Abstract
We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.
Highlights
We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membraneimpermeant PDI inhibitors prevented human immunodeficiency virus type 1 (HIV-1) infection
We show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors
The well established reductive function of surface PDI (13–21) together with the putative presence of disulfide bonding close to the domain of gp120-CD4 interaction (26) suggested that PDI might cleave disulfide bonds in a gp120 molecule bound to the cell surface
Summary
Vol 277, No 52, Issue of December 27, pp. 50579 –50588, 2002 Printed in U.S.A. Inhibitors of Protein-Disulfide Isomerase Prevent Cleavage of Disulfide Bonds in Receptor-bound Glycoprotein 120 and Prevent HIV-1 Entry*. This suggested that inhibitors interfered with virus entry This finding led to the hypothesis that surface PDI associates with the HIV receptor CD4, and through that association, it reaches and reduces a gp120-disulfide bond situated in the region of gp120 that binds to CD4. This hypothesis implied that the cleavage of a loopforming disulfide bond might cause the conformational changes in gp120 that are required to activate the fusogenic function of gp. 4) We showed that gp120 bound to the primary HIV receptor CD4 on target cells undergoes a disulfide reduction that is inhibited by PDI inhibitors (Fig. 3). The following sequence of experiments was carried out to support this hypothesis. 1) We confirmed that HIV-1 target cells are capable of reducing disulfide bonds in proteins that bind to their surface (Fig. 1A). 2) The physical association of PDI and CD4 was demonstrated by coprecipitation of soluble CD4 with affinity-labeled soluble PDI (Fig. 1C) and by coprecipitation of cellular PDI with affinity-labeled cellular CD4 (Fig. 1D). 3) We showed that soluble PDI is capable of reducing disulfide bonds in soluble gp120 and that the PDI-induced opening of a disulfide loop caused conformational changes in gp120 (Fig. 2). 4) We showed that gp120 bound to the primary HIV receptor CD4 on target cells undergoes a disulfide reduction that is inhibited by PDI inhibitors (Fig. 3). 5) We showed that the same inhibitors at the same concentrations prevent HIV entry into target cells (Fig. 4)
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