Biomedical subjects

D Poncet

Publications and source records attributed to D Poncet.

31 records · Page 2Linked to original sources

PubMed1994

Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.

The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.

Baculoviridae↗

PubMed1994

Identification of the nucleic acid binding domain of the rotavirus VP2 protein.

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.

Animals↗

PubMed1993

An immunodominant cytotoxic T cell epitope on the VP7 rotavirus protein overlaps the H2 signal peptide.

C57BL/6 (H-2b) mice were primed with the bovine RF strain of rotavirus to study the induction of CD8+ cytotoxic T lymphocytes (CTLs). These rotavirus-specific CTLs were detected only after in vitro restimulation with the virus. Using a recombinant vaccinia virus we identified the RF VP7 protein as a major target of these CTLs. The response against this protein was obtained also after in vitro restimulation with simian SA11 and human WA strains of rotavirus. Using published Db and Kb allele-specific motifs to predict possible CTL epitopes in the RF VP7 protein, we synthesized and tested 18 predicted peptides of VP7. Only one peptide was able to sensitize target cells at a concentration below 5 x 10(-7) M. This CTL epitope was also induced by immunization with the RF VP7 expressed with a baculovirus vector, and was shown to be immunodominant by its capacity to inhibit, in an unlabelled target assay, the bulk response against cells infected with recombinant vaccinia virus expressing VP7. This CTL epitope overlaps the H2 signal peptide of the protein.

Amino Acid Sequence↗

PubMed1993

Rotavirus protein NSP3 (NS34) is bound to the 3' end consensus sequence of viral mRNAs in infected cells.

Interaction between viral proteins and RNAs has been studied in rotavirus-infected cells. The use of UV cross-linking followed by immunoprecipitation and labeling with T4 polynucleotide kinase allowed us to detect interactions between RNA and nonstructural viral proteins. The RNAs linked to the nonstructural protein NSP3 have been identified as rotavirus mRNAs, and the sequences of the RNase T1-protected fragments have been established. These sequences correspond to the 3' end sequence common to all rotavirus group A genes. We also show that the last 3' nucleotide is cross-linked to the protein and that monomeric and multimeric forms of NSP3 are bound to rotavirus mRNA. The role of NSP3 in rotavirus replication is discussed in the light of our results and by comparison with other RNA-binding proteins of members of the Reoviridae family.

Base Sequence↗

PubMed1991

Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein.

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Animals↗

PubMed1990

A recombinant vaccinia virus expressing the major capsid protein of Simian rotavirus-induced anti-rotavirus antibodies.

cDNA molecules encoding the major structural protein (VP6) of the Simian rotavirus SA11 were inserted under the control of the vaccinia virus 7.5 kDa promoter into the thymidine kinase gene. Synthesis of VP6 was demonstrated by immunoprecipitation of recombinant virus-infected cell. Mice inoculated via several routes with this recombinant vaccinia produce high titers of antirotavirus antibodies lacking neutralizing activity.

Animals↗

PubMed1990

Sequences in rotavirus glycoprotein VP7 that mediate delayed translocation and retention of the protein in the endoplasmic reticulum.

Glycosylation and translocation of the simian rotavirus protein VP7, a resident ER protein, does not occur co-translationally in vivo. In pulse-chase experiments in COS cells, nonglycosylated VP7 was still detectable after a 25-min chase period, although the single glycosylation site was only 18 residues beyond the signal peptide cleavage site. After labeling, glycosylated and nonglycosylated VP7 was recovered in microsomes but the latter was sensitive to trypsin (i.e., the nascent protein became membrane associated) but most of it entered the ER posttranslationally because of a rate-limiting step early in translocation. In contrast with the simian protein, bovine VP7 was glycosylated and translocated rapidly. Thus, delayed translocation per se was not required for retention of VP7 in the ER. By constructing hybrid proteins, it was further shown that the signal peptide together with residues 64-111 of the simian protein caused delayed translocation. The same sequences were also necessary and sufficient for retention of simian VP7 in the ER. The data are consistent with the idea that certain proteins are inserted into the ER membrane in a loop configuration.

Amino Acid Sequence↗

PubMed1989

A plaque hybridization assay for rotaviruses.

A plaque hybridization assay was adapted to rotavirus. In this test cDNA or oligonucleotide probes were used to discriminate between plaques originating from virus carrying genes of bovine and simian origin. Only mRNAs present in infected cells were detected as demonstrated by using oligonucleotides corresponding to both strands. This assay can be used to screen reassortants or mutants of rotaviruses.

Animals↗

PubMed1989

Generation of a helper cell line for packaging avian leukosis virus-based vectors.

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.

Animals↗

PubMed1984

A new method for detection of small modifications in genomic DNA, applied to the human delta-beta globin gene cluster.

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].

Base Sequence↗

PubMed1984

S1-nuclease mapping of the genomic Lepore-Boston DNA demonstrates that the entire large intervening sequence of the fusion gene is of beta-type.

Several reports have suggested but not proven that the large intervening sequence of Lepore delta-beta fusion gene was of beta-type (3-5). A method able to detect rearrangements as small as 4 nucleotide pairs directly into genomic DNA (6) has been applied to the total DNA of a heterozygous Lepore-Boston patient in order to identify accurately the origin of the large intervening sequence of the delta-beta fusion-gene. Hybrid duplexes were formed between genomic Lepore DNA and single-stranded DNA used as probes, then submitted to S1-nuclease treatment. Our data demonstrate that the entire large intervening sequence of the Lepore fusion gene is of beta-type. Moreover, no large modification was detected in any delta- and beta parts of the delta-beta fusion gene.

Base Sequence↗

PubMed1984

Non-random association of the polymorphic Taq I restriction site, located 3 KB 5' to the human delta-globin gene, with the polymorphic Hind III sites within the G gamma- and A gamma-globin genes.

During the study of the DNA from 25 beta-thalassemic subjects from mediterranean origin the polymorphic Taq I restriction site located 3-kb 5' to the human delta-globin gene, was found non-randomly associated to the polymorphic Hind III sites within the G gamma- and A gamma-globin genes. This indicates that the 3' limit of the linkage group of polymorphic restriction sites including the gamma-globin genes is located downstream to the polymorphic Taq I site.

Base Sequence↗

PubMed1983

CG sites and their nucleotide environment in the human gamma-delta-beta-globin gene cluster: distribution, frequency and possible frame for differential methylation.

Available restriction endonucleases including CG dinucleotides in their target sequences (most of them being unable to cut the DNA when the cytosine of the CG sequence is methylated) have been used to map cloned DNA covering the human gamma-delta-beta globin gene cluster. Since the human DNA fragments were cloned in Escherichia coli, only the internal cytosine in the sequence CCAT GG could be methylated. Thus, any recognized "CG enzyme" site can be detected since they are unmethylated. Results show that frequencies of "CG enzyme" sites regularly decrease from the gamma-globin region to the beta-globin region, the latter being very poor in "CG enzyme"' sites. The array of enzymes used here detects 4 times more CG sites than the classical MspI/HpaII system. Examination of previously sequenced parts of the gamma-delta-beta globin gene cluster shows that CG dinucleotides correspond to an average frequency of 1 out of 104 nucleotides in the gamma-globin region and 1 out of 138 nucleotides in the beta-globin region. In the gamma-globin region, 1 CG out of 4 or 5 may be detected by the enzymes used; the detected frequency is less than 1 out of 10 CG in the beta-region. Analysis of nucleotide environment around CG dinucleotides shows occurrence of local differences, the main sequences being CGG in the 5' side flanking the gamma genes and ACG in the corresponding area of the beta gene. The results presented introduce some new considerations about analysis of cytosine methylation which has been previously proposed as playing a role in the control of the activity of gamma, delta and beta genes respectively.

Base Sequence↗