Background: Interaction between HIV gp120 and cell CD4 initiates viral infection of host cells. Results: Only CD4 with reduced disulfides in domain 1 or 2 binds gp120, which inhibits thioredoxin-dependent CD4 dimerization. Conclusion: Cell surface oxidoreductases may prime CD4 for gp120 engagement, and impairment of redox-driven CD4 dimerization by gp120 may compromise CD4 function. Significance: Redox-dependent isomerization of CD4 is critical for HIV entry.

The new structural details on the complex gp41-lipidic epitope will spur new approaches to design gp41-MPER immunogens that might induce broadly neutralizing antibody responses.

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser 60 on the CD4 domain 1 ␣-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys 126 -Cys 196 ), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys 60 and gp120 Cys 126 , and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.The first step of HIV-1 2 entry into host cells involves the interaction between the viral envelope protein (Env) and the host cell receptor CD4. Functionally active (fusogenic) Env exists on the surface of virions as a trimer composed of three heterodimeric gp120-gp41 complexes (1). The latter are established through noncovalent association of monomeric gp120 with membrane-embedded gp41 (2-4), such that gp120 is presented on the exterior of the viral lipid membrane for interaction with CD4. The development of both Env-directed therapeutic compounds (5-10) and Env-based immunogens (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) has been frustrated by the significant sequence heterogeneity known to exist among Envs, even from viruses that are phylogenetically closely related, which limits the cross-reactive breadth of anti-Env antibodies and therapies. Furthermore, Env is extensively glycosylated and conformationally flexible, which compounds the problem of Env ligand accessibility and reactivity. In the background of this extraordinary array of strategies evolved to enable viral escape from effective immune-mediated neutralization, and apart from a very small number of studies that have ...