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.

Protein disulfide isomerase (PDI) is a multifunctional protein with thiol-disulfide redox-isomerase activities. It catalyzes thiol-disulfide interchange reactions on the cell surface that may cause structural modifications of exofacial proteins. PDI inhibitors alter human immunodeficiency virus (HIV) spread, and it has been suggested that PDI may be necessary to trigger HIV entry. This study examined this hypothesis by using cell-to-cell fusion assays, in which the HIV envelope (Env) expressed on the cell surface interacts with CD4(+) lymphocytes. PDI is clustered at the lymphocyte surface in the vicinity of CD4-enriched regions, but both antigens essentially do not colocalize. Anti-PDI antibodies and 2 inhibitors of its catalytic function altered Env-mediated membrane fusion at a post-CD4 cell binding step. The fact that the PDI catalytic activity present on lymphocytes is required for fusion supports the hypothesis that catalysts assist post-CD4 cell binding conformational changes within Env.

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Astrocytes play an active role in the central nervous system and are critically involved in astrogliosis, a homotypic response of these cells to disease, injury, and associated neuroinflammation. Among the numerous molecules involved in these processes are the matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, secreted or membrane-bound, that regulate by proteolytic cleavage the extracellular matrix, cytokines, chemokines, cell adhesion molecules, and plasma membrane receptors. MMP activity is tightly regulated by the tissue inhibitors of MMPs (TIMPs), a family of secreted multifunctional proteins. Astrogliosis in vivo and astrocyte reactivity induced in vitro by proinflammatory cues are associated with modulation of expression and/or activity of members of the MMP/TIMP system. However, nothing is known concerning the intracellular distribution and secretory pathways of MMPs and TIMPs in astrocytes. Using a combination of cell biology, biochemistry, fluorescence and electron microscopy approaches, we investigated in cultured reactive astrocytes the intracellular distribution, transport, and secretion of MMP-2, MMP-9, TIMP-1, and TIMP-2. MMP-2 and MMP-9 demonstrate nuclear localization, differential intracellular vesicular distribution relative to the myosin V and kinesin molecular motors, and LAMP-2-labeled lysosomal compartment, and we show vesicular secretion for MMP-2, MMP-9, and their inhibitors. Our results suggest that these proteinases and their inhibitors use different pathways for trafficking and secretion for distinct astrocytic functions.

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.

The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited ϳ4 unpaired cysteines, and chemically reduced S1 displaying up to ϳ6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens.SARS 3 -CoV is a new human coronavirus that causes a severe acute respiratory syndrome, sometimes with a fatal outcome. Its Spike glycoprotein is the major surface antigen and induces potent neutralizing antibodies (1-8). It is made up of S1 and S2 subdomains (2, 3). The S1 region interacts with angiotensin-converting enzyme 2 (ACE2), the primary cellular receptor (9 -12), L-SIGN (13), and L-SECtin (14), whereas S2 mediates membrane fusion (2, 3). Here, we investigated the functional role of the 20 conserved cysteine residues of mature S1.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 (15-29). 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). 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). 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. the thiol reagent DTNB and bacitracin, ...

During exercise, cardiac oxygen consumption increases and the resulting low oxygen level in the myocardium triggers coronary vasodilation. This response to hypoxia is controlled notably by the vasodilator adenosine and its A 2A receptor (A 2A R). According to the "spare receptor" pharmacological model, a strong A 2A R-mediated response can occur in the context of a large number of receptors remaining unoccupied, the activation of only a weak fraction of A 2A R (evaluated using K D ), which results in maximal cAMP production (evaluated using EC 50 ), and hence in maximal coronary vasodilation. In coronary artery disease (CAD), myocardial ischemia limits adaptation to exercise, which is commonly detected using the exercise stress test (EST). We hypothesized that spare A 2A R is present in CAD patients to correct ischemia. Seventeen patients with angiographically documented CAD and 17 control subjects were studied. We addressed adenosine-plasma concentration and A 2A R-expression at the mononuclear cell-surface, which reflects cardiovascular expression. The presence of spare A 2A R was tested using an innovative pharmacological approach based on a homemade monoclonal antibody with agonist properties. EST was positive in 82% of patients and in none of the controls. Adenosine plasma concentration increased by 60% at peak exercise in patients and in none of the controls (p < 0.01). Most patients (65%), and none of the controls, had spare A 2A R (identified when EC 50 /K D ≤ 0.1) and a low A 2A R-expression (mean: -37% versus controls; p < 0.01). All patients with spare A 2A R had a positive EST whereas the subjects without spare A 2A R had a negative EST (p < 0.05). Spare A 2A R is therefore associated with positive EST in CAD patients and its detection may be used as a diagnostic marker.

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