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B Lebleu

Publications and source records attributed to B Lebleu.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells.

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.

Base Sequence↗
PubMed1995

Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway.

The 2-5A/RNase L system is considered as a central pathway of interferon (IFN) action and could possibly play a more general physiological role as for instance in the regulation of RNA stability in mammalian cells. We describe here the expression cloning and initial characterization of RLI (for RNase L inhibitor), a new type of endoribonuclease inhibitor. RLI cDNA codes for a 68-kDa polypeptide whose expression is not regulated by IFN. Its expression in reticulocyte extracts antagonizes the 2-5A binding ability and the nuclease activity of endogenous RNase L or the cloned 2DR polypeptide. The inhibition requires the association of RLI with the nuclease and is dependent on the ratio between the two proteins. Likewise RLI is coimmunoprecipitated with the RNase L complex by a nuclease-specific antibody. RLI does not lead to 2-5A degradation or to irreversible modification of RNase L. The overexpression of RLI in stably transfected HeLa cells inhibits the antiviral activity of IFN on encephalomyocarditis virus but not on vesicular stomatitis virus. RLI therefore appears as the first described and potentially important mediator of the 2-5A/RNase L pathway.

ATP-Binding Cassette Transporters↗
PubMed1994

Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.

Recently several groups reported a dramatic improvement of reporter gene transfection efficiency using a fusogenic peptide, derived from the Influenza hemagglutinin envelop protein. This peptide changes conformation at acidic pH and destabilizes the endosomal membranes thus resulting in an increased cytoplasmic gene delivery. We describe the use of a similar fusogenic peptide in order to improve the antiviral potency of antisense oligodeoxynucleotides (anti TAT) and oligophosphorothioates (S-dC28) on de novo HIV infected CEM-SS lymphocytes in serum-free medium. We observed as 5 to 10 fold improvement of the anti HIV activities of the phosphodiester antisense oligonucleotides after chemical coupling to the peptide in a one to one ratio by a disulfide or thioether bond. No toxicities were observed at the effective doses (0.1-1 microM). No sequence specificity was obtained and the fusogenic peptide possessed some antiviral activities on its own (IC50: 6 microM). A S-dC28-peptide disulfide linked conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed similar activities as the free S-dC28 oligonucleotide (IC50: 0.1-1 nM). As expected, all the compounds were less potent in the presence of serum but the relative contribution of peptide coupling was maintained.

Amino Acid Sequence↗
PubMed1994

Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells.

Triple helix formation of nucleic acids is the most rational approach to designing site-specific transcription inhibitors. To increase their efficiency, reactive moieties such as psoralen or ethenocytosine have been introduced on the third strand. In transfected cells, these compounds induce a site-specific covalent binding of the third strand to the targeted sequence and efficiently block RNA polymerases. However, the stability of this transcription inhibition has never been checked. We have designed a plasmid containing a triple helix binding site in the coding region of the beta-galactosidase reporter gene and a polymerase chain reaction assay to follow quantitatively the cross-link of a psoralen-derivatized third strand in transfected cells. This assay has revealed that the cross-link was removed within a few hours, leading only to a transitory inhibition of gene expression. Control experiments in DNA repair-deficient cells suggest the implication of repair enzymes in this process.

Base Sequence↗
PubMed1993

2',5'-Oligoadenylate-dependent RNase L is a dimer of regulatory and catalytic subunits.

The subunit composition of RNase L, a key enzyme in the interferon system, has been characterized. RNase L was purified from human Daudi cells on a column of 2-5A-Sepharose and used to immunize Balb/c mice. A specific monoclonal antibody which recognizes a protein of 80 kDa has been isolated. This protein has been characterized and shown to be an RNA-binding protein with nuclease activity which is associated with, but distinct from, the 80-kDa 2-5A-binding protein known previously as RNase L. It is therefore proposed that the 2-5A-dependent RNase L is a complex of two distinct subunits: an 80-kDa RNA-binding protein (i.e. the catalytic subunit) and an 80-kDa 2-5A-binding protein (i.e. the regulatory subunit).

Adenine Nucleotides↗
PubMed1993

Characterization of the nuclear binding sites of oligodeoxyribonucleotides and their analogs.

The intracellular fate of antisense oligodeoxyribonucleotide (oligomers) is poorly understood. Recent observations strongly suggest that after endocytosis and escape from the endocytic compartments, oligomers accumulate in the nuclei of eukaryotic cells by simple diffusion. Isolated nuclei were used to determine the number of these nuclear binding sites and their affinity for phosphodiester, phosphorothioate, and methyl-phosphonate oligomers. These cell-free assays were correlated with intact cell microinjection experiments. Great differences have been observed between these analogs. These data are helpful in understanding the fate of oligomers and the mechanism of their action and could be helpful for a more rational design of antisense molecules.

Base Sequence↗
PubMed1993

Comparative evaluation of seven oligonucleotide analogues as potential antisense agents.

12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).

Antiviral Agents↗
PubMed1992

Poly(L-lysine)-conjugated oligonucleotides promote sequence-specific inhibition of acute HIV-1 infection.

Previously, we have reported that conjugation of antisense oligonucleotides to poly(L-lysine) (PLL) lowers their inhibitory concentration in several biological models. We have now tested these conjugates for inhibition of human immunodeficiency virus type 1 (HIV-1) replication. PLL-conjugated oligonucleotides complementary to the translation initiation site of Tat protein protect cells from the cytopathic effect of HIV-1 in acute infection assays. The EC50 of conjugates is approximately 0.15 microM, which represents a strong reduction in concentration as compared to nonconjugated oligonucleotides (EC50 = 20 microM). In contrast with most reports in the literature, we have observed sequence specific antiviral effects with PLL conjugates. This was particularly noteworthy in antiviral experiments performed with HIV-1 isolates presenting heterogeneity in the 5' end of the tat mRNA sequence. Two mismatches at the target site were sufficient to reduce very significantly the antiviral activity of the conjugates but did not modify the effect of nonconjugated oligonucleotides. Unlike free oligonucleotides, PLL-conjugated ones do not interfere with virus penetration and/or reverse transcription as demonstrated by polymerase chain reaction (PCR) analysis of viral DNA.

Animals↗
PubMed1992

[Control of gene expression by antisense nucleic acids].

The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.

Gene Expression Regulation↗
PubMed1991

Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).

Adenine Nucleotides↗
PubMed1991

Intracellular distribution of microinjected antisense oligonucleotides.

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.

Animals↗
PubMed1991

Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells.

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.

Animals↗
PubMed1991

Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene.

Several groups have reported the use of antisense oligonucleotides to inhibit c-myc gene expression and study its biological role. However high concentrations of free oligonucleotides were generally needed. To lower their concentration and stabilize the antisense effect against c-myc, oligonucleotides were covalently linked to poly(L-lysine) and administered in ternary complexes formed with heparin (100 micrograms/ml). A sequence specific growth inhibition was observed at concentrations lower than 1 microM, while oligonucleotide-poly(L-lysine) conjugates alone were inefficient. Similar results occurred with other polyanionic compounds. Inhibition of proliferation was correlated to a reduction of c-myc protein and to a transient decrease in c-myc mRNA level. However, implication of RNase H in this process could not be demonstrated.

Base Sequence↗
PubMed1991

DNA protein interactions at the interferon-responsive promoter elements: potential for an H-DNA conformation.

The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two B1 and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the B1 complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OL1 were recovered in B2. For each probe, the possibility of the part of the sequence involved in B1 complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secondary structure, the formal existence of a H-DNA structure has not been demonstrated. Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.

2',5'-Oligoadenylate Synthetase↗