The cell surface ofmammalian cells is capable of reductively cleaving disulfide bonds of exogenous membrane-bound macromolecules (for instance, the interchain disulfde of diphtheria toxin), and inhibiting this process with membrane-impermeant sulfhydryl reagents prevents diphtheria toxin cytotoxicity. More recently it was found that the same membrane function can be inhibited by bacitracin, an inhibitor of protein disulfide-isomerase (PDI), and by monoclonal antibodies against PDI, suggesting that PDI catalyzes a thioldisulide interchange between its thiols and the disulfides of membrane-bound macromolecules. We provide evidence that the same reductive process plays a role in the penetration of membrane-bound human immunodeficiency virus (IHV) and show that HIV infection of human lymphoid cells is markedly inhibited by the membrane-impermeant sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid), by bacitracin, and by anti-PDI antibodies. The results imply that HIV and its target cell engage in a thio-disulfide interchange mediated by PDI and that the reduction of critical disulfides in viral envelope glycoproteins may be the initial event that triggers conformational changes required for HIV entry and cell infection. These findings suggest additional approaches to impede cell infection by HIV.The surface of CHO cells has a reductive function capable of cleaving disulfide bonds of various membrane-bound macromolecules, and that cleavage is inhibited by the membraneimpermeant sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and pCMBS (p-chloromercuriphenylsulfonic acid) (1,2). In one instance, cleavage was associated with the reductive activation of diphtheria toxin (DT), a disulfidecontaining protein that binds to a specific surface receptor and must undergo a reductive chain separation to exert its cytotoxicity. Both DTNB and pCMBS prevented DT cytotoxicity (2). More recent work showed (3) that monoclonal antibodies (mAbs) to protein disulfide-isomerase (PDI) and bacitracin, an inhibitor of PDI, cause similar inhibitions. PDI is an oxidoreductase capable of mediating thio-disulfide interchange reactions. It is present mostly in the endoplastic reticulum (4) but has also been detected at the surface of mammalian cells (5,6). Experiments based on the knowledge that DTNB inhibits DT cytotoxicity (2) and on the observation that the structure of the Sindbis virus envelope is maintained by disulfide bonds (7) showed that DTNB causes a significant inhibition of Sindbis infection of BHK cells (8,9). The viral glycoprotein gpl20 by which human immunodeficiency virus (HIV) attaches to its receptor CD4 also contains disulfide bonds (10). The events that follow HIV attachment and lead to viral infection are not yet fully understood (11). We postulated that the plasma membrane of human lymphoid cells would have a reductive function similar to that of CHO cells (3), and we reasoned that the reductive mechanism might cleave disulfides in membranebound gp120 and facilitate viral entry into cells. To dete...

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membraneimpermeant 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 PDI⅐CD4 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.

Evidence had been provided that a disulfidelinked [12511iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thioldisulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI-in particular, bacitracin and anti-PDI antibodies-might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties. (KDEL) sequence (3). It has also been reported to occur at the surface of mammalian cells (4, 5). This paper presents evidence that PDI is indeed responsible for cleaving the disulfides of these membrane-bound macromolecules and shows that cleavage is inhibited by monoclonal anti-PDI antibodies and by bacitracin, an antibiotic known to inhibit both the reductive (6) and the oxidative (7) functions of PDI.MATERIALS AND METHODS Materials. Bacitracin, DTNB, and pCMBS were purchased from Sigma. DT was from List Biological Laboratories, Campbell, CA. Thioredoxin and monobromotrimethylammoniobimane (Thiolyte MQ) were from Calbiochem. Lyphophilized ascites fluid containing anti-PDI monoclonal antibodies RL77, RL90, and HP13 (8) and the parent hybridoma cells were a gift from Charlotte S. Kaetzel, Case Western Reserve University School of Medicine. For some experiments the antibody-containing ascites fluid or conditioned medium was purified by protein G affinity chromatography (Mab Trap G kit from Pharmacia LKB). 125I-Tyn-SS-PDL conjugate was prepared as described (1). The experiments were carried out on Chinese hamster ovary (CHO) cells.Isolation of PDI and Assay of PDI and Thioredoxin. PDI was isolated from calf liver as described by Hillson et al. (9) and was 90-95% pure by electrophoresis. It was assayed as described by Carmichael et al. (10), except that the glutathione concentration was reduced to 250 AM. The amount and radioactivity of 125I-insulin were 50 ,ug and 105 cpm per sample. Under these conditions, PDI had a specific activity of 5200 units/mg in the standard 5-min assay. In the assays of Fig. 1 and Table 1, the reaction time was extended to 3...

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Basic proteins and polyamino acids are taken up by mammalian cells at rates up to 3000 times greater than serum albumin. When given together with serum albumin they increase the albumin uptake by a factor that correlates with their own rate of uptake and can reach more than 50-fold. The lowest threshold of activity detected (10(-10)M)is comparable to the activities of the most potent membrane-active agents.