Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by sequential binding to the primary receptor, CD4, and either of two coreceptors, CCR5 or CXCR4 (1
). CD4 binding to the HIV-1 gp120 exterior envelope glycoprotein results in a change in gp120 conformation that is favorable for CCR5 or CXCR4 binding (42
). Receptor binding is thought to trigger further conformational changes in the HIV-1 envelope glycoproteins, ultimately leading to fusion of the viral and cell membranes.
The binding sites for CD4 and the CCR5 or CXCR4 chemokine receptors on the HIV-1 gp120 glycoprotein are potential targets for intervention. The HIV-1 gp120 glycoprotein is composed of regions conserved among virus strains (C1 to C5) and regions that exhibit significant variation (V1 to V5). The binding site for CD4 has been visualized by x-ray crystallography and includes a highly conserved pocket on the gp120 surface (25
). The gp120 structures involved in chemokine receptor binding include the well-conserved β19 strand and the third variable (V3) loop, which governs chemokine receptor choice (2
). Most of the HIV-1 strains that are transmitted horizontally and that predominate in the first few years of infection utilize CCR5 as a coreceptor (28
). Thus, understanding gp120-CCR5 interaction may facilitate the development of effective therapies and vaccines.
Like all G protein-coupled receptors, CCR5 and CXCR4 are thought to span the membrane seven times. The CCR5 N terminus and second extracellular loop have been shown to be important for the ability of the receptor to support HIV-1 entry (15
). The CCR5 N terminus is electronegative; in addition to being rich in acidic residues, several of the tyrosines in this segment are sulfated posttranslationally (18
). The negatively charged tyrosine sulfates contribute to the efficiency of gp120 binding and HIV-1 entry (8
). Sulfated peptides corresponding in sequence to the CCR5 N terminus bind gp120 glycoproteins from CCR5-using (R5) HIV-1 strains after incubation with soluble CD4 (sCD4) (11
). Studies of gp120 mutants suggest that the binding of the CCR5 N terminus requires sequences in the β19 strand and the base of the V3 loop (12
). Other gp120 sequences near the tip of the V3 loop are thought to contribute to the ability of gp120 to interact with the body of the chemokine receptor (22
). Both sets of interaction are required for functional, high-affinity gp120-CCR5 binding leading to virus entry.
The binding sites for CD4 and chemokine receptor on the HIV-1 gp120 envelope glycoprotein serve as targets for neutralizing antibodies generated during natural infection. Some potent neutralizing antibodies, such as immunoglobulin G1b12, bind near the CD4-binding site of gp120 (38
). Other monoclonal antibodies derived from HIV-1-infected individuals recognize a conserved gp120 structure that is closely related to the chemokine receptor-binding site. The binding of these antibodies to gp120 is induced by CD4 binding; hence, they are designated CD4-induced (CD4i) antibodies (41
). CD4i antibodies block the binding of gp120-sCD4 complexes to the chemokine receptors (42
). CD4i antibodies exhibit various degrees of potency in neutralizing HIV-1. The neutralizing efficacy of CD4i antibodies is limited by steric constraints on antibody binding after the HIV-1 envelope glycoproteins have engaged the CD4 glycoprotein on the target cell surface (27
). Some CD4i antibodies have long complementarity-determining region 3 (CDR3) loops on their heavy chains (8
). These long CDR3 loops may allow CD4i antibodies to access conserved gp120 structures in the β19 strand and V3 base, bypassing the variable gp120 loops that protect these conserved elements. In some cases, sulfated tyrosine residues on the heavy-chain CDR3 loops contribute to the interaction of the CD4i antibody with HIV-1 gp120 (8
). Thus, several of the CD4i antibodies appear to mimic the CCR5 receptor.
One CD4i antibody, 412d, preferentially recognizes gp120 glycoproteins from CCR5-using HIV-1 strains (8
). When a number of gp120 glycoproteins from different HIV-1 variants were examined, a strong correlation was observed between 412d binding and the ability of the gp120 glycoprotein to use CCR5 as a second receptor. Thus, the 412d antibody appears to mimic CCR5 quite closely. Here, we characterize the gp120 epitope for the 412d antibody. To examine whether the structural similarity of the 412d antibody and CCR5 translates into functional similarity, we replaced the CCR5 N terminus by sequences of the 412d heavy-chain CDR3 loop. The ability of these chimeric receptors to support the entry of a variety of HIV-1 strains was tested.