Conformational Changes of gp120 in Epitopes near the CCR5 Binding Site Are Induced by CD4 and a CD4 Miniprotein Mimetic

Abstract

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Interaction with CD4 and Antibodies to CD4-Induced Epitopes of the Envelope gp120 from a Microglial Cell-Adapted Human Immunodeficiency Virus Type 1 Isolate

“…The following MAbs were obtained through the NIH ARRRP: F105 (9, 60) from Marshall Posner and Lisa Cavacini, immunoglobulin IgG1 (IgG1) b12 (7,54,55,65) from Dennis Burton and Carlos Barbas, 17b and 48d (36,(78)(79)(80)(90)(91)(92) from James Robinson, 2G12 (67,81) from Hermann Katinger, and 447-52D (10,(22)(23)(24) from Susan Zolla Pazner. The sheep polyclonal D7324 Ab (47) was purchased from Aalto BioReagents (Dublin, Ireland).…”

“…Binding to CD4 triggers a conformational change in gp120, primarily involving V1/V2 and V3, which results in the exposure of conserved regions previously folded into the core structure (66, 77-79, 88, 91, 92). These CD4-induced (CD4i) regions include discontinuous epitopes recognized by the human neutralizing monoclonal antibodies (MAbs) 17b and 48d, known to interfere with chemokine receptor binding (36,(77)(78)(79)(90)(91)(92). Thus, the CD4i conformational change is thought to expose a highaffinity coreceptor binding site that collocates with these epitopes.…”

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

“…To counteract the high entropy that prevents antibody access to the co-receptor-binding site, one approach is to stabilize the gp120 structure through interaction with CD4. Indeed, binding of gp120 to the host cell CD4 receptor causes the reorientation of the loop regions of gp120 and stabilizes the viral glycoprotein in an intermediate state that exposes the co-receptor-binding site (10,11,(17)(18)(19). Complexes formed between gp120 and CD4 are recognized by a large panel of antibodies, called CD4-induced antibodies, whose epitopes overlap the co-receptor-binding site (20 -23).…”

“…Such CD4 mimetics were developed in our laboratory on the basis of structural similarities between the CDR2-like loop of CD4 and the ␤-hairpin region of a scorpion toxin (32). The last generation of these miniCD4s (27 residues) possesses a CD4-like affinity for gp120 and stabilizes the viral glycoprotein in a CD4-bound-like conformation, exposing both CD4i epitopes and the co-receptor-binding site (19,33,34).…”

Gp120 V3-dependent Impairment of R5 HIV-1 Infectivity Due to Virion-incorporated CCR5

“…The coreceptor-binding site located in the bridging sheet and the V3 loop of gp120 also play a crucial role in interacting with the N-terminal domains of the CCR5 (12-16). Finally, direct interaction between CCR5 and the V3 loop (35-37 amino acid residues) in gp120 induces structural rearrangements of a fusion peptide of gp41, allowing fusion of viral and cellular membranes (14,15,(17)(18)(19).Envelope viruses are known to down-modulate the receptor expression on infected cells to prevent reinfection (20, 21). Post-entry, HIV infection leads to a rapid and potent downmodulation of CD4 molecules expressed at the cell surface.…”