Sylviane Muller

THE ANTI-HIV PSEUDOPEPTIDE HB-19 FORMS A COMPLEX WITH THE CELL-SURFACE-EXPRESSED NUCLEOLIN INDEPENDENT OF HEPARAN SULFATE PROTEOGLYCANS

The HB-19 pseudopeptide 5[Kψ(CH2N)PR]-TASP, ψ(CH2N) for reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4+ cell lines and in primary T-lymphocytes and macrophages. Here, by using an experimental CD4+ cell model to monitor HIV entry and infection, we demonstrate that HB-19 binds the cell surface and inhibits attachment of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with the cell-surface-expressed nucleolin, and eventually results in its degradation. Accordingly, by confocal immunofluorescence microscopy, we demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can prevent the binding of HB-19 to cells and inhibit complex formation with nucleolin. Such a competition between viral particles and HB-19 is consistent with the implication of nucleolin in the process of HIV attachment to target cells. We show that another inhibitor of HIV infection, the fibroblast growth factor-2 (FGF-2) that uses cell-surface-expressed heparan sulfate proteoglycans as low affinity receptors, binds cells and blocks attachment of HIV to permissive cells. FGF-2 does not prevent the binding of HB-19 to cells and to nucleolin, and similarly HB-19 has no apparent effect on the binding of FGF-2 to the cell surface. The lack of competition between these two anti-HIV agents rules out the potential involvement of heparan sulfate proteoglycans in the mechanism of anti-HIV effect of HB-19, thus pointing out that nucleolin is its main target.

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.

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits.

Polyclonal and monoclonal antibodies specific for histones as well as sera directed against synthetic peptides of histones were used to probe the topography of chromatin subunits. In native chromatin, the regions corresponding to residues 130‐135 of H3 and 6‐18 of H2B were found to be exposed and able to interact with antibodies whereas the regions 26‐35 and 36‐43 of H2B and 80‐89 and 85‐102 of H4 were not. In vitro phosphorylation of H3 and H5 in native chromatin or of H3 in H1/H5‐depleted chromatin led to a marked drop in the binding of antibodies specific for residues 130‐135 of H3 and 6‐18 of H2B. Phosphorylation of H1/H5‐depleted chromatin also altered the degree of exposure of certain H2A epitopes but it did not affect the surface accessibility of residues 1‐11 of H2B.

Efficient Binding of Reduced Peptide Bond Pseudopeptides to Major Histocompatibility Complex Class I Molecule

Reduced peptide bond pseudopeptide analogues have been examined for their ability to bind murine class I molecules of the major histocompatibility complex (MHC). Eight pseudopeptide analogues of an antigenic peptide derived from Plasmodium berghei (H-Ser-Tyr-Ile-Pro-Ser-Ala-Glu-Lys-Ile-OH) were obtained by systematically replacing one peptide bond at a time by a reduced peptide bond (CH-NH). The resulting analogues were then tested for their binding to a recombinant single chain SC-Kd class I molecule. The comparative results show that five analogues can efficiently mimic the parent peptide while the introduction of the reduced bond between P3-P4, P7-P8, and P8-P9 is deleterious for SC-Kd binding. The fact that more stable pseudopeptides containing reduced peptide bonds can bind major histocompatibility complex molecules is of great interest for the design of peptidomimetics with potential therapeutical properties. Such peptide analogues may prove useful for the development of peptide-based cytotoxic T lymphocyte vaccines.

Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

The C‐terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130‐135) corresponding to the sequence Ile‐Arg‐Gly‐Glu‐Arg‐Ala (IRGERA) was prepared by solid‐phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121‐135), and it inhibited the H3‐anti H3 reaction in complement fixation, solid‐phase radioimmunoassay, and enzyme‐linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C‐terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non‐acetylated H3, H2A, and H2B with either mono‐, di‐, or tri‐acetylated H4 isolated from cuttle ‐fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C‐terminal) peptide of H3 was considerably decreased when di‐ or tri‐acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.