Mutagenic Stabilization and/or Disruption of a CD4-Bound State Reveals Distinct Conformations of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein

Abstract

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.Over 35 million people are currently infected with human immunodeficiency virus type 1 (HIV-1), the major cause of AIDS (4, 24). The development of a preventive vaccine, which optimally should elicit both virus-neutralizing antibodies and cellular immune responses, is of high priority and urgency (25,32).Neutralizing antibodies must bind the HIV-1 envelope glycoproteins, which mediate the entry of the virus into the target cell (97). The trimeric envelope glycoprotein complex is anchored in the host cell or viral membrane by the gp41 transmembrane glycoprotein, which is noncovalently attached to the gp120 exterior envelope glycoprotein. Most of the surfaceexposed elements of the trimeric envelope glycoprotein complex are contained on the gp120 exterior envelope glycoprotein (52). Comparison of the gp120 glycoproteins from different HIV-1 strains reveals regions of conservation interrupted by long regions of variability (V1 to V5) (46, 80). Intramolecular disulfide bonds in the gp120 glycoprotein result in the incorporation of the first four variable regions (V1 to V4) into large, surface-exposed loops (43, 52). The conserved gp120 regions fold into a core, which contains many of the gp120 elements important for receptor binding (6,65,98).CD4 and the chemokine receptors CCR5 and CXCR4 serve as HIV-1 receptors (14, 17-19, 22, 31). The binding of the HIV-1 gp120 glycoprotein to CD4 contributes to the attachme...

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

“…It has been shown that the replacement of serine 375 by a histidine or tryptophan residue fills the Phe 43 cavity and predisposes Env to assuming a slightly more CD4-bound-like conformation (9). Moreover, it has been shown that sampling of the CD4-bound conformation results in an enhanced exposure of epitopes recognized by anti-cluster A antibodies (14,34).…”

“…It has been shown that some of these conformational rearrangements could be impacted by large alterations in the Phe 43 cavity. The replacement of the well-conserved group M serine at position 375 by a large hydrophobic residue such as tryptophan fills the Phe 43 cavity; this substitution alters the Env conformation by predisposing gp120 to spontaneously assume a state closer to the CD4-bound conformation (9). In agreement with the important role played by Env conformation in the CD4 interaction (1, 9-11), a recent study using the simian-human immunodeficiency virus (SHIV) model in rhesus macaques showed that the replacement of residue 375 by larger hydrophobic or basic amino acids enhanced the affinity of Env for macaque CD4 and allowed better infection of macaque T lymphocytes in culture and in vivo (12), highlighting the importance of this residue for viral replication.…”

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

“…More than 5,000 crystallization trials involving 20 different gp120-ligand combinations were screened (Table S1). Crystals in several space groups could be grown from a deglycosylated HXBc2 gp120 core with intact gp41-interactive region, partially stabilized in the CD4-bound state by T257S and S375W substitutions on gp120 (9). Diffraction to 2.6 and 3.5 Å resolution was observed from orthorhombic and tetragonal crystals, respectively, of the same ternary complex (257/375-stabilized gp120 core, 2-domain CD4, and Fab of antibody 48d); anisotropy and incompleteness, however, reduced the effective resolution of the orthorhombic data to approximately 3 Å (Table S2).…”

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

“…However, the sera could neutralize the homologous JRFL virus weakly with an IC 50 of 4 -8, whereas good neutralization (IC 50 Ͼ50) was obtained only for Tier 1 viruses like SF162 and HxBc2 (19). To reduce the conformational flexibility of gp120 and to stabilize it in the CD4-bound conformation, S375W/T257S mutations were made to fill the Phe-43 cavity (17,18). The mutations improved sCD4 and b12 binding, both in the context of monomeric gp120 and trimeric GCN4-gp120.…”