Biomedical subjects

D Portetelle

Publications and source records attributed to D Portetelle.

At least 73 records · Page 4Linked to original sources

PubMed1993

Enhancement of bovine leukemia virus-induced syncytia formation by di- and tripeptides.

Short hydrophobic peptides were previously shown to inhibit infectivity of para- and orthomyxoviruses. We tested the ability of a series of di- and tripeptides to interfere with cell fusion induced by bovine leukemia virus (BLV). Peptides containing a hydrophobic contribution and/or a positive net charge strongly enhanced syncytia formation induced by BLV on CC81 indicator cells. The size of the multinucleated cells was strongly increased (up to 10-fold) in the presence of the enhancer peptides whereas no effect was observed on the indicator cells in the absence of BLV. The peptides thus amplified the fusion process initiated by BLV envelope glycoproteins. The effect was dose-dependent at concentrations ranging from 20 to 640 microM and did not result from an increased expression of BLV proteins. The peptides did not compete with anti-gp51 monoclonal antibodies for the recognition of eight well-defined epitopes of gp51. We consequently hypothesize that the enhancer peptides interact with the membrane of BLV-producing cells and/or indicator cells and propose a model based on molecular modeling.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.

Amino Acid Sequence↗

PubMed1993

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.

Animals↗

PubMed1993

Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51.

A battery of 19 synthetic peptides was used to characterize efficient neutralizing and helper T-cell epitopes on the bovine leukemia virus (BLV) external envelope glycoprotein gp51. Four of the antipeptide antisera raised in rabbits inhibited the formation of BLV-induced syncytia; these antisera are directed against peptides 64-73, 98-117, and 177-192. Only antisera directed against the 177-192 region also neutralized vesicular stomatitis virus-BLV pseudotypes. This study clearly demonstrates that neutralizing properties can be observed with antibodies raised to regions undescribed so far and included in both the amino-terminal and central parts of the antigen. In addition, some helper T-cell determinants were defined from gp51-immunized mice and from BLV-infected cattle. Although none of the peptides tested behaved as a universal helper T-cell epitope, peptide 98-117 stimulated T-cell proliferation from BALB/c mice and from three infected cows, while peptide 169-188 strongly stimulated T-cell proliferation from one infected cow. Further experiments performed with three peptides overlapping the 169-188 region (177-192, 179-192, 181-192) demonstrated the particular relevance of residue(s) P-177 and/or D-178 in the helper T-cell epitope. These data should assist in the design of an efficient subunit vaccine against BLV infection that contains peptides possessing both B-neutralizing and helper T-cell determinants.

Amino Acid Sequence↗

PubMed1993

Changes in the hypophysial-gonadal axis during the onset of puberty in young bulls.

The objectives of this study were to determine the extent of changes in concentrations of testosterone, growth hormone (GH) and insulin-like growth factor I (IGF-I) concentrations in blood plasma and to characterize the respective plasma-binding proteins of these two peptides during the onset of puberty in male calves. The jugular veins of six male Holstein calves (11-weeks-old) were catheterized and blood was collected every 3 days (one sample every 30 min for 8 h). Hormone concentrations in plasma were determined by specific radioimmunoassay. After incubation with [125I]IGF-I, IGF-I-binding proteins (IGFBPs) were separated by gel filtration; radioactivity was determined in each fraction. Western ligand blotting using radiolabelled hormones as ligand was also used to characterize the IGF-I- and GH-binding proteins in plasma. Puberty was characterized by a rapid (in 1 or 2 days) increase in mean concentrations of testosterone in plasma (from 0.5 to > 2 ng ml-1) and a pulsatile release of the hormone. During puberty, IGF-I concentrations also increased rapidly in 8-10 days from +/- 50 ng ml-1 to > 150 ng ml-1, whereas concentrations of GH in plasma remained relatively stable during the experimental period. A significant correlation was observed between IGF-I and testosterone concentrations (r = 0.77; P < 0.001) throughout the experimental period. Three different IGF-I-binding protein fractions with apparent molecular masses of > 200, 150-170 and 45-65 kDa were found in plasma using gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Presence of growth hormone-binding proteins in cattle plasma and milk.

The presence of GH-binding proteins (GHBPs) in the plasma of adult cattle was investigated using Sephadex G-200 filtration, Western ligand blotting and Western blotting. The changes in the concentration of GHBP in the plasma of dairy half-sister heifers during the first year of life as well as the presence of GHBP in milk were also investigated. When analytical chromatography (on a 1.6 x 100 cm column) was performed, five peaks of recombinant bovine GH (rbGH)-associated radioactivity were revealed in cattle plasma; the first peak, which appeared near the void volume, was presumed to represent aggregates, the second (M(r) 290 kDa) and the third peaks (M(r) 75 kDa) corresponded to specific rbGH-GHBP complexes; the last two peaks representing free 125I-labelled rbGH and Na[125I]. Western ligand blotting revealed multiple GHBPs. Three major bands were observed at approximately 190, 58 and 31 kDa; an excess of unlabelled hormone blocked the binding of 125I-labelled rbGH. Minor non-specific binding bands were also detected in cattle plasma with molecular weights between 40 and 136 kDa. One monoclonal antibody (8H7) produced against synthetic peptide (amino acids 54-63 of the extracellular domain of the bovine GH receptor) specifically interacted with 190 and 58 kDa bands while the 31 kDa band was not recognized. Finally, Western ligand blots were performed to evaluate the changes in plasma GHBP during the first year of life in 55 dairy half-sister heifers and to identify GHBP in milk. In plasma, the intensity of the 31 kDa band varied greatly between animals while the other specific bands remained stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Monoclonal antibodies to bovine growth hormone potentiate hormonal activity in vivo by enhancing growth hormone binding to hepatic somatogenic receptors.

Administration of GH complexed with monoclonal antibodies (MABs) potentiates the in vivo actions of the hormone. In particular, growth and serum IGF-I concentrations of GH-treated hypophysectomized rats are increased by concomitant injection of anti-GH MABs. Among 37 anti-bovine GH (bGH) MABs, we selected one MAB with the most potentiating effects to investigate the mechanisms responsible for this phenomenon. Hypophysectomized rats were killed 18 h after a single s.c. injection of bGH (100 micrograms/rat), alone or complexed with increasing doses of MAB (4, 40, 400 micrograms/rat; MAB:bGH molar ratio: 0.005, 0.05, 0.5). IGF-I was measured by radioimmunoassay in acid-extracted sera and livers, whereas liver IGF-I mRNA was quantified by Northern blot hybridization. The in vivo occupancy of liver somatogenic (GH) receptors was derived from the determinations of total and free 125I-labelled bGH binding to liver homogenates treated with 4 mol MgCl2/l or water. Injection of MAB-bGH complexes enhanced body weight gain and raised serum IGF-I, liver IGF-I and liver IGF-I mRNA more than bGH alone (1.6-, 6-, 10- and 7-fold increases at the highest dose of MAB, compared with bGH alone; P < 0.001). These potentiating effects of the MAB were dose-dependent and significant potentiation of the growth response was already observed with the lowest dose of MAB. In vivo occupancy of liver GH receptors was markedly higher 18 h after injection of MAB-bGH complexes than after bGH alone, and this effect was also dose-dependent (receptor occupancy of 28%, 37% and 83% after 4, 40 and 400 micrograms of MAB respectively compared with 6% after bGH alone; P < 0.05, 0.05 and 0.001 respectively). In contrast, the in vitro binding of 125I-labelled bGH to liver homogenates was decreased in the presence of high doses of MAB. We conclude that low amounts of MABs complexed with bGH potentiate the stimulation by the hormone of liver IGF-I synthesis and secretion in a dose-dependent manner. These effects are mediated, at least in part, through changes in hormone-receptor interaction in vivo, leading to enhanced and/or prolonged binding of bGH to its somatogenic receptors.

Animals↗

PubMed1993

Dynamics of mitotic activity and expression of viral proteins gp51 and p24 of bovine leukemia virus producing cells.

The dynamics of expression of viral proteins gp51 and p24, virus particle production and mitotic activity of a highly productive bovine leukemia virus clone of the fetal lamb kidney (FLK) cell line were studied. The period of the highest protein production (20-44 h after incubation) was established by the indirect immunofluorescence method using specific monoclonal antibodies. It was followed by a complete formation of the cell monolayer and the most intensive period of the mitotic activity determined by the 3H-thymidine labeling method. After the active synthesis of viral protein, the formation of mature viral particles and their shedding in the intercellular spaces was established electron-microscopically and by the syncytia induction test. A similar comparative study was carried out with a BLV producing short-term lymphocyte culture (STLC).

Animals↗

PubMed1992

ELISA test, a new method to detect and quantify isoproturon in soil.

An indirect competitive Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed to quantify isoproturon (a commonly used phenylurea herbicide) in soil samples. To produce the specific antibodies needed for detection of isoproturon, an isopropylanilin analogue was covalently linked to a proteic carrier and the resulting conjugate used as immunogen. Hybridomas were obtained from spleen cells of mice immunized with this conjugate. One specific monoclonal antibody (MAb) was selected and used to the ELISA allowing the detection of small amounts of herbicide (20-250 micrograms l-1). Cross-reactivities with other current phenylureas appear to be insignificant. Samples of six soils were mixed with several quantities of herbicide (to reach final concentrations ranging between 0.15 and 1 microgram g-1) and extracted either with methanol to determine the total residue or with a solution of CaCl2, to establish the herbicide available from the soil solution. The extracts were analyzed simultaneously by gas chromatography and ELISA in these conditions, correlation coefficients between both methods were never lower than 93.5%. Organic matter and ionic strength appeared to be the most important parameters interfering the ELISA test. To avoid these drawbacks, standard curves were established for the ELISA by spiking isoproturonless soils extracts. In these conditions, correlations between both methods were considered as sufficient. ELISA of untreated samples were carried out to verify the isoproturon availability curves established previously.

Antibodies, Monoclonal↗

PubMed1992

The 19-27 amino acid segment of gp51 adopts an amphiphilic structure and plays a key role in the fusion events induced by bovine leukemia virus.

Previous results indicate that the external glycoprotein gp51 of bovine leukemia virus plays an important role in the process of cell fusion induced by bovine leukemia virus (Bruck, C., Mathot, S., Portetelle, D., Berte, C., Franssen, J. D., Herion, P., and Burny, A. (1982) Virology 122, 342-352; Vonèche, V., Portetelle., D., Kettmann, R., Willems, L., Limbach, K., Paoletti, E., Ruysschaert, J. M., Burny, A., and Brasseur, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3810-3814) and suggest that a region encompassing residues 23 and 25 of gp51 is involved in this process (Portetelle, D., Couez, D., Bruck, C., Kettmann, R., Mammerickx, M., Van der Maaten, M., Brasseur, R., and Burny, A. (1989) Virology 169, 27-33; Mamoun, R., Morisson, M., Rebeyrotte, N., Busetta, B., Couez, D., Kettmann, R., Hospital, M., and Guillemain, B. (1990) J. Virol. 64, 4180-4188). X-ray diffraction studies performed on envelope glycoproteins of influenza virus indicate that the NH2-terminal part of the external glycoprotein lies very close to the fusion peptide. The same overall structure seems to exist in human immunodeficiency virus as suggested by site-directed mutagenesis followed by syncytia induction assays. Our theoretical studies indicate that a segment expanding between residues 19 and 27 of gp51 probably adopts an amphipathic beta-strand structure. We hypothesize that the amphipathic 19-27 structure of gp51 plays an important role in the process of membrane fusion by interacting with the fusion peptide or with another region of gp30. Mutational analysis disrupting the amphipathy of the 19-27 region strongly altered the fusogenic capacity of the gp51-gp30 complex.

Amino Acid Sequence↗

PubMed1992

Fusogenic segments of bovine leukemia virus and simian immunodeficiency virus are interchangeable and mediate fusion by means of oblique insertion in the lipid bilayer of their target cells.

Modified bovine leukemia virus (BLV) glycoproteins were expressed by using vaccinia virus recombinants, and their fusogenic capacities were examined by a syncytia-formation assay. This analysis indicates that (i) both BLV envelope glycoproteins gp51 and gp30 are necessary for cell fusion; (ii) insertion of the N-terminal segment of gp30 (fusion peptide) into the lipid bilayer in an oblique orientation, as predicted by computer conformational analysis, results in fusogenic capacities higher than insertion in a perpendicular or parallel orientation; and (iii) replacement of the BLV fusion peptide with its simian immunodeficiency virus counterpart does not modify the fusogenic capacity of the BLV glycoprotein.

Amino Acid Sequence↗

PubMed1992

Mutations in the bovine leukemia virus Tax protein can abrogate the long terminal repeat-directed transactivating activity without concomitant loss of transforming potential.

The bovine leukemia virus Tax protein transactivates gene expression directed by the viral long terminal repeat (LTR) and contributes to immortalization of primary cells. Theoretical analysis of the protein sequence revealed the presence of a putative zinc finger structure at its amino end. Selected mutations in that region completely abolished transactivation, demonstrating its importance for LTR-directed gene regulation. However, these mutations did not interfere with the ability of tax to bind zinc or to contribute to immortalization of primary cells. Thus, transactivation of bovine leukemia virus LTR and target cell transformation are independent functions of Tax and involve different functional domains of the protein.

Animals↗

PubMed1992

In vivo transfection of bovine leukemia provirus into sheep.

Bovine leukemia virus is horizontally transmitted mainly through infected cells by direct blood transfer. In this report, a cloned bovine leukemia virus (BLV) provirus was examined for its infectivity by direct inoculation into sheep. One hundred micrograms of plasmid DNA containing a complete provirus was mixed with a cationic liposome solution and injected intradermally into five sheep at three different locations. Seroconversion occurred 1 to 2 months after injection as demonstrated by immunodiffusion, indirect ELISA (for the gp51 envelope protein), and blocking ELISA (for gp51 and the major capsid protein, p24). These results demonstrate that BLV infection can be efficiently initiated by direct transfection into sheep. This approach should thus facilitate investigation of the involvement of BLV genetic determinants in the induction of leukemia in ruminants.

Animals↗

PubMed1992

Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies.

A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs). Four recombinant phages with inserts of about 2-5 kbp were isolated. One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30. This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells. The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs. Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260. All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51. The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.

Amino Acid Sequence↗

PubMed1992

A cyclic AMP-responsive DNA-binding protein (CREB2) is a cellular transactivator of the bovine leukemia virus long terminal repeat.

To gain insight into the cellular regulation of bovine leukemia virus (BLV) trans activation, a lambda-gt11 cDNA library was constructed with mRNA isolated from a BLV-induced tumor and the recombinant proteins were screened with an oligonucleotide corresponding to the tax activation-responsive element (TAR). Two clones (called TAR-binding protein) were isolated from 750,000 lambda-gt11 plaques. The binding specificity was confirmed by Southwestern (DNA-protein) and gel retardation assays. Nucleotide sequence analysis revealed that TAR-binding protein is very similar to the CREB2 protein. It contains a leucine zipper structure required for dimerization, a basic amino acid domain, and multiple potential phosphorylation sites. A vector expressing CREB2 was transfected into D17 osteosarcoma cells. In the absence of the tax transactivator, the CREB2 protein and the cyclic AMP-dependent protein kinase A activate the BLV long terminal repeat at a basal expression level: trans activation reached 10% of the values obtained in the presence of tax alone. These data demonstrate that CREB2 is a cellular factor able to induce BLV long terminal repeat expression in the absence of tax protein and could thus be involved in the early stages of viral infection. In addition, we observed that in vitro tax-induced trans activation can be activated or inhibited by CREB2 depending on the presence or absence of protein kinase A. These data suggest that the cyclic AMP pathway plays a role in the regulation of viral expression in BLV-infected animals.

Animals↗

PubMed1991

Iodoacetamide treatment of bovine leukemia virus glycoprotein gp51 enhances the western blotting reactivity of anti-peptide antibodies.

Rabbit polyclonal antibodies were raised against synthetic peptides of the bovine leukemia virus envelope glycoprotein gp51 and tested against the full size protein by the Western blotting technique. We show that acetylation of gp51 by iodoacetamide either maintains or significantly increases the antigen-antibody reaction and conclude therefrom that the reactive potential of an anti-peptide antibody may require acetylation of the sulfhydryl groups of the blotted protein.

Acetylation↗

PubMed1991

Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein.

Six sequential epitopes (A, B, B', D, D', E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142-161 (MCF4), B and B' in peptide 195-205, D and D' in peptide 218-237 (MCF6), and A in peptide 249-268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51.

Amino Acid Sequence↗

PubMed1991

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection.

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (ATI) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 x 10(3) sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.

Animals↗