted for CD4 is an extracellular region of four tandem domains having homology with members of the Ig superfamily, followed by a transmembrane domain and a cytoplasmic region (2, 3) . HIV binds CD4 via the major envelope glycoprotein, gp120, and recent genetic studies have localized the high affinity gp120 binding site on CD4 to -12 amino acids within the NH2-terminal, Ig-like (V1) domain (4-6). Similarly, residues have been defined in the COOH-terminal half of gp120 that are critical for CD4 binding (7,8). It is clear that the interaction between CD4+ human cells and cells expressing HIV envelope is sufficient to induce synrytium formation (9-11), and that virus entry is by pH-independent fusion ofthe virus and cell membranes (12, 13) . Little else is known, however, about the events subsequent to binding that lead to membrane fusion .Recently, evidence from two studies suggests that CD4 may play a role in cell-cell fusion (synrytium formation) additional to envelope binding . In the first study, single residue substitutions made in the CDR-3 analogous loop of the V1 domain of CD4 resulted in mutant molecules that were unable to facilitate synrytium formation, even though they mediated both virus binding and infectivity to an extent comparable with wild-type CD4 (14). In the second, chemical mutagenesis of a CD4+ T cell line resulted in CD4 molecules that bound gp120 but mediated synrytium formation poorly (15). Although the mechanism of inhibition of cell fusion was not elucidated in either study, the findings indicate that CD4 participates in events subsequent to gp120 binding that are required for membrane fusion. mAbs reactive with CD4/V1 are highly efficient at inhibiting the binding of gp120 to CD4, and thereby prevent infection and synrytium formation (6,(16)(17)(18)(19) . In contrast, a mAb reactive with the third or fourth CD4 domains has been shown not to inhibit gp120 binding (18,19) . To investigate whether regions of CD4 oth...

Analysis of the human immunodeficiency virus type 1 envelope protein interaction with the CD4 host cell receptor

“…These results indicate that if oligomerization of CD4 is a prerequisite for HIV infection a membrane anchor beyond the H/V-binding region of CD4 is required to allow the oligomerization to occur. Several studies suggest that CD4 may not only serve as a passive attachment site, but may also play an important role in the fusion process (Camerini & Seed, 1990;Celada et al, 1990;Healey et al, 1990). HIV infection is blocked in vitro by recombinant sCD4 (Deen et al, 1988;Fisher et al, 1988;Hussey et al, 1988).…”

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

“…The murine mAb against human CD4, Q4120 (IgG 1 ) (Healey et al, 1990) was obtained from Dr. Q. Sattentau through the Centralised Facility for AIDS Reagents at the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). The recombinant human mAb 2G12 (IgG 1 ) against HIV gp120 (Buchacher et al, 1994) was obtained from Dr. H. Katinger through the same program.…”

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

“…Antibodies used in this study were as follows: anti-CD4, Q4120 (Healey et al, 1990) was obtained from the AIDS Reagent Project of the United Kingdom Medical Research Council (Potters Bar, UK); anti-CXC chemokine receptor 4 (CXCR4), 12G5 (Endres et al, 1996) was provided by Dr. James Hoxie (University of Pennsylvania, Philadelphia, PA); anti-CC chemokine receptor 5 (CCR5), MC-5 (Signoret et al, 2000) was provided by Dr. Matthias Mack (Medizinische Poliklinik, Ludwig-Maximilians-University of Munich, Munich, Germany); anti-HA, 12CA5 was provided by Dr. David Drechsel (University College London, London, UK); rabbit anti-green fluorescent protein (GFP) was provided by Dr. David Shima (Imperial Cancer Research Fund, London, UK); anti-CD63, 1B5 was prepared in house (see below); anti-Lgp120, rabbit antibody against human LAMP1 was provided by Dr. Sven Carlsson (University of Umeå, Umeå, Sweden); anti-transferrin receptor H68.4 was purchased from Zymed Laboratories (San Francisco, CA); rabbit antibody against the ␥ subunit of AP-1 was provided by Dr. Margaret Robinson (University of Cambridge, Cambridge, UK); anti-vesicular stomatitis virus G-protein, P5D4 was provided by Dr. Thomas. Kreis (University of Geneva, Geneva, Switzerland); anti-HCMV-IE1, MAB810 (Chemicon, Temecula, CA); rabbit anti-mouse labeled with Alexa Flour-594 (Molecular Probes, Eugene, OR).…”