Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

Abstract

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Significant Redox Insensitivity of the Functions of the SARS-CoV Spike Glycoprotein

“…The sample was then analyzed by SDS-PAGE (8%). After blotting onto a nitrocellulose filter (Schleicher & Schuell) and blocking with phosphate-buffered saline (PBS), pH 7.4, 2% casein, 0.5% Tween 20, the filter was processed using streptavidin-coupled peroxidase (1:500, 30 min, 25°C) and diaminobenzidine, as described previously (26,30). Alternatively, staining of the filter was performed using Amido Black to assess the presence of contaminant proteins in the sample.…”

“…For HIV, gentle chemical reduction of Env efficiently prevents viral infectivity by inhibiting the capacity of the surface subunit (SU) to bind its ligands on the target cell surface (15,19). In contrast, after receptor binding, fusion mediated by the transmembrane subunit occurs solely following reduction of SU by a cell-surface protein-disulfide isomerase (PDI) activity (19,(25)(26)(27). For these reasons, reducing or free sulfhydryl reagents and nonpermeant inhibitors of PDI (e.g.…”

“…On one hand, manipulation of the native disulfide network of mature envelopes at the virus surface using reducing or alkylating reagents has important consequences for their subsequent capacity to carry out the virus/cell interaction process (15, 16, 19, 20, 23, 24, 26, and 28). On the other hand, natural and specific thiol/disulfide rearrangements occur within several viral envelopes to trigger conformational changes that insert the fusion peptide into the cell surface leading to virus entry (20,21,23,(25)(26)(27). For HIV, gentle chemical reduction of Env efficiently prevents viral infectivity by inhibiting the capacity of the surface subunit (SU) to bind its ligands on the target cell surface (15,19).…”

“…A variety of evidence has shown that the capacity of the viral envelope glycoproteins to mediate virus/cell membrane fusion depends on a precise thiol/disulfide balance within the viral surface complex (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). On one hand, manipulation of the native disulfide network of mature envelopes at the virus surface using reducing or alkylating reagents has important consequences for their subsequent capacity to carry out the virus/cell interaction process (15, 16, 19, 20, 23, 24, 26, and 28).…”

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

“…Error bars, SEM. There is considerable evidence in the literature that molecules other than CD4 and chemokine receptors, such as ceramide (Finnegan et al, 2004), glycosphingolipids (Puri et al, 1999;Fantini et al, 2000), and protein disulfide isomerases (Fenouillet et al, 2001;Gallina et al, 2002;Barbouche et al, 2003;Markovic et al, 2004), affect efficiency of fusion and/or infection. Such molecules or other factors may strongly influence the transfer of aqueous contents from virus to the cell interior.…”