FR2725214A1 - NOVEL COMPOUNDS FOR PREPARING ANTI-INFECTIOUS AGENTS - Google Patents

Abstract

Nucleotide sequences capable of coding for polypeptides having RNAase L inhibitory activity are disclosed. Said nucleotide sequences and said inhibitors are useful for developing antiviral agents.

Description

TITLE: NOWEAUX COMPOUND USES
FOR THE PREPARATION OF ANTI-INFECTIOUS AGENTS.

 The subject of the invention is novel compounds which can be used for the preparation of anti-infective agents.

 It is known that the body's natural defense against viral infections involves interferons, which act through different mediators, including the 2-5A / RNase L system.

 The treatment of cells with interferons (hereinafter abbreviated as IFN) induces many genes among which those of 2-SA synthetases. These enzymes, activated by double-stranded RNA (cellular or viral), polymerize ATP into a series of 2'-5 'phosphodiester-linked oligoadenylates, hereinafter referred to as "2-5A".

This 2-5A activates, at nanomolar concentrations, a latent endoribonuclease RNase L, which possesses in particular the property of cleaving the mRNAs at the UpNp (N = U or A) sequences.

 In viral infection, activation of the 2-5A / RNase L system leads to the selective degradation of viral mRNAs, resulting in inhibition of viral growth.

 This selectivity of an enzyme, RNase L, having little sequence specificity is due to a localized activation of the system, which activation would take place at the double stranded viral structures such as the HIV sequence TAR.

 The 2-5A / RNase L system could be involved in other situations such as cell growth and differentiation. This via the degradation of so-called unstable messengers carrying in 3 'AUUUA potential unstabilized sequences of RNase L (Shaw and Kamen, 1986, Cell, 46, 659-667).

 The role of the 2-5A system, however, is limited during infection with certain viruses, such as encephalomyocarditis virus (EMCV), vaccinia, herpes or HIV, which reduces the anti-inflammatory effect. viral interferons.

 It is therefore conceivable to have the means to effectively fight against an infection with such viruses.

 The work of the inventors in this field has led them to demonstrate that RNase L is inhibited by a cellular protein.

 The invention is more particularly based on the demonstration, on the one hand, of a mechanism for regulating RNase L by this cellular protein and, on the other hand, of the induction of this cellular protein by viruses.

 The invention thus offers a new particularly effective and specific approach for developing new antiviral strategies in pathological situations.

 It is therefore an object of the invention to provide, as new chemicals, DNA sequences capable of encoding polypeptides having inhibitory properties to RNase L, as well as the corresponding antisense sequences. , the corresponding RNA sequences and the cDNA sequences.

 It also aims to provide said polypeptides, as new chemicals, and a method for obtaining them according to the techniques of genetic engineering, so as to have these products in sufficient quantities for biological applications, and in purified form.

 According to yet another aspect, the invention relates to the application of these various products to develop compositions or products with an anti-viral effect or to enhance the effect of interferons.

The invention aims, as new products, the nucleotide sequences, isolated from their natural environment, capable of hybridizing with at least one fragment of the sequence SEQ ID No. 1 or SEQ ID No. 2 which codes for a polypeptide capable of inhibiting RNase
L.

 The sequence SEQ ID No. 1 is given at the end of the description, with the other sequences mentioned in the document called "Sequence Listing".

 This hybridization can be carried out under stringent conditions, but also under relaxed conditions.

 The invention relates in particular to nucleotide sequences, isolated from their natural environment, corresponding to at least a portion of the sequences SEQ ID No. 1 and SEQ ID No. 2, capable of coding for an RNase L inhibitor.

 Such sequences are further characterized in that they are capable of encoding, performed according to standard recombinant DNA techniques, for polypeptides having RNase L inhibitory activity, comprising at least one amino acid linkage. in the sequence SEQ ID No. 3.

 The invention relates to the nucleic acid sequences corresponding to the open reading frame ranging from position 118 to 1914 in SEQ ID No. 1.

 These sequences are further characterized in that they comprise two P-loop type motifs.

 Also within the scope of the invention are the antisense sequences corresponding to the sequences defined above, the mRNA sequences and their antisense, and the corresponding cDNAs.

 These different nucleotide sequences are further characterized in that they are devoid of mammalian DNA, infectious agents, prions and other natural product materials.

 It relates more particularly to the above nucleic acid sequences, inserted into an expression vector for polypeptides having an RNase L inhibitory activity.

 The invention also aims, as new chemicals, polypeptides having an inhibitory effect vis-à-vis ribonuclease.

The polypeptides according to the invention are characterized in that they possess an RNase L inhibitory activity and are constituted by a sequence of amino acids in the sequence coded by
SEQ ID No. 1 or SEQ ID No. 2.

These polypeptides are further characterized by a sequence of amino acids in SEQ ID No. 3 or in
SEQ ID NO: 4.

 The invention relates in particular to polypeptides as defined above having, in their sequence of amino acids, a sequence homologous with the CX2CX2CX3C: 4Fe4S-ferredoxin motif and / or a high number of thiol groups and / or a high number of leucine motifs.

 The polypeptide corresponding to the open reading frame in the sequence SEQ ID No. 1 comprises 599 amino acids, has a molecular weight evaluated at 67.515 kDa and an isoelectric point estimated at 8.7.

 The polypeptides of the invention are further characterized in that they are as obtained by expression, in a suitable host cell, according to recombinant DNA techniques, of an expression vector comprising a DNA sequence. consisting of a sequence of nucleotides in the sequence SEQ ID No. 1 or SEQ ID No. 2, recovery of the expressed polypeptide and purification.

 The polypeptides within the meaning of the present invention are of several types.

It may be fragments or derivatives obtained by genetic and / or chemical modification from the sequences SEQ ID No. 1 or SEQ ID No. 2, or sequences
SEQ ID NO: 3 or SEQ ID NO: 4, since they retain at least partly the activity of inhibiting RNase L.

 By modification of genetic and / or chemical nature is meant any mutation, deletion, substitution, addition and / or modification of one or more nucleotides.

 These modifications make it possible, if desired, to adapt the sequences defined above to the expression in a type of vector or host, to facilitate their cellular penetration or to increase the inhibition activity.

 The inhibitory activity of these polypeptides can be demonstrated according to various protocols described in the examples.

 Advantageously, the polypeptides defined above can be obtained in a form freed from any contaminant and in sufficient quantity for the biological applications described below.

 To obtain the polypeptides of the invention, as illustrated by the examples, a cDNA library is screened with a 2-5A labeled, for example, with a radioactive element; the desired gene is isolated, then introduced into an expression vector and a host cell is transfected with this vector. After expression of the polypeptide with inhibitory activity of the desired RNase, the latter is recovered after lysis of the cells, and subjected to at least one purification step.

 For the production of the polypeptide, prokaryotic systems, such as bacteria, or eukaryotes, such as baculoviruses, insect cells and yeasts, advantageously as commercially available, will be used.

 For the purification step, conventional biochemistry techniques may be used, for example isoelectrofocusing and / or commercially available vectors including sequences for affinity, antibody or nickel purifications, for example.

The invention also relates to the expression vectors containing at least one of the nucleotide sequences defined above subjected to the control of a suitable promoter, in particular the vectors corresponding to at least a portion of SEQ ID No. 1 or
SEQ ID No. 2, or their antisense sequences.

 The host cells transfected with these vectors and which comprise the expressed recombinant proteins also form part of the invention. The cells used for the transfections advantageously correspond to those mentioned above, available commercially.

The study of the properties of the polypeptides defined above made it possible to demonstrate, as already pointed out, an inhibitory effect of RNase L and, consequently, of its dependent activation of 2-5 A. These polypeptides bind to RNAse and inhibit its enzymatic activity (see Example 4, Figure 5 and Example 5, Figure 6)
It is recalled that RNase L was first described as a molecular complex of about 200 kDa (Slattery et al., 1979, Proc Natl Acad Sci USA, 76, 4778-4782). However, its molecular weight varies with the conditions of analysis used as the protein concentration (Floyd-Smith et al., J. Biol Chem 257, 8584 8587), and the origin of the cells (Bisbal et al, Eur J .

Biochem. 179, 595-602).

 Zhou et al (1993, Cell, 72, 753-765) published the cloning of an 80D protein called 2DR, which binds 2-5A and cleaves poly (U) after translation into glass in the reticulocyte lysate. rabbit, but not in the wheat germ lysate.

Recently, thanks to a specific monoclonal antibody, the inventors have characterized an RNA-binding protein associated with the 25A-binding protein of 80 KD. The inventors have therefore proposed a model where RNase
High molecular weight L is in fact a complex composed of at least two proteins: the 2-5A binding protein (2-5ABP) and the RNA binding protein (RNABP) (Salehzada et al., 1993, J. Biol. Chem., 268, 7733-7740).

 The polypeptides of the invention specifically associate with 2-5ABP and RNABP as shown by their coimmunoprecipitation with MAb3 monoclonal antibody (Example 5, Figure 6).

 The tests carried out with the polypeptides of the invention, of which illustrative examples are given below, have shown that they are capable of inhibiting the RNase L activity, both in cell extracts (Example 4, FIG. 5). only in intact cells (Example 9, Figure 10), as well as the 2-5A binding activity of the 2DR protein expressed in a cell-free system (Example 4, Figure 5).

The observed inhibition is dependent on the ratio of RNase L to polypeptide and does not result in 2-5A degradation or stable modification of the
RNase L.

 They are not regulated by interferons but are associated with RNase L and co-precipitate with the RNase L complex when put in contact with a monoclonal antibody specific for RNase L.

 They therefore constitute regulators of the 2-5A / RNase L system, which corresponds to one of the routes of action of interferons in mammalian cells.

 The polypeptides of the invention are advantageously used to stabilize the RNAs in cell extracts during molecular biology techniques (Example 8, FIG. 9).

More particularly, the invention relates to the use of the polypeptides defined above as target agents for detecting products capable of inhibiting their own inhibitory effect and thus making it possible to regulate the functioning of the 2-SA / RNase system.
L, and thereby increase the action of interferons.

The invention therefore relates to a method for screening molecules or substances for detecting in the latter an activity directed against the inhibitory activity of the
RNase L polypeptides of the invention.

 This method is characterized in that a polypeptide of the invention is brought into contact with a molecule or substance to be tested, and the reactivation of RNase L, again able to bind 2-5A, or the inhibition is detected. of its inhibitor.

To highlight the activity of RNase
It is advantageous to proceed as described in Example 4.

 Products capable of inhibiting the inhibitory activity with respect to RNase L of the polypeptides of the invention are polyclonal and monoclonal antibodies. These antibodies are also covered by the invention as new products.

 These antibodies directed against the polypeptides of the invention are advantageously prepared according to conventional techniques.

Another application of the polypeptides of the invention consists in the development of products capable of preventing the production of RNase inhibitors.
L by infectious agents or to neutralize the inhibitors produced by these agents.

Viruses, such as EMCV, induce inhibitors of RNase L, this induction can be inhibited by interferon when the action of the latter is able to restore the activity to the 2-SA / RNase system
L. But when the 2-5A / RNase system becomes unbalanced, interferon can no longer exert its anti-viral effect and the viral infection progresses.

 The invention thus provides means, with the polypeptides defined above, to search for active products vis-à-vis the inhibitors of RNase L induced by viruses.

 The invention is particularly directed to the use of antisense nucleotide sequences defined above for transfecting mammalian cells (see Example 9, Figure 10 and Example 11 Figure 12 A to F).

 The antisense DNA may be contained in different vectors such as retroviruses or recombinant viruses modified to be usable in the mammal by operating according to conventional techniques of genetic engineering.

 The first step is to obtain transgenic animals in which the gene for the RNase L inhibitor has been rendered inactive, or on the contrary will be overexpressed.

The procedure is carried out according to the techniques described in "Manipulating the mouse embryo." A Laboratory Manual,
Hogan B et al, Cold Spring Harbor Laboratory, 1986, Ed.

Cold Spring Harbor. For example, DNA may be microinjected directly into the pronucleus of fertilized eggs (see Constantitini and Lacy, 1981, Nature, 294, pp. 92-94). The embryo can also be infected with a retroviral vector (Jaenisch, 1976, Proc Nat Nat Sc 1973, 1260-1264). This technique can also allow the inactivation of the gene (Jaenish et al., 1983, Cell, 32, 209-216). Another technique for inactivating the gene may be microinjection of a gene encoding antisense RNA (Izant and Weintraub, 1985, Science, 229-345-352).

These animals allow the study of their sensitivity to viruses inducing the inhibitor of the
RNase L, like the EMCV, with or without an IFN treatment. By taking samples from the animal, the level of virus produced is determined and, by comparing with control animals, the evolution of the infection is followed.

 In another step, the experiments are carried out on another animal model susceptible to infection equivalent to the HIV system in humans for selectively expressing inhibitor antisense in the cells infected with the virus.

 It is carried out either by driving viruses or retroviruses by targeting CD4, or by obtaining a selective expression of the antisense in the infected cells only, this being under the control of the natural promoter of the inhibitor gene. of RNase L which is induced by the virus itself.

 The invention thus provides the means for studying the evolution of an infection in these animal models.

 The vectors used in the above model are contained in pharmaceutical compositions in association with an inert carrier. These compositions are advantageously in the form of sterile, isotonic solution or are preserved in freeze-dried form.

 These solutions are administered as sprays intranasally, or orally, intravenously or intramuscularly.

Other features and advantages of the invention are given in the following examples which relate to sequence inhibitory polypeptides.
SEQ ID No. 3 or SEQ ID No. 4, respectively called RLI and
H2ABP.

In these examples, reference is made to Figures 1 to 12, where
FIG. 1 represents the constructions of
CDNA coding for the polypeptides according to the invention,
FIG. 2, the nucleotide sequence of the RLI polypeptide cDNA and the corresponding amino acid sequence,
FIGS. 3A and 3B, Northern blot analysis of HeLa cells, whether or not treated with interferon, hybridized with different probes,
FIGS. 4A to 4C, autoradiographs after SDS-PAGE of RLI cDNA translation products and 2DR polypeptide in two different translation systems, and results of hybridization with 2-5ApCp probe.

FIGS. 5A to 5C, the inhibition effects of RLI on RNase L in a rabbit reticulocyte system or the human Daudi or Hela cells,
FIG. 6, the association by immunoprecipitation with a monoclonal antibody specific for RNase L, of RLI associated with 2DR or
RNase L,
FIG. 7, the stability of 2-5A in the presence of RLI,
FIG. 8, the absence of degradation or stable modification of RNase L by RLI,
FIG. 9, RNA stabilization in a reticulocyte extract in the presence of RLI,
FIGS. 10A and 10B, the response to interferon of the cells transfected by the directional construction of H2ABP inhibiting RNase L,
FIG. 11, induction of RLI mRNA by EMCV,
FIGS. 12A to 12C, RNase activity
L during EMCV infection of empty vector-transfected HeLa cells, the sense construct
H2ABP inhibiting RNase L or the antisense construction of
H2ABP inhibiting RLI, in the presence or absence of interferon,
- Figures 12D, E, F, the same cells but after infection with VSV.

 First of all, the products and methods used in the examples given to illustrate the invention are indicated below.

I - Products and methods
Cell and cell extracts
Human Daudi cells are used as mRNA sources because of their high level of 2-5A binding activity. These cells are cultured in suspension and maintained in RPMI 1640 medium supplemented with 10% (v / v) fetal calf serum, 60
IU / ml penicillin and 50 μg / ml streptomycin.

 HeLa cells are cultured in the same medium with 2 mM glutamine.

 The cells are treated with human interferon a / ss (IFN a / p Hu abbreviated).

 The cell extracts are prepared as described by Salehzada et al in J. Biol. Chem., 1993, 268, pages 7733-7740. Briefly, the cells are resuspended in 2 volumes of hypotonic buffer, broken in a homogenizer and centrifuged at 10,000 g.

Isolation of cDNA clones
The technique used to isolate the RLI cDNA clones is given in Example 1.

For the 2DR cDNA, PCR is carried out with primers determined from the sequence published by
Zhou et al in Cell, 1993, 72, 753-765.

 RNase L radio-binding and radiocovalent labeling on affinity of RNase L.

 The radio-binding assay and the covalent labeling method were carried out using, as sources of RNase L, S10 cell extracts, rabbit reticulocyte lysates or wheat germ extracts (with or without the different translated proteins). cloned).

 2-5A4-3'-t32P] pCp (3000 Ci / mmol) is used as the probe (see Bayard et al., Biochemistry, 1986, 25, 3730-3736).

RNA analysis
Total cellular RNA is prepared using the lithium chloride-guanidine thiocyanate method according to the method of Cathala et al in DNA, 1983, 4, 327-333.

The Northern blot hybridizations (passive transfer of nucleic acids on membranes) are carried out according to the standard techniques as described by Sambrook et al in Molecular Cloning: a laboratory manual, 1982 (Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York).

 The probes are synthesized by multi-primer method (Ready-to-go kit, Pharmacia).

After auto-radiography, the mRNAs are quantified by image analysis using the program.
Bioimage on a Millipore image analysis station (Sun Station).

 Each lane is standardized with the GAPDH probe (glyceraldehyde-3-phosphate dehydrogenase).

II Examples
Example I: RLI cDNA
(shown in Figure 1)
. Isolation of cDNA clones
A cDNA library of Daudi ZAP cells is established. Screening with 2-5A4-3 '- [32P] pCp (3000 Ci / mmol) as a radiolabeled probe was performed using the technique developed for renaturation of RNase L activity on a filter. 10 mM IPTG nitrocellulose filters are impregnated, dried and applied to phage plates for 3 hours at 37 ° C. The filters are then rinsed with saturated buffer A with 5% (v / v) of skimmed milk and rinsed again with buffer A without milk, followed by incubation for about 14 hours at 4 ° C in buffer A with 2-5A4-3 '- [32P] pCp (5x105 dpm / ml). The composition of Buffer A is as follows: 10 mM Hepes, pH 7.5, 80 mM KCl, 5 mM Mg (OAc) 2, 1 mM EDTA, 5% (v / v) glycerol, mM of p-mercaptoethanol.

 After washing in buffer A, the filters are self-radiographed on films.

 A positive clone containing a 2861 bp insert called H2ABP is thus isolated (FIG. 2). This insertion comprises an ATG initiator at position 125 (see FIG. 2). It is translated as a 39 kDa protein into a rabbit reticulocyte lysate or wheat germ extract. It presents an open reading frame from the first nucleotide.

 An internal primer determined from the H2APB sequence is used to isolate by anchored PCR a complete 3568 bp cDNA encoding the RLI (see FIG. 1).

 Briefly, dG nucleotides are added to one end of a polydT cDNA with a transferase terminal.

This cDNA (0.5 μg) was amplified by PCR (30 cycles) in a final volume of 50 μl using a 5 'polydC primer with PstI and BamH1 sites (5'TTTCTGCAGGATCCCCCCCCCCCCCC) and an internal primer (5').
CACTTAGATCATGTTCCACCACAAT 3 ') as shown in FIG.

 This 924 bp cDNA fragment, designated PCR in Figure 1, which overlaps the 217 bp H2ABP clone, is cloned into the BglII site. The complete RLI cDNA contains additional 707bp on the 5 'side of H2ABP.

Nucleotide sequence analysis (shown in Figure 2)
Sequencing of H2ABP in pSK (pBluescript II SK, Stratagene) was performed according to the Sanger dideoxy nucleotide sequencing method (T7 sequencing kit, Pharmacia) after the 3 'step deletions.

 These deletions are generated by nuclease digestion with exonuclease III-S1, followed by refilling with Klenow polymer DNA (Pharmacia).

 The entire cDNA sequence is sequenced according to the Sanger method and internal primers. This sequence with the corresponding amino acid sequence is shown in FIG.

 The RLI cDNA has a 5 'non-coding region of 118 nucleotides, an open reading frame extending to nucleotide 1914 and a long 3' untranslated region of 1654 bp.

 The open reading frame encodes a polypeptide of 599 amino acids, of molecular weight 67.515.

 The ATG codon at the beginning of the open reading frame has the characteristics of a strong initiator codon, that is, a purine in the -3 position and a G in the +4 position.

 The RLI cDNA contains two ATP / GTP repeats or P-loop motifs: one from residues 110 to 117 and the other from residues 379 to 386 (see empty rectangles in Figure 2).

 In the 3 'non-coding region, the polyadenylation signal sequences AATAAA are underlined and the ATTTA sequences which may correspond to instability are underlined by two lines.

In the amino acid sequence, between amino acids 55 and 66, there is a complete homology of RLI with the motif CX2CX2CX3C: 4Fe4S
Ferredoxine (see rectangle with black squares in Figure 2).

There is also an internal repeat of 128 amino acids with a homology of 53% starting at amino acids 200 and 440. (These parts are underlined in bold in the figure)
The gray rectangles correspond to a potential PCK phosphorylation site.

 RLI has a high number of thiol groups and is very rich in leucine residues.

 Its estimated isoelectric point is 8.7.

Example: Regulation of the expression of RLI mRNA by interferon (illustrated in FIG. 3)
In a first series of experiments, HeLa cells were treated with 103 U / ml IFN a / p Hu for 2, 4, 6, 8, 10, 18 and 24 hours. Total cytoplasmic RNA is extracted from the cells and analyzed by Northern blotting. The same experiments are performed on untreated HeLa cells. The RNAs (20 μg) are fractionated on a 1.2% (w / v) agarose gel, and hybridized with an RLI cDNA probe, with a human probe encoding an IFN-induced 15 kDa protein. , or with a human GAPDH probe (glyceraldehyde3-phosphate dehydrogenase). The filter is autoradiographed for 5 h in the probe test
GAPDH and for 12 h in tests with other probes.The results obtained are given in Figure 3A. Examination of this figure shows that the RLI cDNA hybridizes with two 3.5 kb and 2.8 kb cellular mRNAs in HeLa cells. The same result is obtained in Daudi and CEM cells.

 It is found that treatment with human interferon a / p, even at high concentrations, does not regulate the two mRNAs that hybridize with the RLI clones.

 The duration of interferon treatment does not result in a quantitative variation of these mRNAs.

 The cells, however, normally respond to treatment with interferon. Indeed, operating under the same conditions, a 15 kDa protein already described as being induced by an interferon treatment (see Blomstrom et al., J. Biol Chem., 1986, 261, 8811-8816) is induced. .

To study the differences between mRNAs of 2.8 and 3.5 bp, another series of experiments consisting of a transfer analysis were performed.
Northern total cytoplasmic RNA is extracted from HeLa cells. The RNAs (20 μg) were fractionated on 1.2% (w / v) agarose gel and hybridized with two RLI cDNA probes described in Figure 1, ie a 762 bp probe (A in Figure 3B), specific for the 5 'end of RLI, and a 764pb probe (B in Figure 3B) showing its 3' end.

 Probe A is found to hybridize with both mRNAs while Probe B hybridizes only with the longer mRNA. These two mRNAs therefore differ in their 3 'untranslated region. The similarity between the sizes of the two natural mRNAs and those of the cloned cDNAs corresponds to a pure coincidence.

 Example 3: In Vitro Synthesis of the mRNA Encoding RLI and 2DR and These Polypeptides

(shown in Figure 4)
Transcription of cDNAs
Transcription was performed with T3 polymerase in the presence of m7G (5 ') ppp (5') G according to the manufacturer's instructions (Promega).

 The RNA is analyzed and quantified by electrophoresis on 1.2% (w / v) agarose gels in TBE buffer (50 mM Tris pH 8, 50 mM boric acid, 1 mM EDTA).

RNA is extracted with phenol / dichloromethane (v / v) and precipitated with ethanol,
250 mM NaCl.

Translation of mRNAs
The in vitro translation is carried out in the presence of 35S methionine in two cell-free translation systems, namely in rabbit reticulocyte lysates (lanes 1 and 2 of Figure 4A) (with or without dog pancreatic microsomes), where the system 2-S / RNase L is present, or in wheat germ extracts (lanes 3 and 4), which does not have this system. The procedure is as specified by the manufacturer (Promega).

 The products are analyzed by SDS-PAGE and autoradiographed. In FIG. 4A, the results obtained after translation of cDNA of RLI (lanes 2 and 4) and of 2DR (lanes 1 and 3) are reported.

 Expression of the complete RLI cDNA in wheat germ extracts or rabbit reticulocyte leads to a single 68 kDa polypeptide as expected (Fig. 4, lanes 2 and 4).

 Translation of the 2DR cDNA yields a single 80 kDa polypeptide (Figure 4A, lanes 1 and 3). The level of expression is slightly lower in the wheat germ extract relative to the reticulocyte lysate for both clones.

 There is no posttranslational modification of these polypeptides when translated in the presence of pancreatic dog microsomal membranes).

 In another series of experiments, various extracts were analyzed by SDS-PAGE, Western blot and hybridization with the MAb3 antibody, specific for the RNase L complex. The autoradiographs obtained are given in FIG. 4B, and runway 1 corresponds to the extracts. of rabbit reticulocyte without mRNA, lane 2 to these extracts after translation of 2DR and lane 3 after translation of RLI, lane 4 to wheat germ extracts without mRNA, lane 5 to extracts after translation of 2DR and the lane 6 after translation of RLI.

 By comparing lanes 1 on the one hand and 2 and 3 on the other hand, it is found that only the 80 kDa polypeptide (RNABP) is revealed in the reticulocyte extract either before or after translation (compare lane 1 tracks 2 and 3).

 Similarly, the monoclonal antibody recognizes no protein in the wheat germ extract where the 2-5A / RNase L system does not exist and therefore RNABP is absent.

 In a third series of tests, the 2DR and RLI translation products are deposited on nitrocellulose sheets. The products are incubated with 2-5ApCp probe, according to the protocol used to screen the ZAP library. Then autoradiography analyzes are carried out. Autoradiographs are given in Figure 4C, where "O" is an experiment in wheat germ or rabbit reticulocyte without RNase L, 2DR or RLI; "RLI" to experiments with 0.8 x 10-15 moles of RLI in both wheat germ and 2DR reticulocyte extracts in experiments with 1.3 x 10-15 moles of 2DR in wheat germ or reticulocyte extracts.

 There is a slight binding of RLI at 2-5 ApCp in the wheat germ extract.

 Surprisingly, a decrease in 2-5ApCp binding by endogenous RNase L is observed when RLI is expressed in a rabbit reticulocyte lysate.

 An increase in the binding of 2-5ApCp is however clearly observed when 2DR is expressed in wheat germ extracts or those of rabbit reticulocytes (FIG. 4c).

This example shows that RLI binds only 2-5A weakly compared to 2DR. In addition, when
RLI and 2DR are present at the same time in the reticulocyte extract, there is an inhibition of the binding of 2-5A by endogenous 2DR (RNase L).

 Example: Demonstration of the inhibitory effect of RLI without RNAase L according to the dose.

(illustrated in Figure 5)
inhibition of RNase binding at 2-5
AT.

 Three series of experiments conducted in this study are reported below.

 a) Translations are carried out into reticulocyte or wheat germ extracts of mRNA encoding luciferase, RLI or 2DR.

 Different concentrations of the translation products are added to the reticulocyte or wheat germ extracts, as well as to an extract in which the 2DR mRNA has already been expressed.

 Thus, RLI (or luciferase), endogenous RNase L and 2DR expressed in vitro are different.

 The binding of 2-5 A to RNase L of these extracts is investigated in radiolabelling or covalent labeling assays.

 The results are given in Figure 5A expressed in% of 2-5 bound ApCp (100% corresponding to the binding obtained when 2DR alone is expressed).

correspond à un extrait réticulocyte + luciférase ;

à un extrait réticulocyte + RLI

à un extrait réticulocyte + 2DR + RLI ;

à un germe de blé + 2DR + RLI.In this figure, the curve

corresponds to a reticulocyte + luciferase extract;

to a reticulocyte extract + RLI

to a reticulocyte extract + 2DR + RLI;

to a wheat germ + 2DR + RLI.

 Expression of RLI in reticulocyte extracts led to a large, dose-dependent decrease in radiolabelling and radio-covalent binding of endogenous 2-5ApCp RNase L in the reticulocyte lysate (Figure 5A; Figure 5C, lanes 1 and 2).

 As expected, the expression of 2DR alone, in wheat germ or reticulocyte extracts, increases binding to 2-5ApCp in both assays (Figure 5A, Figure 5C lane 3).

 Coexpression of RLI leads to inhibition of 2-5ApCp binding by 2DR which is performed according to the radiolabel assay or radiocovalent assay (Figure 5A, Figure 5C lanes 3 and 5).

 No inhibition of endogenous RNase L in reticulocyte extract or expressed 2DR is observed after addition of a protein unrelated to the process under study such as luciferase (Figure 5A, Figure 5C, tracks 3 and 4). what serves as a witness.

 The same phenomenon is observed with other cell extracts.

 By way of example, the addition of RLI to extracts of Daudi cells or HeLa cells inhibits the binding of RNase L to 2-5ApCp (FIG. 5B).

 It is interesting to note that the translation of RLI into wheat germ extracts does not inhibit the binding of 2DR to 2-5ApCp (Fig. 5A).

 This difference between the two translation systems may reflect differences in the post-translational modifications of the protein.

 Inhibition of 2DR activity by RLI does not result from competition for 2-5A.

 Indeed, inhibition of 2DR binding to 2-5ApCp is not modified when performing the experiment with increasing concentrations of 2 SApCp since the ratio between 2DR and RLI is not modified.

 It will be noted with interest that the truncated initial clone H2ABP has the same properties as the complete clone. It inhibits the binding of endogenous RNase L, or 2DR expressed invitro, to 2-5ApCp.

 This example clearly shows that RLI inhibits the binding of RNase L to 2-5A in different cellular systems. In addition, it appears that this inhibition is not due to inhibition between the two proteins for 2-5A binding.

 Note also that FIG. 5A illustrates that the activation of the complex depends on the stoichiometry between RLI and RNase L, which is of interest for studying the involvement of RNase L in normal situations such as growth. or differentiation, but also in pathological cases such as viral infection or cancers.

 Example 5 Study of the Association of RLI with RNAase L or 2 DR (illustration in FIG. 6).

 After translation, in the presence of 35 S methionine, RLI and 2 DR mRNAs into wheat germ or rabbit reticulocyte lysates, immunoprecipitation is performed with the monoclonal antibody MAb3.

 For immunoprecipitation, cell extracts or incubations of translation products are incubated for 3 hours at room temperature with a dilution of 1/1000 MAb3 or control antibody.

The RNase L-antibody complexes are incubated for about 14 hours at 4 ° C with Protein A-Sepharose (Pharmacia) and recovered by centrifugation.The beads are washed several times with 10 mM Tris buffer (pH 7.4). ), 0.5% (v / v) aprotinin, 1% (v / v) NP40, 2 mM EDTA, 150 mM
NaCl, and resuspended in a buffer volume
300 mM Tris (pH 8.9), 5% (w / v) SDS, 5% (v / v) p-mercaptoethanol, 20% v / v glycerol.

 Protein A-Sepharose is heated for 5 minutes. at 95 ° C and the immunoprecipitated proteins are analyzed by SDS-PAGE 10% (w / v).

 The gel is dried and subjected to autoradiography.

The results are given in FIG. 6. It can be seen that, on lanes 2, 4 and 5, after translation into the reticulocyte, there is immunoprecipitation of the proteins,
RLI lane 2, 2DR lane 4, or co-immunoprecipitation of both, lane 5. This immunoprecipitation is via the RNABP association, a protein recognized by the antibody and present in the reticulocyte lysate.

On the other hand, when these proteins are translated into the wheat germ (lanes 1 and 3), hence the
RNABP is absent, no immunoprecipitation is observed.

 Thanks to the use of a specific antibody, it has been well demonstrated in this example that the RLI protein associates well with the protein that it inhibits, 2DR or RNase L.

Example: Stability of 2-5 ApCp (shown in Figure 7)
The reticulocyte extracts are supplemented or not with RLI or 2DR mRNA and incubated for 1 hour at 30 ° C under translation conditions.

 2-5 ApCp is added to these extracts, and the whole is incubated at 4 ° C for 15 minutes.

(as for the radio link) or at 37 ° C at different periods of time as shown in Figure 7.

 A buffer containing 50% (v / v) formamide, 1% (v / v) bromophenol blue and 1% (v / v) xylene cyanol is added.

The samples are boiled for 3 minutes and centrifuged at 10,000 g. The supernatants were loaded onto 20% (w / v) polyacrylamide gels containing 7M urea. The samples were analyzed by electrophoresis at 1100-1600 volts until a 15 cm staining was obtained. bottom of the gel with bromophenol blue. The wet gel is exposed to a film
Kodak.

 No significant degradation was observed in the reticulocyte + RLI extracts, either at +4 ° C (compare lanes 1 and 2) or at 37 ° C (compare lanes 4 to 9).

 This example shows that inhibition of 2-5A binding is not due to its degradation by RLI.

Example 7 Reactivation of RNase L after Separation from RLI (illustrated in FIG. 8)
The RLI mRNA is translated into a reticulocyte extract.

 The operation is carried out under the same conditions as those illustrated by Example 4 and FIG. 5A or C, but the fixation of 2-5ApCp on RNase L is carried out after RNase L has been separated from RLI by gel fractionation. SDS.

 By comparing lanes 1 and 2, it is found that the amount of RNase L is the same for lane 1 (reticulocyte alone) or lane 2 (reticulocyte + RLI).

 The example therefore shows that the RLI does not inhibit RNase L by degradation of the latter or irreversible modification.

Example 8 Stabilization of mRNAs in the Presence of RLI (illustrated in FIG. 9)
The rabbit reticulocyte extracts are incubated for 1 h at 30 ° C under translation conditions (1) without added mRNA (retic), (2) by adding RLI mRNA (retic + RLI), (3) adding 2DR mRNA (retic + 2
DR), (4) by adding 2DR and RLI mRNA (retic + 2DR +
RLI). Then 2-5A4 is added at 100 nm (+) or no (-) and the extracts are again incubated for 30 min at 30 ° C.

 The RNAs are isolated and analyzed on a 1.2% (w / v) agarose gel. RNAs and their degradation products are quantified by image analysis.

 This example shows that, in a reticulocyte extract, the RNAs are stabilized in the presence of RLI and this even by overexpressing 2DR.

Example 9: RLI activity in intact cells (shown in Figure 10)
Expression vectors and transfections
The human H2ABP cDNA, which encodes a truncated active form of RLI, is directionally subcloned into pcDNARIneo (Invitrogen) according to the standard methods described by Sambrook et al in the above reference. H2ABP / pcDNAIneo or the plasmid pcDNAneoI (each at 7 μg) are transfected into HeLa cells by co-precipitation with calcium phosphate. Stable transfectants are selected by culturing the cells in the presence of 1 mg / ml G418 (Gibco / BRL).

Individual clones were isolated for analysis of the expression of the transfected cDNA. The clone expressing the highest levels of cDNA is chosen to continue studies
Anti-viral activity of interferons.

 The pcDNAIneo or H2ABP / pcDNAIneo cells are inoculated at the rate of 105 cells per well. These cells are treated 24 hours later with different dilutions of IFN a / p Hu for 20 hours.

 The cells are then infected with vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) at a multiplicity of infections (moi) of 1.0 for 1 hour at 37 ° C in medium. RPMI supplemented with fetal calf serum at 5% (v / v) The unadsorbed virus was removed by 3 washes with RPMI containing 10% fetal calf serum (v / v).

 Virus titers are determined 18 hours later as described by Milhaud et al. Ann. Virol, 1983, 134 E 405-416.

 This example shows that overexpression of RLI in HeLa human cells inhibits some of the anti-viral effect of interferon for EMCV and not VSV (compare Figures 10A to 10B).

Example 10: Induction of RLI mRNA by EMCV (illustrated in FIG. 11)
HeLa cells are infected with a me

of 10 by EMCV or VSV, after or without treatment with IFN a / ss Hu at 1000 U / ml.

 The RNAs are then extracted from these cells at different post-infection times as described in Figure 11.

 After Northern Blot analysis of these messengers by operating as indicated above and quantification, FIG. 11 shows that induction of RLI mRNA is observed by infecting with EMCV and not with VSV and that this induction is partly reversed. by pre-treatment with interferon.

Example 11: Study of RNase Inhibition
L in sense or antisense transfectants of RLI in human HeLa cells (shown in Figure 12A to
F).

Human HeLa cells were transfected with pcDNAI neo expression vectors (HeLa W4),
H2ABP sense / pcDNAIneo (HeLa 7.3) and H2ABP antisense / pcDNAIneo (HeLa 8.2) according to the technique described in the previous example.

 Each clone was then infected with EMCV or VSV at one m.o.i. of 1 after or not having been treated with IFN a / s Hu at 1000 U / ml.

 With respect to EMCV infection (Fig. 12A), inhibition of IL-5 binding to RNase L increased during infection, mediated by interferon.

For HeLa 7,3 cells, expressing more than
RLI, inhibition of 2-5A binding is earlier and stronger (Figure 12B).

For HeLa 8.2 cells expressing antisense RLI, therefore less than RLI, during infection inhibition of 2-5A binding to RNase is not observed
L.

 FIGS. 12D, E, F show the same transfectants infected with VSV, this virus not inducing the inhibitor (as illustrated by FIG. 11 of the previous example), no variation in 2-5A binding to RNase L during infection.

SEQUENCE LIST (1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: NATIONAL INSTITUTE FOR HEALTH AND
MEDICAL RESEARCH
(B) STREET: 101 Tolbiac Street
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75654 cedex 13
(A) NAME: NATIONAL CENTER FOR SCIENTIFIC RESEARCH
(CNRS)
(B) STREET: 3 rue Michel Ange
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75794 cedex 16
(ii) TITLE OF THE INVENTION: new compounds usable for the
preparation of anti-infective agents
(iii) NUMBER OF SEQUENCES: 6
(iv) COMPUTER-READABLE FORM:
(A) SUPPORT TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 3568 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) Hypothesis: No
(iii) ANTI-SENSES: NO
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 118..1914
(xi) DESCRIPTION OF THE SEQUENCE:

SEQ ID NO: 1:
CCGGTCCTGA GACACGCTGT GTGGCTGAAA AGTGAAGGCA AGAGCTCATT TGGCCTCTGT 60
GCTCCCCTCC GCAAGGGATC GTTTCTCCAG AAGAGCTGGA TATTCTTTCG CCCAGTT 117
ATG GCA GAC AAG TTA ACG AGA ATT CGT ATT GTC AAC CAT GAC AAA TGT 165
AAA CCT AAG AAA TGT CGA AGC GAA TGC AAA AGT ATA TGT CCT GTA GTT 213
CGA ATG GGA AAA TTA TGC ATA GAG GTT ACA CCC CAG AGC AAA ATA GCA 261
TGG ATT TCC GAAT ACT CTT TGT ATT GGT TGT GGT ATC TGT ATT AAG AAA 309
TGC CCC TT GGC GCC TTA TCA ATT GTC AAT CTA CCA AGC AAC TTC GAA 357
AAA GAA ACC ACA CAT CGA TAT TGT GCC AAT GCC TTC AAA CTT CAC AGG 405
TTG CCT ATC CCT CGT CCA GGT GAA GTT TTC GGA TTA GTT GGA ACT AAT 453
GGT ATT GGA AAG TCA GCT GCT TTA AAA ATT TTA GCA GGA AAA CAA AAG 501
CCA AAC CTT GGA AAG TAC GAT GAT CCT CCT GAC TGG CAG GAG ATT TTC 549
ACT TAT TTC CGT GGA TCT GAA TTA CAA AAT TAC TT ACA AAG ATT CTA 597
GAA GAT CTA GAC AAA CGC ATC ATC AAA CCT CAAT TAT GTA CGC AGA TTC 645
CTA AGG CTG GCA AAG GGG ACA CTC GGA TCT ATT TTC GAC CGA AAA GAT 693
GAA ACA AAG ACA CAG GCA ATT GTA TCT CAG CAG CTT GAT TTA ACC CAC 741
CTA AAA GAA CGA AAT GTT GAAT GAT CTT TCA GGA GGA AGG TTC CAG AGA 789
TTT CGT TGT CGT GTC GTT TGC ATA CAG AAA CGT GAT ATT TTC ATC TT 837
GAT GAG CCT TCT AGT TAC CTA GAT CTG AAC CAG CGT TTA AAG CGT CGT 885
ATT ACT ATA CGA TCT CTA ATA AAT CCA GAT AGA TAT ATC ATT CTC CTC 933
GAA CAT GAT CTA GTA TTA GAC TAT GTG GCC TTC ATC TGC TCT 981
TTA TAT GGT GTA AGC AGC CGC TAT GGA GTT ACT GTC ACT ATC CCT TTT AGT 1029
GTA AGA GAA CCC ATA AAC ATT TTT TTC GAT GGC TAT GTT CCA ACA GAA 1077
AGA GST TCA CTT GTT TTC AAA CTC CGA GAC ACA 1125
GCA AAT GAA GAA GAA GTT AAA AAG ATC TGT ATG TAT AAA TAT CCA GGA 1173
ATG AAG AAA AAA ATG GGA GAA TTT GAG CTA GCA ATT GTA CGT GGA GAG 1221
TTT ACA GAT TCT GAA ATT ATC GTC ATC GTC GGG GAA AAT GGA ACG GGT 1269
AAA ACG ACA TT ATC AGA ATG CTT CGT GGA AGA CTT AAA CCT GAAT GAA 1317
GGA GGA GAA GTA CCA GTT CTA AAT CTG AGT TAT AAC CCA AGC AAA ATT 1365
AGT CCC AAA TCA ACT GGA AGT GTT CCC CAG TTA CTA CAT GAA AAC ATA 1413
AGA GAT CGT TAT ACT CAC CCA CAA TTT CTC ACC GAT GTA ATG AAC CCT 1461
CTG CAA ATT GAA AAC ATC ATT GAT CAA GAG GTC CAG ACA TTA TCT GGT 1509
GGT GAA CTA CAG CGA GTA CGT TTA CGC CTT TGC TTG GGC AAA CCT GCT 1557
GAT GTC TAT TTA ATT GAAT GAA CCA TCT GCA TAT TTG GAT TCT GAG CAA 1605
AGA CTG ATG GCA GCT CGA GTT GTC AAA CGT TTC ATA CTC CAT GCA AAA 1653
AAG ACA GCC TT GTT GTG GAA CAT GAC TTC ATC ATG GCC ACC TAT CTA 1701
GCG GAT CGC GTC ATC GTT TT GAT GGT GTT CCA TCT AAG AAC ACA GTT 1749
GCA AAC AGT CCT CAA ACC CTT TTG GCT GGC ATG AAT AAA TT TTG TCT 1797
CAG CTT GAA ATT ACA TTC AGA AGA GAT CCA AAC AAC TAT AGG CCA CGA 1845
ATA AAC AAA CTT AAT TCA ATT AAG GAT GAA GAA CAA AAG AAG AGT GGA 1893
AAC TAC TTT TTC TTG GAT GAT TAGACTGACT CTGAGAATAT TGATAAGCCA 1944
TTTATTAAAA GGAGTATTTA CTAGAATTTT TTGTCATATA AAACTTGAAT CAGGATTTTA 2004 TGCCCCACAT ACTCTGGAAC TTGAAGTATA ATATACTTAA TATAACATAA AAAGCCAGTT 2064
GGGTTCTAAA TTGTAGTTGA AACACAGAAA ATGCCACTTT TCTGTTCCTG AAGAGGCTCT 2124
TTTGTGCATA ATATTCTAAA ATGAAGACAT TTCAAGCTAT ACAAATTACT TCCAAGTTTT 2184 CATGATGTAT GGGAAGATTT TCAGTAGGTG TATTATATTC ACGGTACCAA ATGCTGACCA 2244
GTGTTGCTCC ATTTTTTAAA TCTTGAAAAG GGTTTCTGTA CTTACCTGGT TTGCCAAGTA 2304 TGCCAGTGTA ATGAAACTGC CCTTATTTTA AAAGCCAGTC AAAGATTCCA CTGATTGACA 2364
TTTGATAAAT AAACATCAGG ATTATGTTTA TTGTTTGTTT TCAGTCTTTG CACTATATTA 2424 CCAGTATATG GTTTCCGAGG AAGATTATCT ACTGCAAAAC ACCACTGTTG GAAAAATAGG 2484 TATTTTTAAA TTGTTTTTAA TCCTTTTTTG GTGCTTTTAA ACATGTTTAA GCAAAAACCA 2544
ATTCAGTCCA TTCCCCGCAA AAAACCCCTA ACTTTACTCT GAACTTTTTT TGTTTTTGCA 2604
TTCCATGAGG TTCTGTATTC AGTCATTCTC TAGGTAATGT CATTTTTGTA CACATATATT 2664
TATATAATCA CTGATTGAGA TTAGGAAAA AGCATTTCTA AAGAATATTT GCTTCCCTTA 2724
GAACTACAGA CTCGAAATCT TTAAAGATGG TGCCTAAGCA TCTATGTATT TTTTTTAAGT 2784 TCCACAGATT TTTCTGTTGG GCAGGCCAAG GATTATAAAC CACTTCCCTA AAGGCAACAT 2844
TAATGCAAAA GTCCCCAGAT GGCAATACAA AGTATCCCCT GGTACCACAT ATATTCATTT 2904
GTGAGTTTGG ATATAGAGCA CATTATCTAA ACCATTTTGT AGTTCCAAAA ACCCATCTAA 2964
ATTTCTTGAG TTCCTGAATT TTGAACAGGA TTACCTGGAG CCTGGAGCCA CTTTAAGTTG 3024
TACTTCTGAC TAAACTGGAA TTATGAGTGA GGAAGAGTGT TTACTAAATA AATGACTGGG 3084 GCAAGCAAAA TTGAGGAGGA AATTAGAAAC TGTTTGACAA ACTTTAAGAG CTACTTGAAA 3144 TAACAGAAGT CTTGATTAAT ATGCAAATAA TGGCTAGAAA GTATGGTTTA ACTGGACCCT 3204
ATTATGCCTT TTAAAAATAA TTTCAGTAAC CCATAAATAC ATGTTGTAAA AAATTCAAAT 3264 ATACAGAATG GAATAAAAAA ATGATCTCCC TTTATTACCC TCCCAAAGGT TACCAGCGTT 3324 TGAATTTAAT AATGTATATT CTTTCATGCT TTTTTCTGTG CACTTACCTA AGTGTGAATA 3384
TGTAAAGGGT TTGTTTTGTA TACAAATGGG ATTATACTAA AATAAGTAAT GCCTATTTTT 3444
AAGGATAGGT TAAATTTGTG AATGATCATT TCAAATATAT TGAATAAAAT AAGCAAAAGC 3504
TATTGTTATT TACTGATCCT GAAAAAAAAA to AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 3564
YYYY 3568
(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 2861 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) Hypothesis: No
(iii) ANTI-SENSES: NO
(ix) ADDITIONAL CHARACTERISTIC:
(A) NAME / KEY: CDS
(B) LOCATION: 2..1207
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
G GCA ATT GTA TGT CAD CAG CTT GAT TTA ACC CAC CTA AAA GAA CGA 46
AAT GTT GAAT GAT CTT TCA GGA GGA GAG TTG CAG AGA TT GCT TGT GCT 94
GTC GTT TGC ATA CAG AAA GCT GAT ATT TTC ATG TT GAT GAG CCT TCT 142
AGT TAC CTA GAT GTC AAG CAG CGT TTA AGG GCT ATT ACT ATA CGA 190
TCT CTA ATA AAT CCA GAT AGA TAT ATC ATT GTG GTG GAA CAT GAT CTA 238
AGT GTA TTA GAC TAT CTC TCC GAC TTC ATC TGC TGT TTA TAT GGT GTA 286
CCA AGC GCC TAT GGA GTT GTC ACT ATG CCT TT AGT GTA AGA GAA GGC 334
ATA AAC ATT TTT TTG GAT GGC TAT GTT CCA ACA GAA AAC TTG AGA TTC 382
AGA GAT GCA TCA CTT GTT TT AAA GTG GCT GAG ACA GCA AAT GAA GAA 430
GAA GTT AAA AAG ATG TGT ATG TAT AAA TAT CCA GGA ATG AAA AAA AAA 478
ATG GGA GAA TT GAG CTA GCA ATT GTA GCT GGA TT GAG ACA GAT TCT 526
GAA ATT ATG GTG ATG CTG GGG GAA AAT GGA ACG GGT AAA ACG ACA TT 574
ATC AGA ATG CTT GCT GGA AGA CTT AAA CCT GAT GAA GGA GGA GAA GTA 622
CCA GTT CTA AAT GTC AGT TAT AAG CCA AGC AAA ATT AGT CCC AAA TCA 670
ACT GGA AGT GTT CGC CAG TTA CTA CAT GAA AGA ATA AGA GAT GCT TAT 718
ACT CAC CCA CAA TT GTG ACC GAT ATG GTA ATG CCT CTG CAA ATT GAA 766
AAC ATC ATT GAT CAA GAG GTG CAG ACA TTA TCT GGT GGT GAA CTA CAG 814
CGA GTA CGT TTA CGC CTT TGC TTG GGC AAA CCT GCT GAT GTC TAT TTA 862
ATT GAAT GAA CCA TCT GCA TAT TTG GAT TCT GAG CAA AGA CTG ATG GCA 910
GCT CGA GTT GTC AAA CGT TTC ATA CTC CAT GCA AAA AAG ACA GCC TTT 958
GTT GTG GAA CAT GAC TTC ATC ATG GCC ACC TAT CTA GCG GAT CGC GTC 1006
ATC GTT TT GAT GGT GTT CCA TCT AAG AAC ACA GTT GCA AAC AGT CCT 1054
CAA ACC CTT TTG GCT GGC ATG AAA TT TT TTG TCT AGC CTT GAA ATT 1102
ACA TTC AGA AGA GAT CCA AAC AAC TAT AGG CCA CGA ATA AAC AAA CTT 1150
AAT TGA ATT AAG GAT GAA GAA GAA AAG AGA GGA AAC TAC TT TTC 1198
TTG GAT GAT TAGACTGACT CTGAGAATAT TGATAAGCCA TTTATTAAAA 1247
GGAGTATTTA CTAGAATTTT TTGTCATATA AAACTTGAAT CAGGATTTTA TGCCCCACAT 1307
ACTCTGGAAC TTGAAGTATA ATATACTTAA TATAACATAA AAAGCCAGTT GGGTTCTAAA 1367
TTGTAGTTGA AACACAGAAA ATGCCACTTT TCTGTTCCTG AAGAGGCTCT TTTGTGCATA 1427
ATATTCTAAA ATGAAGACAT TTCAAGCTAT ACAAATTACT TCCAAGTTTT CATGATGTAT 1487
GGGAAGATTT TCAGTAGGTG TATTATATTC ACGGTACCAA ATGCTGACCA GTGTTGCTCC 1547
ATTTTTTAAA TCTTGAAAAG GGTTTCTGTA CTTACCTGGT TTGCCAAGTA TGCCAGTGTA 1607
ATGAAACTGC CCTTATTTTA AAAGCCAGTC AAAGATTCCA CTGATTGACA TTTGATAAAT 1667
AAACATCAGG ATTATGTTTA TTGTTTGTTT TCAGTCTTTG CACTATATTA CCAGTATATG 1727
GTTTCCGAGG AAGATTATCT ACTGCAAAAC ACCACTGTTG GAAAAATAGG TATTTTTAAA 1787
TTGTTTTTAA TCCTTTTTTG GTGCTTTTAA ACATGTTTAA GCAAAAACCA ATTCAGTCCA 1847
TTCCCCGCAA AAAACCCCTA ACTTTACTCT GAACTTTTTT TGTTTTTGCA TTCCATGAGG 1907
TTCTGTATTC AGTCATTCTC TAGGTAATGT CATTTTTGTA CACATATATT TATATAATCA 1967
CTGATTGAGA TTTAGGAAAA AGCATTTCTA AAGAATATTT GCTTCCCTTA GAACTACAGA 2027
CTCGAAATCT TTAAAGATGG TGCCTAAGCA TCTATGTATT TTTTTTAAGT TCCACAGATT 2087
TTTCTGTTGG GCAGGCCAAG GATTATAAAC CACTTCCCTA AAGGCAACAT TAATGCAAAA 2147
GTCCCCAGAT GGCAATACAA AGTATCCCCT GGTACCACAT ATATTCATTT GTGAGTTTGG 2207 ATATAGAGGA CATTATCTAA ACCATTTTGT AGTTCCAAAA ACCCATCTAA ATTTCTTGAG 2267
TTCCTGAATT TTGAA QGGA TTACCTGGAG CCTGGAGCCA CTTTAAGTTG TACTTCTGAC 2327 TAAACTGGAA TTATGAGTGA GGAAGAGTGT TTACTAAATA AATGACTGGG GCAAGCAAAA 2387
TTGAGGAGGA AATTAGAAAC TGTTTGACAA ACTTTAAGAG CTACTTGAAA TAACAGAAGT 2447
ATGCAAATAA CTTGATTAAT TGGCTAGAAA GTATGGTTTA ACTGGACCCT ATTATGCCTT 2507
TTAAAAATAA TTTCAGTAAC CCATAAATAC ATGTTGTAAA AAATTCAAAT ATACAGAATG 2567
GAATAAAAAA ATGATCTCCC TTTATTACCC TCCCAAAGGT TACCAGCGTT TGAATTTAAT 2627
AATGTATATT CTTTGATGCT TTTTTCTGTG CACTTACCTA AGTGTGAATA TGTAAAGGGT 2687
TTGTTTTGTA TACAAATGGG ATTATACTAA AATAAGTAAT GCCTATTTTT AAGGATAGGT 2747
TAAATTTGTG AATGATCATT TCAAATATAT TGAATAAAAT AAGCAAAAGC TATTGTTATT 2807
TACTGATCCT GAAAAAAAAA AMAAAAAAAA ALAAAAAPSA AAAALAAASA AAAA 2861 (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 599 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
Met Ala Asp Lys Leu Thr Arg Ala Island Val Asn Island His Asp Lys Cys
1 5 10 15
Lys Pro Lys Lys Cys Arg Gln Glu Lys Cys Lys Ser Cys Pro Val Val
20 25 30
Arg Met Gly Lys Leu Cys Island Glu Val Thr Pro Gln Ser Lys Island Ala
35 40 45
Trp Ile Ser Glu Thr Leu Cys Ile Gly Cys Gly Ile Cys Ile Lys Lily
50 55 60
Cys Pro Phe Gly Ala Leu Ser Val Asn Leu Leu Ser Asn Leu Glu
65 70 75 80
Lys Glu Thr Thr His Arg Tyr Cys Ala Asn Ala Phe Lys Leu His Arg
85 90 95
Pro Pro Leu Pro Pro Gly Glu Leu Val Leu Gly Val Leu Gly Thr Asn
100 105 110
Gly Ile Gly Lys Ala Ala Ala Leu Lys Leu Ala Gly Lys Gln Lys
115 120 125
Pro Asn Leu Gly Lys Tyr Asp Asp Pro Pro Asp Trp Gln Glu Ile Leu
130 135 140
Thr Tyr Phe Arg Gly Ser Glu Leu Gln Asn Tyr Phe Thr Lys Ile Leu 145 150 155 160
Glu Asp Asp Leu Lys Ala Ile Lys Island Gln Tyr Val Ala Arg Phe
165 170 175
Leu Arg Leu Ala Lys Gly Thr Val Gly Ser Ile Leu Asp Arg Lys Asp
180 185 190
Glu Thr Lys Thr Gln Ala Val Cys Gln Gln Island Leu Asp Leu Thr His
195 200 205
Leu Lys Glu Arg Asn Val Glu Asp Leu Ser Gly Gly Glu Leu Gln Arg
210 215 220
Phe Ala Cys Ala Val Val Cys Island Gln Lys Ala Asp Phe Island Met Phe 225 230 235 240
Asp Glu Pro Ser Ser Tyr Leu Asp Val Lys Gln Arg Leu Lys Ala Ala
245 250 255
Ile Thr Ile Arg Ser Leu Ile Asn Pro Asp Asp Arg Tyr Ile Val Val
260,265,270
Glu His Asp Leuk Leu Ser Val Leu Asp Leuk Tyr Ser Asp Asphe Ile Cys Cys
275 280 285
Leu Tyr Gly Val Ser Ser Ala Tyr Val Gly Val Thr Met Pro Phe Ser
290,295,300
Val Arg Glu Gly Ile Asn Ile Phe Leu Asp Asp Gly Tyr Pro Val Thr Glu 305 310 315 320
Asn Leu Arg Phe Arg Asp Ala Ser Leu Val Phe Lys Val Ala Glu Thr
325 330 335
Ala Asn Glu Glu Glu Val Lys Lys Met Cys Lys Tyr Lys Tyr Pro Gly
340,345,350
Met Lys Lily Lys Met Gly Glu Phe Glu Leu Ala Island Val Ala Gly Glu
355 360 365
Phe Thr Asp Ser Glu Ile Met Val Met Leu Gly Glu Asn Gly Thr Gly
370,375,380
Lys Thr Thr Phe Arg Island Met Leu Ala Gly Arg Leu Lys Pro Asp Glu
385 390 395 400
Gly Gly Glu Pro Val Val Asn Val Val Ser Tyr Lys Pro Gln Lys Ile
405 410 415
Ser Pro Lys Ser Thr Gly Ser Val Arg Gln Leu Leu His Glu Lys Ile
420,425,430
Arg Asp Ala Tyr Thr His Pro Gln Phe Val Thr Val Val Met Lys Pro
435 440 445
Leu Gln Island Glu Asn Island Asp Asp Gln Glu Gln Val Thrn Leu Ser Gly
450 455 460
Gly Glu Leu Gln Arg Val Arg Leu Arg Leu Leu Leu Gly Lys Pro Ala
465 470 475 480
Asp Val Tyr Leu Asp Glu Pro Asp Ser Ala Tire Leu Asp Ser Glu Gln
485 490 495
Arg Leu Met Ala Ala Arg Val Val Lys Arg Phe Ile Leu His Ala Lys
500 505 510
Lys Thr Ala Phe Val Val Glu His Asp Phe Ile Met Ala Thr Tyr Leu
515 520 525
Ala Asp Arg Val Val Val Phe Asp Val Gly Pro Val Ser Lys Asn Thr Val
530,535,540
Ala Asn Ser Pro Gln Leu Leu Leu Ala Gly Met Asn Lys Phe Leu Ser
545 550 555 560
Gln Leu Glu Thr Thread Arg Arg Pro Asn Asn Arg Arg Arg
565 570 575
Asn Island Lys Leu Asn Ser Lys Island Asp Val Glu Gln Lily Lys Ser Gly
580 585 590
Asn Asp Tyr Phe Leu Asp Asp
595 (2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 402 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Ala Ile Val Cys Gln Gln Asp Leu Leu Leu Leu Glu Arg Asn
15 10 15
Val Glu Asp Leu Ser Gly Gly Glu Leu Gln Arg Phe Ala Cys Ala Val
20 25 30
Val Cys Island Gln Lys Ala Asp Phe Island Met Phe Asp Glu Ser Pro Ser
35 40 45
Tyr Leu Asp Val Lys Gln Arg Leu Lys Ala Ala Island Thr Island Arg Ser
50 55 60
Leu Ile Asn Pro Asp Arg Tyr Ile Val Val Glu His Asp Asp Ser Ser
65 70 75 80
Val Leu Asp Tyr Tire Leu Ser Asp Phe Ile Cys Cys Leu Tire Gly Val Pro
85 90 95
Ser Ala Tyr Gly Val Thr Val Met Pro Phe Ser Arg Val Glu Gly Ile
100 105 110
Asn Ile Phe Leu Asp Asp Gly Tyr Pro Val Thr Glu Asn Leu Arg Arg Arg
115 120 125
Asp Ala Ser Leu Val Phe Lily Val Ala Glu Thr Ala Asn Glu Glu Glu
130 135 140
Val Lys Lys Met Cys Met Tyr Lys Tyr Pro Gly Met Lys Lys Lys Met 145 150 155 160
Gly Glu Phe Glu Leu Ala Island Val Ala Gly Glu Phe Thr Asp Ser Glu
165 170 175
Ile Met Val Met Gly Glu Leu Asn Gly Thr Gly Lys Thr Thr Phe Ile
180 185 190
Arg Met Leu Ala Gly Arg Leu Lys Asp Asp Glu Gly Gly Glu Pro Val
195 200 205
Val Leu Asn Val Ser Tire Lys Pro Gln Lys Island Ser Pro Lys Ser Thr
210 215 220
Gly Ser Val Arg Gln Leu Leu His Glu Lilies Isle Arg Asp Ala Tyr Thr 225 230 235 240
His Pro Gln Phe Val Thr Val Val Met Lys Pro Leu Gln Glu Island Asn
245 250 255
Ile Asp Asp Gln Glu Gln Val Gln Leu Leu Gly Gly Glu Leu Gln Arg
260,265,270
Val Arg Leu Arg Leu Cys Leu Gly Lys Pro Ala Asp Val Tyr Leu Ile
275 280 285
Asp Glu Pro Ser Ala Leu Leu Asp Ser Glu Arg Gln Leu Met Ala Ala
290,295,300
Arg Val Val Lys Arg Phe Island Leu His Ala Lily Lys Thr Ala Phe Val 305 310 315 320
Val Glu His Asp Phe Island Met Ala Thr Tire Leu Ala Asp Arg Val Ile
325 330 335
Val Phe Asp Gly Val Ser L Pro Asn Thr Val Ala Asn Pro Ser Gln
340,345,350
Thr Leu Leu Ala Gly Met Asn Lys Phe Leu Ser Gln Leu Glu Ile Thr
355 360 365
Phe Arg Arg Asp Pro Asn Asn Tyr Arg Pro Arg Asn Ile Lys Leu Asn
370,375,380
Ser Ile Lys Asp Glu Val Glu Lys Ser Gly Asn Tyr Phe Phe Leu 385 390 395 400
Asp Asp

Claims (29)

 1 / nucleotide sequences, isolated from their natural environment, characterized in that they are capable of hybridizing with at least one fragment of the sequence SEQ ID No. 1 or SEQ ID No. 2 which encodes a polypeptide capable of to inhibit RNase L.  2 / nucleotide sequences, isolated from their natural environment, characterized in that they correspond to at least part of the sequences SEQ ID No. 1 and SEQ ID No. 2, capable of encoding an RNase L inhibitory polypeptide.  3 / nucleotide sequences, isolated from their natural environment, characterized in that they are capable of encoding, implemented according to standard recombinant DNA techniques, for polypeptides having an RNase L inhibitory activity, comprising at least less a sequence of amino acids in the sequence SEQ ID No. 3.  4 / sequence of nucleotides according to claim 2 or 3, characterized in that it corresponds to the open reading frame from position 118 to 1914 in SEQ ID No. 1.  5 / sequence according to claim 4, characterized in that it comprises two patterns of the P loop type.  6 / The mRNA sequences corresponding to the sequences according to one of claims 1 to 5.  7 / The antisense sequences corresponding to the sequences according to one of the preceding claims.  8 / The cDNAs correspond to the sequences according to one of claims 1 to 7.  9 / nucleic acid sequences according to one of the preceding claims inserted into an expression vector for polypeptides having RNase L. inhibitory activity.  10 / Polypeptides, characterized in that they possess an RNase L inhibitory activity and are constituted by a sequence of amino acids in the sequence encoded by SEQ ID No. 1 or SEQ ID No. 2.  11 / Polypeptides according to claim 10, characterized by a sequence of amino acids in SEQ ID No. 3 or in SEQ ID No. 4.  12 / Polypeptides according to claim 10 or 11, characterized in that they possess in their sequence of amino acids a sequence homologous with the CX2CX2CX3C: 4Fe4S-ferredoxin motif and / or a high number of thiol groups and / or a high number of leucine motifs.  13 / A polypeptide according to claim 10, characterized in that it corresponds to the amino acid sequence encoded by the open reading frame in the sequence SEQ ID No. 1, which comprises 599 amino acids, and that has an estimated molecular weight of 67.515 kDa and an isoelectric point estimated at 8.7.  14 / Polypeptides according to one of claims 10 to 13, characterized in that they are as obtained by expression, in an appropriate host cell, according to recombinant DNA techniques, an expression vector comprising a DNA sequence consisting of a sequence of nucleotides in the sequence SEQ ID No. 1 or SEQ ID No. 2, recovery of the expressed polypeptide and purification.  15 / Method for obtaining the polypeptides according to one of claims 10 to 14, characterized in that it comprises screening a cDNA library with a 25A labeled for example with a radioactive element, the isolation of the desired nucleic acid sequence, its introduction into an expression vector, the transfection of a host cell with this vector, and then after the expression of the RNase-inhibiting polypeptide encoded by the said nucleic sequence, the recovery of this last, after lysis of the cells, and its purification.  16 / expression vectors containing at least one of the nucleotide sequences according to one of claims 1 to 8, subjected to the control of a suitable promoter.  17. The host cells transfected with the vectors according to claim 16 and which comprise the expressed recombinant proteins.  18 / Use of the polypeptides according to one of claims 10 to 14 as regulatory agents of the 2-5A / RNase L system.  19 / The use of the polypeptides according to one of claims 10 to 14 as target agents for detecting products capable of inhibiting their own inhibitory effect and thus making it possible to regulate the operation of the 2-5A / RNase L system, and hence of increase the action of interferons.  20 / Products capable of inhibiting the RNase L inhibitory effect of the polypeptides according to one of claims 10 to 14, characterized in that they are polyclonal or monoclonal antibodies directed against these polypeptides.  21 / A method of screening molecules or substances for detecting in them an activity directed against the inhibitory activity of the RNase L polypeptides according to one of claims 10 to 14, characterized in that the polypeptide is contacted with a molecule or substance to be tested, and that the reactivation of RNase L, again capable of binding 2-5A, or the inhibition of its inhibitor, is detected.  22 / Use of the polypeptides according to one of claims 10 to 14, to find active products vis-à-vis virus-induced RNase L inhibitors, capable of neutralizing them.  23 / Use of antisense nucleotide sequences according to claim 7 for transfecting mammalian cells to prevent the production of RNase L inhibitors by infectious agents.  24 / A pharmaceutical composition containing vectors such as retroviruses or recombinant viruses modified for use in the mammal, containing an antisense nucleotide sequence according to claim 7.  25 / A pharmaceutical composition containing vectors such as retroviruses or recombinant viruses, modified for use in the mammal, containing a nucleotide sequence according to one of claims 1 to 5, these vectors being in association with a pharmaceutically inert vehicle.  26 / A pharmaceutical composition containing vectors such as retroviruses or recombinant viruses, modified to be usable in the mammal, containing a nucleotide sequence according to claim 6, these vectors being in association with a pharmaceutically inert vehicle.  27 / A pharmaceutical composition containing vectors such as retroviruses or recombinant viruses, modified for use in the mammal, containing a nucleotide sequence according to claim 8, these vectors being in association with a pharmaceutically inert vehicle.  28 / A pharmaceutical composition containing vectors such as retroviruses or recombinant viruses, modified to be usable in the mammal, containing a nucleotide sequence according to claim 9, these vectors being in association with a pharmaceutically inert vehicle.  29 / Pharmaceutical composition according to one of claims 24 to 28, characterized in that it is in the form of a sterile solution, isotonic or is stored in freeze-dried form.