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R Kettmann

Publications and source records attributed to R Kettmann.

At least 73 records · Page 4Linked to original sources
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

Bovine leukemia virus, an animal model for the study of intrastrain variability.

Intradermal injection of a cloned bovine leukemia virus (BLV) provirus (pV344) into sheep allowed direct evaluation of intrastrain variability. A sheep was injected with pV344 DNA mixed with DEAE-dextran and became persistently infected with BLV strain 344. After 18 months, DNA was extracted from peripheral blood leukocytes from a single 0.5-ml blood sample. The long terminal repeat (LTR) and the env gene were amplified by using the polymerase chain reaction, cloned, and sequenced. Nineteen independent LTR clones (0.6-kb inserts) and 16 env clones (1-kb inserts) were analyzed. The in vivo rate of nucleotide change was 0.009%/year (two mutations out of 14,464 bp in 1.5 years), corresponding to only one amino acid change in the env gene. Five point mutations (all transitions), corresponding to a modification rate of 0.034%/year (five mutations out of 9,709 bp in 1.5 years), were identified in the LTR. As a control for Taq DNA polymerase errors, the same procedure using pV344 plasmid DNA was carried out. Out of 9,944 bp sequenced, three point mutations were found (i.e., one misincorporation in 3,315 nucleotides). These data demonstrate the extremely low level (or absence) of intrastrain variability of BLV in vivo. Consequently, BLV persistence in the infected host does not seem to result from an escape mutant strategy, in sharp contrast with the high mutation rates observed in the lentivirus family. The lack of genetic variation supports the possibility of successful vaccine against BLV and probably against the related human T-cell leukemia viruses.

Animals↗
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↗
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↗
PubMed1992

Nucleotide sequence of bovine interleukin-6 cDNA.

We report the cloning of bovine interleukin-6 (IL-6) cDNA. The clone was isolated from a bovine-leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library cloned in the bacteriophage lambda gt11. The cDNA encodes a full length IL-6 protein made of 208 amino acids with 65, 53, 42 and 42% homology to published sequences of porcine, human, mouse and rat IL-6, respectively. The significance of IL-6 expression in a BLV-induced tumor is briefly discussed.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the bovine interleukin-6 gene promoter.

We report the cloning and sequencing of a 1252 base pairs (bp) DNA fragment containing the bovine interleukin-6 (IL-6) gene promoter. This fragment was isolated from a bovine genomic library constructed in the lambda GEM11 vector. Comparison with human, murine and rat IL-6 gene promoters reveals a high degree of conservation of the 200 bp immediately upstream of the RNA CAP site. This region contains nucleotide stretches matching with consensus sequences recognized by transcription factors, including NF-KB, CREB and NF-IL6. A potential AP-1 binding site is found 284 nucleotides upstream of the RNA CAP site. The bovine IL-6 gene promoter cloned upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene was shown to be active in bovine and ovine cells.

Animals↗
PubMed1992

Sequence of bovine interleukin 7.

We report the cloning and the sequencing of a cDNA coding for the mature bovine interleukin 7 (IL-7). The clone was isolated from a bovine leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library. The 5' non-coding sequence and the sequence of the signal peptide were obtained from a clone isolated from a bovine genomic library. The entire bovine IL-7 protein is 176 amino acids long and shows 75 and 65% homology to published sequences of human and murine IL-7, respectively.

Amino Acid Sequence↗
PubMed1991

The bovine leukemia virus tax gene contains an enhancer sequence.

Transactivator proteins of the bovine leukemia (BLV) and human T-lymphotropic (HTLV) viruses increase long terminal repeat (LTR)-directed viral gene expression and act as immortalizing oncogenes in tissue culture. We report here that the BLV tax gene sequence contains an enhancer-like activity. The X long open reading frame was cloned up-stream of the beta-globin promoter linked to the chloramphenicol acetyltransferase (CAT) gene. In the presence of tax sequences, up to sevenfold enhancement of CAT expression was observed. A computer-assisted homology search revealed the presence of a consensus enhancer core sequence (GTGTTGTTGGTTG) into the third exon of the 2.1-kb X mRNA. These studies demonstrate that the tax gene contains a transcriptional enhancer which could be involved in early viral gene expression in vivo.

Animals↗
PubMed1991

Characterization of Two Gene Transcripts Modulated by Cytokinins in Micropropagated Apple (Malus domestica [L.] Borkh) Plantlets.

The micropropagation of apple (Malus domestica [L.] Borkh) cultivars is usually achieved by axillary bud stimulation and requires an exogenous cytokinin supply. Two cDNA libraries were constructed from mRNA isolated from plantlets grown in vitro on medium with or without benzyladenine. One cDNA clone (pSD3), corresponding to transcripts more abundant in plantlets grown on medium containing cytokinin than on medium lacking the hormone, was isolated. It corresponds to a mRNA of about 1,800 nucleotides which codes for a proline-rich protein with a predicted mass of 31,000 daltons. Its accumulation is restricted to roots and stems of in vivo grown apple plantlets and to stems of microcuttings cultivated on medium without cytokinin. Furthermore, it accumulates to comparable levels in leaves and stems when plantlets are grown on medium containing benzyladenine. A second cDNA clone (pSD4), corresponding to transcripts down-regulated in the presence of cytokinin in the culture medium, was also characterized. Its corresponding mRNA is about 700 nucleotides in length and encodes a protein highly homologous to the precursor of the 10-kilodalton polypeptide of the photosystem II from spinach. This mRNA accumulates specifically in leaves of apple plantlets and is more abundant in leaves of plantlets grown in the absence of cytokinin compared with plantlets grown in the presence of benzyladenine.

Journal Article↗
PubMed1991

Nucleotide sequence of the bovine cyclic-AMP responsive DNA binding protein (CREB2) cDNA.

The bovine cyclic AMP responsive binding protein cDNA (CREB2) was isolated from a lambda-gt11 cDNA expression library using a 32P labelled oligonucleotide corresponding to the 21 bp enhancer sequence present in the BLV LTR. The deduced amino acid sequence revealed that CREB2 contains a leucine zipper structure (residue 295 to 316), a basic amino acid domain (residue 268 to 291) and several potential phosphorylation sites.

Amino Acid Sequence↗