Josette Svab

The Anti-HIV Cytokine Midkine Binds the Cell Surface-expressed Nucleolin as a Low Affinity Receptor

The growth factor midkine (MK) is a cytokine that inhibits the attachment of human immunodeficiency virus particles by a mechanism similar to the nucleolin binding HB-19 pseudopeptide. Here we show that the binding of MK to cells occurs specifically at a high and a low affinity binding site. HB-19 prevents the binding of MK to the low affinity binding site only. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicated that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, is the domain that binds MK. The specific binding of MK to cells is independent of heparan sulfate and chondroitin sulfate expression. After binding to cells, MK enters cells by an active process. Interestingly, the cross-linking of surface-bound MK with a specific antibody results in the clustering of surface nucleolin along with glycosylphosphatidylinositol-linked proteins CD90 and CD59, thus, pointing out that MK binding induces lateral assemblies of nucleolin with specific membrane components of lipid rafts. Our results suggest that the cell surface-expressed nucleolin serves as a low affinity receptor for MK and could be implicated in its entry process.

Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells.

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4+ cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kψ(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kψ(CH2N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kψ(CH2N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4+ cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KΨ(CH2N)PR]-TASP construct, Ψ(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KΨ(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KΨ(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KΨ(CH2N)PR]-TASP, the p95·TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KΨ(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KΨ(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Preparation and characterization of polyclonal antibodies specific for the 69 and 100 K-dalton forms of human 2–5A synthetase

Recently, the existence of 40-, 46-, 69- and 100- kDa forms of 2,5-oligoadenylate (2-5A) synthetase have been established in interferon-treated human cells. Using monoclonal antibodies specific for 69- and 100- kDa forms of 2-5A synthetase, we purified these proteins by immunoaffinity chromatography and raised murine polyclonal antibodies. All immunized mice developed antibodies (anti-69 or anti-100 kDa form) which were characterized by their capacity to immunoprecipitate [35S] cysteine labeled proteins from interferon-treated cells or identify these proteins by electrophoretic transfer immunoblot analysis of extracts from control and interferon-treated cells. The 69 and 100 kDa 2-5A synthetases were induced in different types of human cells, such as Daudi, BJAB, HeLa and differentiated HL-60 cells. These enzymes were not detectable nor induced in MRC5 and undifferentiated HL-60 cells.

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections.

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2-5-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).

Characterisation of the interferon-mediated protein kinase by polyclonal antibodies

Interferon-treated human cells show an enhanced level of a double-stranded (ds) RNA-dependent protein kinase activity which is manifested by the phosphorylation of an endogenous 72,000 molecular weight protein (p72K kinase). By the use of murine polyclonal antibodies against this p72K kinase, here we have characterized the protein kinase activity associated with immune complexes precipitated from extracts of interferon-treated cells. Precipitation of the p72K kinase by the polyclonal antibodies results in the formation of a complex in which the kinase activity is manifested by phosphorylation of the 72K protein. This phosphorylation, however, is independent of dsRNA. Such immunoprecipitates can also phosphorylate exogenous substrates, calf thymus histones and the alpha subunit of protein initiation factor eIF2.

Purification of human leukocyte interferon to homogeneity with the use of a monoclonal antibody: Detailed technique and parameters

Summary We had previously described the isolation of anti-human leukocyte interferon monoclonal antibody HBA and its use in the purification and radioimmunoassay of α interferon. The detailed procedure for this purification is described here. Human leukocyte interferon can be purified to homogeneity after two passages on an immunoadsorbant constructed with the monoclonal antibody HBA. The specific activity of the purified interferon is ≥2×10 8 IU/mg of protein. Analysis of the purified proteins by SDS-gel electrophoresis and silver-staining shows that the purified interferon consists of three species of polypeptides of different molecular weights: 18,000 for the major species and 21,000–27,000 for the minor ones. By this simple and rapid technique, it is possible to obtain pure human leukocyte interferon with good recovery (70%) from crude or partially purified preparations. Furthermore, this method could be applied to the large-scale preparation of homogeneous interferon.

Interferon-induced enzymes in mice and in volunteers in response to glucocorticoids

Summary The induction of 2-5A-synthetase has been reported in lymphoid cells treated with glucocorticoids. However, we have been unable to reproduce these results. On the other hand, in the present work, we show that this enzyme, in conjunction with double-stranded RNA-dependent protein kinase, is enhanced in mice treated with hydrocortisone and in volunteers treated with β-1-24 tetracosatide, a synthetic corticosteroid-stimulating hormone. Dose-response experiments carried out in mice indicated that the enhacement of splenic 2-5A-synthetase was observed 48 h after the injection of hydrocortisone and was independent of the dose (25–300 μg/mouse). Administration of mice with anti-mouse (α+β) inteferon immunoglobulins 6 h after injection of hydrocortisone blocked the enhancement of splenic 2-5A-synthetase. These results suggest that the enhancement of 2-5A-synthetase is due to the production of interferon as a consequence of hydrocortisone treatment. In volunteers, the levels of 2-5A-synthetase and protein kinase in peripheral blood mononuclear cells were enhanced 3 and 2 days after the injection of β-1-24-tetracosatide, respectively. The peak of urinary cortisol was 24 h after the injection of β-1-24-tetracosatide. Circulating interferon was not detectable either in mice or in volunteers with high levels of interferon-induced enzymes. This is probably due to the production of very low levels of interferon and the fact that circulating interferon has a very short half-life.