FR3017879A1 - CELLS AND METHODS FOR SELECTING ACTIVE COMPOUNDS AGAINST GAMMA HERPES VIRUS INFECTION - Google Patents

Abstract

An in vitro method for selecting active compounds against infection with a gamma herpesvirus, comprising the steps of: a) culturing yeast cells in the genome of which at least one copy of a nucleic acid has been inserted rendering said cells capable of expressing a recombinant protein comprising at least a [GMP] moiety and at least a [REPORTER] moiety, wherein: [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, and the yeast cells being cultured in the presence of a candidate compound, b ) measuring the level of production of said recombinant protein by the cells cultured in step a), and c) comparing the level of production of said recombinant protein measured in step b) with a value of reference.

Description

FIELD OF THE INVENTION The present invention relates to the field of the identification of antiviral compounds, and in particular the identification of active compounds against infection by a gamma herpes virus.

PRIOR ART The herpes virus family represents a group of double-stranded DNA viruses which is widely distributed within the animal kingdom. Herpes viruses are the second leading cause of viral diseases in humans, just after diseases caused by infections with influenza viruses. Certain pathologies caused by herpes viruses, and in particular gamma herpes viruses, have important consequences for the lives of patients. By way of illustration, infection with Epstein-Barr virus (EBV), which is a gamma herpesvirus, is the causative agent of Burkitt's lymphoma, nasopharyngeal carcinoma and infectious mononucleosis. However, there is currently no therapeutic treatment against EBV. Only therapeutic tools of the vaccine type are currently under development. Another illustration concerns infections with gamma herpes virus HHV8 (also called KSHV), which cause Kaposi syndrome. The HHV8 virus is associated with lympho-proliferative disorders associated with the AIDS disease. For this other gamma herpes virus, the therapeutic arsenal is also limited or nonexistent. As an illustration of treatments against cancers induced by KSHV, mention may be made of surgery or local irradiation, anti-herpes treatment with ganciclovir, HAART treatment in patients with HIV, or liposomal anthracyclines such as daunorubicin, doxorubicin, or combinations of doxorubicin / bleomycin / vinblastine or bleomycin / vinblastine. These liposomal anthracyclines may also be associated with anti-angiogenic therapy with IL-12. Herpesviruses include subfamilies of Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae (or gamma herpesvirus). One of the main features of herpes viruses is their ability to establish long-lasting latent infection in hosts. Latent infections are established after an initial stage of primary infection, the latent infection being characterized by the persistence of the virus in specific host cells in the absence of detectable production of infectious virus. Two fundamental features of these latent infections are (i) the maintenance of the viral genome in the infected host cell and (ii) the ability of the virus to escape detection by the immune system. Gamma herpes viruses establish latent infections in B or T lymphocytes. The mechanisms contributing to the maintenance of the latency of this herpesvirus subfamily have been well characterized. Studies on the latent infection of gamma herpes viruses have highlighted the essential role of a category of viral proteins, called "stability", designated GMP (for "Genome Maintenance Protein"). The GMP protein (s) play an essential role (i) in the persistence of the viral genome in latently infected cells and (ii) in the transmission of the viral genome to the daughter cells during cell divisions. In addition, recent studies have revealed the mechanisms that allow the GMP viral protein to be expressed in latently infected cells and simultaneously escape detection by CD8 + cytotoxic T lymphocytes from the host organism. During the latent infection phase, downregulation of viral gene expression is an essential feature that allows gamma herpes viruses to escape surveillance of the immune system. However, because of the central role of the GMP protein in the long-term latency phase, any escape strategy of control by the immune system must involve the GMP protein itself. In view of current knowledge, the overall effect of escape properties to immune system control involving GMP proteins is a conserved regulatory mechanism in gamma herpes viruses in general. The most numerous studies concerning the mechanisms of action of GMP proteins relate to Epstein-Barr virus (or EBV), for which the GMP protein is designated EBNA1 (for "Epstein-Barr Nuclear Antigen 1"). Those skilled in the art may particularly refer to Blake ("Immune evasion by gamma herpes virus genome maintenance proteins" Journal of General Virology (2010) 91, 829-846) for an introduction to the so-called "GMP" proteins. . Like all GMPs of gamma herpes viruses, EBNA1 protein is essential for maintaining viral persistence. However, despite the strong antigenicity of the EBNA1 protein and the presence of antigenic peptide-specific T cells derived from EBNA1, the immune system does not detect and destroy EBNA1-expressing cells. The stealthiness of the EBNA1 protein is due to the inhibition of the translation of the mRNA encoding EBNA1 by a cis-acting mechanism (referred to as "cis-inhibition" or "cis inhibition"), which mechanism is induced by a repeated amino acid sequence, called the GAr domain (or "Glycine-Alanine repeat"). "Portion [GAr]" is understood to mean a Glycine-Alanine repeat sequence, which consists of a series of Alanines, separated by one, two or three glycines located at the N-terminal portion of EBNA1 of the Epstein- Barr (see Levitskaya et al., "Inhibition of antigen processing by the internal region of the Epstein-Barr virus nuclear antigen-1; Nature (1995); 375, 685-8). For reference, the peptide sequence of the EBNA1 protein (B95-8 strain) of Epstein-Barr virus, as published by Baer et al. (Nature, 1984, vol 310) is represented by the sequence SEQ ID No. 1. Also for reference, the complete peptide sequence of the GAr domain of the EBNA1 protein of sequence SEQ ID No. 1, also called "239 GAr" because of a length of 239 amino acids, is represented by the sequence SEQ ID No. 2. Thus, according to one embodiment, the portion [GAr] may be a peptide of sequence SEQ ID No. 2, also called "239GAr", which is encoded by a nucleic acid of sequence SEQ ID No. 3. It is therefore known that the GAr domain contained in the GMP EBNA1 protein controls the translation of its own mRNA, thus minimizing the production of EBNA1-derived peptides which can be presented on the surface of the infected host cells in association with an antigen of Class I of the CMI-I. Obtaining an effective CD8 + T cell-mediated immune response against EBV-infected cells in which the GAr domain of the EBNA1 protein has been deleted demonstrates the importance of the GAr domain in the strategy of the virus. inhibit the presentation of EBNA1-derived antigens to cells of the immune system (Apcher et al., 2010, PLoS Pathog, Vol 6: e100151; Levitskaya et al., 1995, Nature, Vol 375: 685-688; Yin et al., 2003, Science, Vol 301: 1371-1374).

As mentioned above, the existing therapeutic tools against pathologies caused by gamma herpes viruses are today very limited. Essentially, it is the development of vaccine strategies, whose effectiveness is uncertain because of the stealthiness of these viruses that allows them to escape the control of the viral infection by the immune system, mainly by CD8 + T cells. There is therefore a need for the availability of active substances useful against infection with a gamma herpes virus, including methods for selecting such active substances.

In particular, there is a need for the implementation of screening methods to identify new compounds that may affect the ability of these gamma herpes viruses to escape the host's immune system. There is a particular need to identify compounds that can treat and / or prevent EBV-associated pathologies, such as EBV-associated cancers. The object of the invention is to satisfy these needs. SUMMARY OF THE INVENTION The present invention provides an in vitro method of selecting active compounds for infection with gamma herpesvirus, comprising the steps of: a) culturing yeast cells in the genome of which at least one copy of a nucleic acid making these cells capable of expressing a recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER], wherein: [GMP] signifies a polypeptide comprising any or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, and the cells yeast being cultured in the presence of a candidate compound, b) measuring the level of production of said recombinant protein by the cells cultured in step a), and c) comparing the level of production of said The present invention provides in particular an in vitro method for the selection of active compounds against a gamma herpesvirus infection, comprising the following steps: a) a recombinant protein measured in step b) with a reference value; culturing yeast cells in the genome of which at least one copy of a nucleic acid has been inserted, making these cells capable of expressing a recombinant protein of following formula (I): NH2- [TAG1], [GMP] - [REPORTER] [TAG2] y-COOH (I), in which: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1 - [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such said yeast cells, the yeast cells being grown in the presence of a candidate compound, b) measuring the level of production of the protein of formula (I) by the cells cultured in step a), and c) comparing the level of production of the protein of formula (I) ) measured in step b) with a reference value. In some embodiments of the method, the recombinant protein comprises a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of Epstein-Barr virus. The invention also relates to an in vitro method of selecting active compounds against Epstein-Barr virus infection comprising the following steps: 1) culturing mammalian cells having the following characteristics: (i)) said cells express a MHC Class I antigen, and (ii) said cells express a recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER], wherein: - [GMP] means a polypeptide comprising all or part of the amino acid sequence of an Epstein-Barr virus stability protein, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells, mammalian cells being cultured in the presence of a candidate compound, 2) measuring the expression level of the class I MHC-associated recombinant protein on the surface of cells cultured in step a), and 3) comparing the level ofexpressing the recombinant protein measured in step b) with a reference value. The invention also relates to the in vitro method of selection above (or "second method"), wherein the candidate compound is selected, or preselected, by the implementation of the first method. The invention also relates to a yeast cell in the genome from which has been inserted at least one copy of a nucleic acid making these cells capable of expressing a recombinant protein as described above. The invention also relates to a mammalian cell in the genome from which has been inserted at least one copy of a nucleic acid making these cells capable of expressing a recombinant protein as described above, as well as a MHC antigen. The invention also relates to cells in the genome from which a single copy of a nucleic acid has been inserted, making these cells capable of expressing a recombinant protein as described above. The invention also relates to selection methods, as described above, and implementing said cells. DESCRIPTION OF THE FIGURES Figure 1 illustrates the development of a GAr-mediated translation inhibition test in yeast cells.

Figure IA: Schemes showing nucleic acid constructs encoding fusion proteins comprising the Ade2p enzyme whose N-terminus is fused to a specified length GAr domain. pADH: promoter of the ADH gene; HA: polynucleotide encoding the HA peptide; xxGAr: polynucleotide encoding a GAr domain having "xx" amino acids in length; FIG. 1B. SDS PAGE and Western Blot gel analysis of the protein extracts of the clone cells ADE2, ade24, 2IGArADE2, 43GArADE2 and 235GArADE2; Figure IC: SDS PAGE and Western Blot gel analysis of the protein extracts of the clone cells ADE2, ade2A and Papio-30GArDE2; Figure ID: Determination of half-life time of HA-Ade2p and HA-43GAr-Ade2p fusion proteins by the cycloheximide flushing method. Representative results from five independent tests. Figure 1E: Relative levels of HA-ADE2, ade24, 43GArADE2 and Papio-30GArDE2 mRNAs using the qRT-PCR technique. This diagram represents the average of the results of three independent tests. Figure 2 illustrates the effect of GAr domain on translation into yeast cells is independent of the promoter used. The polynucleotide encoding the 235 GAr domain (full-length GAr domain) fused to the ADE2 gene inhibits the translation of its own mRNA when it is expressed under the control of the strong constitutive pTEF promoter. Figure 2A: Schemes showing the nucleic acid constructs tested; Figure 2B: SDS PAGE and Western Blot gel analysis of protein extracts of ADE2, ade2A and 235GArADE2 clone cells whose expression is under the control of the pTEF promoter. Figure 3 illustrates the results of the pulse-chase test showing that the 43GAr domain inhibits Ade2p synthesis but has no effect on the stability of Ade2p. Yeast cells expressing HA-43GAr-ADE2 or HA-ADE2 were incubated with radiolabelled methionine / cysteine mixture for 15 minutes ("pulse" step), followed by a large excess of a mixture of methionine / non-cysteine. radiolabeled has been added (hunting step or "chase").

Figure 3A: At the times indicated, protein extracts were prepared; the proteins were immunoprecipitated using an anti-HA antibody and then analyzed by the SDS-PAGE method, followed by an autoradiography step using a "Storm Phosphorimager®" radioactive material display apparatus.

FIG. 3B: the same protein extracts as those analyzed in FIG. 3A were analyzed by SDS-PAGE and by Western blot using anti-HA antico rs. Figure 4 illustrates that doxorubicin (DXR) specifically interferes with the effect of the GAr domain on yeast translation. The ade2A strain expressing the HA-43GAr-ADE2 construct was plated on rich medium, and then increasing concentrations of 5-fluorouracil (5FU) or doxorubicin (DXR) were loaded onto the filters. Figures 4A and 4B: Relative levels of HA-ADE2 or HA-43GAr-ADE2 mRNAs that were untreated or treated with the indicated concentrations of 5FU (FIG. 4A) or DXR (FIG. using the qRTPCR technique. Figures 4A and 4B show the average of the results of three independent tests. FIGS. 4C and 4D: The same cell extracts as those used for FIGS. 4A and 4B were analyzed by SDS-PAGE and Western blot in order to determine the relative levels of 43 GA-Ade2p or of Ade2p with respect to actin which was used as a charge indicator. Figures 4C and 4D show the effect of the indicated concentrations of 5FU or DXR on the level of HA-43GAr-Ade2p compared to HA-Ade2p.

Figure 5 illustrates that DXR specifically stimulates antigen presentation from the 235GAr-Ova construct in a T-cell based assay, and increases the level of expression of the EBNA1 protein. HEK 293 T Kb cells stably expressing the class I Kb MHC molecule were transfected with plasmids encoding chicken ovalbumin alone (OVA - left-hand portion of Figures 5A, 5B and 5C), or coding for ovalbumin whole-range fused chicken GAr (235GAr-OVA - right-hand portion of Figures 5A, 5B and 5C).

Figure 5A: Expression levels of OVA and 235GAr-OVA proteins in HEK 293 T Kb cells. Figure 5B: Presentation of class I MHC restricted OVA derived antigens was estimated after treatment of cells with increasing concentrations of DXR for 24 hours, then measuring f3Gal activity in reporter B3Z hybridoma T cells. CD8 +. The results were normalized by comparison with the presentation of the exogenous SL8 peptide. Figure 5C: Presentation of class I MHC-restricted OVA derived antigens was estimated after treatment of cells with increasing concentrations of 5FU for 24 hours and then measuring the activity (3Gal in T cells of the reporter hybridoma B3Z CD8 + The results were normalized by comparison with the exogenous SL8 peptide presentation Figure 5D: Similarly, EBNA1 and EBNA14 protein levels in HEK 293 T cells were determined after 24 hours of treatment with the indicated concentrations. Actin was used as a load control Figure 5E: Burkitt Raji lymphoma cells endogenously expressing an EBNA1 protein were transfected with a cDNA encoding an EBNA1 protein whose GAr domain is longer; which allowed the detection of endogenous EBNA1 and exogenous EBNA1 in the same cells after treatment with DXR for 8 hours. was used as a load control The results were compared by one-way analysis of variance analysis with Dunnett's multiple comparison test (*** means p <0.001; ** means p <0.01).

Figure 6 illustrates the effect of DXR and its chemical analogues on the suppression of GAr mediated translation in yeast and on the induction of p53 and p21 expression in mammalian cells. Figure 6A: Representation of chemical structures of doxorubicin and its analogues.

Figure 6B: yeast screening box with derivatives of doxorubicin. FIG. 6C: The genotoxic effect of DXR and its specified analogues by Western blot was estimated using as visualization data the activation of the expression of the p53 tumor suppressor gene and one of its p21 target genes in A549 cells. Figure 7 illustrates the effect of DXR and its chemical analogues on antigen presentation in T-cell based assays. The effect of daunorubicin (Figure 7A), valrubicin (Figure 7B), etoposide (Figure 7C) and epirubicin (Figure 7D) on antigen presentation using the T-cell based assay, as described in Figure 5. The results were compared by analysis of variance to a "one-way analysis of variance" factor) with Dunnett's multiple comparison test (*** means p <0.001, ** means p <0.01). DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for the selection of active substances against infection with a gamma herpes virus.

The term "process for the selection of active substances against infection with a gamma herpes virus" also means methods that can influence the ability of these gamma herpes viruses to escape the host's immune system. The present invention therefore also provides applicable methods for the selection of compounds capable of treating and / or preventing EBV-associated pathologies, such as EBV-associated cancers. The Applicant has devised a method for the selection of active substances against infection with a gamma herpes virus, which process uses recombinant yeast cells. More specifically, the Applicant has designed recombinant yeast cells capable of expressing recombinant polypeptide constructs comprising all or part of a "stability protein" of a gamma herpes virus, called GMP protein (for "Genome Maintenance Protein"), the expression of these polypeptide constructs in said recombinant yeast cells being easily detectable or quantified, in particular by detecting a "reporter" type peptide or polypeptide. The Applicant has thus shown that a correlation existed between (i) the modulation of the level of expression of the recombinant constructs by these yeast cells, under the effect of a candidate compound and (ii) the aptitude of said candidate compound to act against infection by a gamma herpesvirus, especially in a mammalian cell. Advantageously, and as shown in the examples, a mammalian cell may be a human cell.

In particular, the Applicant has shown that, in the recombinant yeast cells which have been designed, the complete GMP protein or the domain containing the repeated sequences of the latter responsible for the stealth of the virus, acts by inhibiting the translation of its own mRNA. as has been shown in human cells. Illustratively, this GMP protein may be the GAr domain of Epstein-Barr virus (EBV) EBNA1 protein. In other words, it is provided according to the invention, with the recombinant yeast cells defined in the present description, a eukaryotic cell model mimicking the human physiology of a gamma herpes virus, and more specifically mimicking the mechanisms used by the gamma herpes virus in human cells to escape the control of a viral infection by the immune system. Thus, the recombinant yeast cells that have been designed by the Applicant can be used to select compounds of therapeutic interest, intended for the treatment of infections with gamma herpes viruses. Mammalian cells expressing a class I MHC antigen and capable of expressing recombinant polypeptide constructs comprising all or part of a GMP protein (for "Genome Maintenance Protein") of a mammalian cell expressing MHC class I antigen are also provided according to the invention. gamma herpes virus, the expression of these polypeptide constructs in said mammalian cells can also be detected or quantified, in particular by a peptide or a "reporter" type polypeptide.

Within the meaning of the invention, the term "gamma herpesvirus" is understood to mean any virus belonging to the genera Lymphocryptovirus, Rhadinovirus, Macavirus or Percavirus. According to a preferred embodiment, the Lymphocryptovirus may be Epstein-Barr virus or KSHV virus.

The complete GMP protein, which includes the repetitive sequences responsible for the stealth of the virus, works by inhibiting the translation of its own mRNA (referred to as cis inhibition).

Thus, according to a particular embodiment, the GMP protein of gamma herpes virus, or part thereof [GMP], which is included in the recombinant protein expressed by the cells of the invention, is encoded by a nucleic acid whose sequence corresponds to that present in the strain of said gamma herpes virus.

In this respect, and still according to this embodiment, it is understood that the nucleic acid encoding said recombinant protein preferably comprises a nucleic acid corresponding to that present in the strain of said gamma herpes virus. The mammalian and recombinant yeast cells are suitable for carrying out the selection methods detailed below. Advantageously, the yeast cells and the mammalian cells of the invention are genetically modified by insertion into their genome of at least one copy of a nucleic acid rendering these cells capable of expressing a recombinant protein comprising at least a part [ GMP] and at least one part [REPORTER].

Preferably, these cells are genetically modified so as to insert into their genome a single copy of a nucleic acid rendering these cells capable of expressing a recombinant protein of the invention, which comprises at least one [GMP] part and at least one part [REPORTER]. The insertion into the genome of said nucleic acid in a single copy has a number of advantages, in particular a better control of the expression of the reporter gene, the absence of overexpression phenomena and the reduction of the toxicity of said insertions. The lines obtained are thus more stable, allowing for greater reproducibility when they are used in the selection processes of the invention.

SELECTING METHODS The present invention relates to an in vitro method for selection of active compounds against infection with a gamma herpes virus, comprising the steps of: a) culturing yeast cells in the genome of which has been inserting at least one copy of a nucleic acid rendering said cells capable of expressing a recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER], wherein: [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, and the yeast cells being grown in the presence of a candidate compound, b) measuring the level of production of said recombinant protein by the cells cultured in step a), and c) comparing the level of production of said p recombinant rotin measured in step b) with a reference value. A "recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER]" within the meaning of the invention, also includes a "recombinant protein comprising a single portion [GMP] and a single portion [REPORTER].

According to some embodiments, said recombinant protein may therefore comprise a sequence comprising a polypeptide of formula (Ia) or (Ib): NH2- [GMP] - [REPORTER] -COOH (Ia) or NH2- [REPORTER] - [ GMP] -COOH (lb).

According to a particular embodiment, said recombinant protein comprises at least one part [GMP] and at least one part [REPORTER], the part [GMP] being placed in N-terminal of the part [REPORTER]. According to this embodiment, the [GMP] portion may in particular be the [GAr] portion of the EBNA1 protein of the Epstein-Barr virus.

Advantageously, said recombinant protein may further include one or more additional peptides of "tag" or "tag" type, capable of binding to a ligand. These additional peptides may in particular be used as detection means, in order to quantify the concentration of recombinant protein in said cell.

It is clear that such "tag" peptides can be present both in N-terminal and C-terminal.

According to an exemplary embodiment, a "tag" peptide (or [TAG]) can be grafted at the N-terminus of the construct. In certain particular embodiments, the recombinant protein comprises a portion [TAG] (including [TAG1] or [TAG2]), said portion [TAG] being a peptide selected from the following list: an HA peptide, a poly-histidine peptide , a polyglutamate peptide, a Calmodulin peptide, a FLAG peptide, and a c-myc peptide. A recombinant protein of the invention may therefore comprise, zero, one or more parts [TAG], N-terminal or C-terminal of the construction. According to an exemplary embodiment, the recombinant protein comprises an N-terminal HA peptide of the construct. Advantageously, the presence of a portion [TAG] to the recombinant proteins expressed by the cells of the invention allows to verify, for example by Western Blot, the expression variation of said recombinant protein. A recombinant protein according to the invention may be under the control of the pADH promoter, or promoter of the ADH gene. A recombinant protein according to the invention may be under the control of the pTEF promoter, or promoter of the TEF gene. In particular, the present invention provides an in vitro method for selection of active compounds against gamma herpesvirus infection, comprising the following steps: a) culturing yeast cells in the genome from which it has been inserted; at least one copy of a nucleic acid rendering said cells capable of expressing a recombinant protein of following formula (I): NH 2 - [TAG 1] - [GMP] - [REPORTER] - [TAG 2] y -COOH (I), Wherein: [TAG1] and [TAG2] mean a peptide capable of binding to a ligand, - x and y, the same or different, represent an integer equal to 0 or 1, - [GMP] means a polypeptide comprising any or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, yeasts being cultured in the presence of a candidate compound, b) measuring the to the production of the protein of formula (I) by the cells cultured in step a), and c) comparing the production level of the protein of formula (I) measured in step b) with a reference value .

According to a preferred embodiment, the [GMP] polypeptide comprises the GAr domain of the EBNA1 protein of Epstein-Barr virus, or a fragment thereof. Thus, the present invention also relates to an in vitro method for selection of active compounds against infection with a gamma herpesvirus, comprising the following steps: a) cultivating yeast cells in the genome of which has been inserting at least one copy of a nucleic acid rendering said cells capable of expressing a recombinant protein comprising at least a portion [GAr] and at least a portion [REPORTER], wherein: [GAr] means a polypeptide comprising all or part of the amino acid sequence of the GAr domain of the EBNA1 protein of Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, and the yeast cells being grown in the presence of a candidate compound, b) measuring the level of production of said recombinant protein by the cells cultured in step a), and c) comparing the level of production of said protein recombinant measured in step b) with a reference value.

Thus, the present invention also relates to an in vitro method for selection of active compounds against infection with a gamma herpesvirus, comprising the following steps: a) cultivating yeast cells in the genome of which has been inserting at least one copy of a nucleic acid rendering said cells capable of expressing a recombinant protein of the following formula (II): NH 2 - [TAG] - [GMP] - [REPORTER] -COOH (II), in which: [TAG] means a peptide capable of binding to a ligand, - x is an integer equal to 0 or 1, - [GMP] means a polypeptide comprising all or part of the amino acid sequence of a protein of stability of genome of a gamma herpesvirus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, the yeast cells being cultured in the presence of a candidate compound, b) measuring the level of production of the protein of formula (II) by the cells lules grown in step a), and c) compare the level of production of the protein of formula (II) measured in step b) with a reference value. In some embodiments, the invention also relates to screening methods as described above, wherein said recombinant protein comprises a [GMP] portion comprising all or part of the amino acid sequence of a stability protein genome of a gamma herpesvirus selected from Lymphocryptovirus, Rhadinovirus, Macavirus and Percavirus. In particular, said recombinant protein comprises a part [GMP] comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus selected from the KSHV virus, the Epstein virus -Barr (EBV) and papio virus (HVP), especially KSHV and EBV. The expression "the recombinant protein comprises ..." or "the nucleic acid comprises ..." is also understood as "the recombinant protein is constituted by / of ..." and "the nucleic acid is constituted by / of ... ". Preferably, said recombinant protein comprises a [GMP] portion comprising all or part of the amino acid sequence of an Epstein-Barr virus genome stability protein. Even more preferentially, said recombinant protein comprises a part [GAr] comprising all or part of the GAr domain of the EBNA1 protein of the Epstein-Barr virus. In some embodiments, the recombinant protein comprises a peptide comprising from 8 to 250 continuous amino acids of a genome stability protein of a gamma herpes virus, which includes from 8 to 250 continuous amino acids of a genome stability of a gamma herpes virus, for example from 10 to 250, for example from 15 to 250 amino acids, which includes from 20 to 60 amino acids, which also includes 21 amino acids, 43 amino acids, 235 acids amino and 239 amino acids.

Within the meaning of the invention, a peptide comprising from 8 to 250 continuous amino acids of a genome stability protein of a gamma herpes virus includes a peptide comprising 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 ,,,,, , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 , 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 , 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 , 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190 , 19 1, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 continuous amino acids of a genome stability protein of a gamma herpes virus. In some embodiments, the recombinant protein comprises a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of Epstein-Barr virus EBNA1 protein.

It is thus understood that, within the meaning of the invention, and according to this embodiment, a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of Epstein-Barr virus includes a peptide comprising 8, 9 , 10, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 , 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 11, 35, 59, 83, 12, 13, 14, 36, 37 , 38, 60, 61, 62, 84, 85, 86, 106, 107, 124, 125, 15, 39, 63, 87, 108, 126, 127, 145, 163, 181, 199, 217, 235, 128 , 146, 164, 182, 200, 218, 236, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 1 77, 178, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 237, 238 or 239 continuous amino acids of the GAr domain in question. 143, 161, 179, 197, 215, 233, 144, 162, 180, 198, 216, 234, In certain particular embodiments, the recombinant protein comprises a peptide comprising from 15 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of Epstein-Barr virus, and preferably from 20 to 60 amino acids. Thus, according to one embodiment, the recombinant protein comprises a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus of sequence SEQ ID No. 2. Thus, according to another embodiment, said recombinant protein is encoded by a nucleic acid, which comprises a sequence coding for a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus. The presence of a portion [REPORTER] in the recombinant protein expressed by the cells of the invention will thus make it possible to quantify the level of production of said recombinant protein, by determining the level of expression of this part [REPORTER]. A [REPORTER] part therefore corresponds to a peptide or a protein having a characteristic allowing it to be observed in the laboratory. Thus a portion [REPORTER] may correspond to the product of one of the many "reporter" genes known to those skilled in the art, such as GFP (or "green fluorescent protein"), or enzymes whose action will directly cause or indirectly the appearance of a colored product, such as the gene coding for β-galactosidase. It is commonly accepted that a "reporter" gene is a gene whose product (peptide or protein) has a characteristic allowing it to be observed in the laboratory.

Preferably, a portion [REPORTER] thus allows a rapid quantification, by visualization in the culture medium, of the product formed. In some particular embodiments, the recombinant protein comprises a [REPORTER] moiety selected from a group comprising a fluorescent protein and an enzyme. Advantageously, the [REPORTER] portion may consist of an enzyme capable of inducing in the culture medium a colored reaction allowing the selection of colonies by quantifying the level of expression of the recombinant protein. In some particular embodiments, the recombinant protein therefore comprises a [REPORTER] portion consisting of the enzyme Ade2p. According to this embodiment, the recombinant protein preferably comprises a [REPORTER] portion consisting of the enzyme Ade2p of Saccharomyces cerevisiae. The enzyme Ade2p is encoded by the yeast ADE2 reporter gene, and relates to a phosphoribosylaminoimidazole carboxylase, which is involved in the adenine biosynthesis pathway. Yeast cells in which the ADE2 gene has been mutated or deleted (ade24 strains) form red colonies in a rich medium, due to the accumulation of phosphoribosylaminoimidazole (AIR), the substrate of Ade2p, which becomes red after oxidation. Conversely, cells expressing a sufficient amount of Ade2p will form white colonies. An intermediate amount of Ade2p leads to the formation of pink colonies, the intensity of which is proportional to the amount of Ade2p produced. For example, yeast cells can be cultured in the following media: YPD [1% (w / w) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose], 1 YPD [0.5% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose]. Solid culture media may contain 2% (w / v) agar. According to a particular embodiment, the recombinant protein preferably comprises a part [REPORTER] consisting of the enzyme Ade2p, and the yeast cells are yeast cells having a non-functional ADE2 gene, for example of genotype ade2-1. or ade221). The yeast strains used can therefore be derived from the strain "W303 WT": MATa, leu2-3,112 trp 1-1 canl-100 ura3-1 ade2-1 his3-11,15.

According to a particular embodiment, the genotype of the ade2A strain is the following: MATa, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 :: his5s. In this regard, and according to a preferred embodiment, the yeast cells are Saccharomyces cerevisiae or Schizosaccharomyces pombe cells, preferably Saccharomyces cerevisiae cells, and preferably of MATA leu2-3, 112 trp-1 canl genotype. It is therefore understood that a preferred strain of the invention is a strain which does not produce a protein comprising, said part [REPORTER], which therefore includes both (i.alpha. ) strains that are naturally incapable of producing said protein, and (ii) strains rendered incapable of doing so, for example following a mutation or a deletion. When the enzyme Ade2p is used as part [REPORTER], a strain according to the invention will therefore not produce the enzyme Ade2p endogenously. According to one embodiment, when the enzyme Ade2p is used as part [REPORTER], step b) processes can be carried out by determining the color of the cells cultured in step a). As illustrated in the examples, the selection process described above made it possible to select, from a vast collection of candidate compounds, several compounds of interest capable of modulating the expression of a gamma GMP protein. herpes viruses, in particular compounds capable of reducing the ability of a GMP protein of gamma herpes virus to inhibit the translation of its own mRNA, and thus exposing the cells infected with the gamma herpes virus in question to the cytotoxic action of CD8 + T cells of the host. It is shown in the examples that the compounds selected by the method described above allow the presentation of the viral GMP protein considered on the surface of the infected host cells in association with a class I MHC molecule, said host cells becoming targets. for CD8 + T cells of the host. Thus, the invention also relates to an in vitro method for the selection of active compounds against an infection with Epstein-Barr virus comprising the following steps: 1) cultivating mammalian cells having the following characteristics: i)) said cells express a MHC Class I antigen, and (ii) said cells express a recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER], wherein: [GMP] signifies a polypeptide comprising all or part of the amino acid sequence of a stability protein of the genome of gamma herpesvirus, preferably Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells, the mammalian cells being cultured in the presence of a candidate compound, 2) measuring the expression level of the class I MHC-associated recombinant protein on the surface of the cells grown in step a), and 3) compare the level of expression of the recombinant protein measured in step b) with a reference value. According to a particular embodiment, the invention therefore also relates to a method as described above, wherein said recombinant protein is a recombinant protein of formula (I): NH2- [TAG1] ,, - [GMP] - [REPORTER] - [TAG2] y-COOH (I), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1, [GMP] means a polypeptide comprising all or part of the amino acid sequence of a stability protein of the gamma herpesvirus genome, preferably Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells. According to an alternative embodiment, the invention relates to a method as described above, wherein said recombinant protein is a recombinant protein of formula (II): NH2- [TAG] - [GMP] - [REPORTER] -COOH (II), wherein: [TAG] means a peptide capable of binding to a ligand, - x represents an integer equal to 0 or 1, - [GMP] means a polypeptide comprising all or part of the sequence of amino acid of a stability protein of the gamma herpesvirus genome, preferably Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells. As seen above, the recombinant protein may comprise a portion [GMP] of variable size, for example having a length ranging from 8 to 250 continuous amino acids of a genome stability protein of a gamma herpes virus, preferably of the virus. Epstein-Barr. In particular, the recombinant protein may comprise a [GMP] portion having a length ranging from 8 to 250 amino acids, in particular from 15 to 250 amino acids, which includes from 20 to 60 amino acids.

Advantageously, the selection methods using recombinant yeast cells and mammalian cells can be carried out separately, sequentially or in combination. Illustratively, a selection process using mammalian cells can thus be carried out with respect to one or more candidate compounds previously selected by a selection method using the recombinant yeast cells of the invention. According to one embodiment, the invention therefore relates to a method as described above, the candidate compound being selected by the implementation of the in vitro selection process using recombinant yeast cells.

According to one particular embodiment, the invention relates to a method of selection using mammalian cells as described above, in which the recombinant protein comprises a portion [REPORTER] which comprises an antigen recognized specifically by lymphocyte cells. T CD8 +. Examples of such antigens are known to those skilled in the art.

For example, antigens produced by proteolysis and recognized by the major histocompatibility complex (MHC) class I, such as antigens produced by proteolysis of ovalbumin. For reference, the sequence of chicken ovalbumin is a sequence SEQ ID No. 4. Examples of antigens according to the invention may be peptides obtained by proteolysis of ovalbumin such as the peptide NH2-SIINFEKL-COOH (SEQ ID No. 5) and sold by the company AnaSpec, Inc. According to a particular embodiment the invention relates to a method of selection using mammalian cells as described above, wherein the recombinant protein comprises a portion [REPORTER] which is an ovalbumin fragment comprising the peptide NH2-SIINFEKL-COOH (SEQ ID No. 5). According to this embodiment, the invention therefore relates to an in vitro method for selecting active compounds as described above, step b) being carried out according to the following steps: b1) cultivating the mammalian cells obtained at the end of step a) in the presence of CD8 + T lymphocyte cells specifically recognizing the peptide NH2-SIINFEKL-COOH (SEQ ID NO: 5), and b2) measuring the level of proliferation of CD8 + T lymphocyte cells cultured in step 1 ) 1). According to this embodiment, the CD8 + T lymphocyte cells cultured in step b1) express a detectable recombinant protein, preferably an enzyme or a fluorescent protein. Selection methods employing mammalian cells can be carried out from particular mammalian cells such as cells of the HEK293 T kb line and Raji cells. The cells of the HEK293 T kb line are derived from human embryonic kidney cells which stably express class I murine MHC Kb antigen. Raji cells are tumor cells derived from type III Burkitt lymphoma. and infected with EBV.

All the selection methods of the invention are capable of taking, as a reference value in step c), a reference value chosen from: (i) a value obtained in step b) when the step a) is carried out in the absence of a candidate compound, (ii) a value obtained in step b) when step a) is carried out in the presence of a compound capable of inducing an overproduction of the recombinant protein, and iii) a value obtained in step b) when step a) is carried out in the presence of a compound capable of blocking the production of the recombinant protein.

It is therefore understood, with respect to these models, that the ability of a candidate compound to modulate the production of the recombinant protein comprising the [GMP] portion of a stability protein of a gamma herpes virus, is indicative of its faculty. modulating the ability of said gamma herpes virus to escape the immune system of the host. Thus, the ability of a candidate compound to increase the production of said recombinant protein is indicative of its ability to render cells infected with a gamma herpes virus visible to the immune system, particularly during the latent phase of infection. . The ability of a candidate compound to increase the production of said recombinant protein may also be indicative of its ability to treat and / or prevent diseases associated with infection with said gamma herpes virus. CELLS OF YEASTS AND VIAMI VIALS It is also provided, according to the invention, mammalian cells and yeasts as such, able to allow the implementation of the selection methods of the invention. Naturally, the cells and variants of these cells used in said methods are also considered as such below. The invention thus relates to a yeast cell in the genome from which at least one copy of a nucleic acid has been inserted, making these cells capable of expressing a recombinant protein comprising at least one [GMP] part and at least one part [ REPORTER], wherein: [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, in particular Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cell. In particular, the invention relates to a yeast cell in which said recombinant protein is a recombinant protein of the following formula (I): NH2- [TAG1] - [GMP] - [REPORTER] - [TAG2] y-COOH (I ), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1, - [GMP] signifies a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, in particular Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells. According to one embodiment, the recombinant protein comprises a part [TAG1] and / or [TAG2], said portion [TAG1] and / or [TAG2] being a peptide chosen from the following list: an HA peptide, a poly-peptide, histidine, a poly-glutamate peptide, a Calmodulin peptide, a FLAG peptide and a c-myc peptide. According to a particular embodiment, the recombinant protein comprises a portion [GMP] having a length ranging from 8 to 250 amino acids, preferably from 20 to 60 amino acids. According to a preferred embodiment, the [GMP] portion corresponds to all or part of the [GAr] domain (or "part [GAr]") of the EBNA1 protein of the EpsteinBarr virus. According to a particular embodiment, the recombinant protein comprises a portion [REPORTER] selected from a group comprising a fluorescent protein and an enzyme.

According to this embodiment, the recombinant protein comprises a portion [REPORTER] consisting of the enzyme Ade2p Saccharomyces cerevisiae. The invention also relates to a mammalian cell, preferably a human cell, having the following characteristics: (i) said cell expresses a MHC Class I antigen, and (ii) said cell expresses a recombinant protein comprising at least a portion [GAr] and at least a portion [REPORTER], wherein: [GAr] means a peptide comprising all or part of the continuous amino acid sequence of the GAr domain of Epstein-Barr Virus EBNA1 protein, and [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cell. According to a particular embodiment, the invention relates to a mammalian cell, preferably a human cell, having the following characteristics: (i) said cell expresses a class I MHC antigen, and (ii) said cell expresses a recombinant protein comprising at least a portion [GAr] and at least a portion [REPORTER], wherein: - [GAr] means a peptide comprising at least 8 amino acids of the continuous amino acid sequence of the GAr domain of the EBNA1 protein of the Epstein-Barr, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cell. According to a particular embodiment, the invention relates to a mammalian cell having the following characteristics: (i) said cell expresses a class I MHC antigen, and (ii) said cell expresses a recombinant protein of formula (I) following: NH2- [TAG1] ,, - [GAr] - [REPORTER] - [TAG2] y -COOH (I), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, x and y, which are identical or different, represent an integer equal to 0 or 1; [GAr] denotes a peptide comprising at least 8 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus, and [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cell. Thus, according to a particular embodiment, the invention relates to a mammalian cell having the following characteristics: (i) said cell expresses a class I MHC antigen, and (ii) said cell expresses a recombinant protein of formula (I ) following: NH2- [TAG1] ,, - [GAr] - [REPORTER] - [TAG2] y -COOH (I), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand x and y, which are identical or different, represent an integer equal to 0 or 1; [GAr] denotes a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cell.

GMP DOMAINS A protein or a GMP ("Genome Maintenance Protein") polypeptide, also called "GMP part", corresponds to all or part of a viral protein that plays a role in maintaining the latent phase of a gamma herpes virus.

Depending on the gamma herpes virus considered, this or these GMP proteins will generally have one or more repeated sequences. By way of example, mention may be made of the EBNA1 protein (Epstein-Barr virus) of sequence, and the LANA1 protein (Kaposi sarcoma associated herpes virus or KSHV) of sequence.

By way of example, and in a nonlimiting manner, three LANA1 proteins of different sizes have been described: a protein of 1129 amino acids of sequence SEQ ID No. 6, another (called "long form" of LANA1) of 1162 amino acids of sequence SEQ ID No. 7, and another of 1003 amino acids of sequence SEQ ID No. 8. Also by way of reference, the 1162 amino acid LANA1 protein of sequence SEQ ID No. 6 is encoded by a nucleic acid of sequence SEQ ID No. 9. The 1003 amino acid LANA1 protein of sequence SEQ ID No. 8 is encoded by a nucleic acid of sequence SEQ ID No. 10.

These GMP proteins predominantly comprise one or more variable sequences of variable size, constituting a central domain, which is responsible for maintaining the latent phase of said gamma herpes virus. The "GAr domain" of the EBNA1 protein of the Epstein-Barr virus and the "DEQPRLV-rich" domain of the LANA1 protein of the KSHV virus are particularly noteworthy. The term "GAr domain", or "part [GAr]", is understood to mean a glycine-alanine repeat sequence, which consists of a series of Alanines, separated by one, two or three glycines located at the N-terminal portion of EBNA1. Epstein-Barr virus (see Levitskaya et al., "Inhibition of antigen processing by the internal region of the Epstein-Barr virus nuclear antigen-1; Nature (1995); 375, 685-8). The term "DEQPRLV-rich" or "part [DEQPRLV]" is understood to mean a series of repeated sequences, which consist of a series of Aspartates, Glutamates, Glutamines, Prolines, Arginines, Leucines and / or of Valines localized on the LANA1 protein of the KSHV virus. For reference, the sequence of the "DEQPRLVrich" domain corresponding to the long form of the LANA1 protein of the KSHV virus is a protein sequence SEQ ID No. 17, which is encoded by a nucleic acid of sequence SEQ ID No. 18. This "DEQPRLV-rich" domain can also be divided, in the literature, into several subdomains, such as CR1-2-3, "DEQ-rich" or "EQL-rich" type domains. Within the meaning of the invention, a "DEQPRLV-rich" domain is therefore likely to comprise all or part of these subdomains. In one embodiment, the "DEQPRLV-rich" domain comprises the entire repeat sequence comprised in the LANA1 protein, which relates to a peptide of sequence SEQ ID No. 6, 7 or 8 ,. According to a preferred embodiment, the recombinant protein comprises at least one portion [GAr], said portion [GAr] being of variable size. According to a particular embodiment, the portion [GAr] may be a peptide of sequence SEQ ID No. 11, also called "21GAr", which is encoded by a nucleic acid of sequence SEQ ID No. 12. According to this particular embodiment, the portion [GAr], called "21GAr", can therefore be a peptide encoded by a nucleic acid of sequence SEQ ID No. 12.

According to one embodiment, the portion [GAr] may be a peptide of sequence SEQ ID No. 13, also called "43GAr", which is encoded by a nucleic acid of sequence SEQ ID No. 14. According to this particular embodiment, the portion [GAr], called 43GAr, may therefore be a peptide encoded by a nucleic acid of sequence SEQ ID No. 14. According to one embodiment, the portion [GAr] may be a peptide of sequence SEQ ID No. 15, also called "235GAr", which is encoded by a nucleic acid of sequence SEQ ID No. 16. According to this particular embodiment, the portion [GAr], called 235 GAr, can therefore be a peptide encoded by a nucleic acid of sequence SEQ ID No. 16. For reference, the sequence "21 GAr" corresponds to the N-terminal part of the sequence "43 GAr", which is included in the sequence "235 GAr". According to a preferred embodiment, the portion [GAr] may be a peptide "21 GAr", "43 GAr" and / or "235 GAr". In a still preferred mode, the [GAr] portion may be a "43 GAr" peptide and / or "235 GAr" peptide. NEW THERAPEUTIC USES It is recalled that infections with gamma herpes viruses have already been caused by the occurrence of several types of diseases, and in particular of several types of cancer. It is also recalled that infection with Epstein-Barr virus (EBV) is a causative agent of Burkitt's lymphoma, nasopharyngeal carcinoma and infectious mononucleosis. EBV is also associated with some forms of Hodgkin lymphoma. The implementation of the selection methods of the invention has already made it possible to propose a new therapeutic use of compounds hitherto known for other indications. In this respect, the identification of the mode of action of compounds on the lytic cycle of gamma herpes viruses makes it possible to consider both their use on new types of pathology, but also on distinct patient populations. Among the compounds identified by the Applicant, mention may be made of doxorubicin (also known as hydroxydaunorubicin, otherwise known under the name Adriamycin), which is widely used in chemotherapy to treat cancers such as lymphomas, carcinomas, sarcomas, or breast cancers. It is an antibiotic of the family of anthracyclines hitherto known to be an intercalator of DNA. Doxorubicin is especially used to treat cancers related to the Epstein-Barr virus. Those skilled in the art may in particular refer to the review by Zimmermann & Trappe (Therapeutic options in post-transplant lymphoproliferative disorders, Ther Adv Hematol 2011, 2 (6): 393-407). Surprisingly, it therefore appears that the multi-chemotherapies containing doxorubicin or its derivatives cause the activation of the lytic phase of the Epstein-Barr virus via the GAr domain of the EBNA1 protein of the Epstein Barr virus. Thus, the invention also relates to the therapeutic use of the compounds identified by the in vitro selection methods described above, for treating and / or preventing the diseases associated with infection with gamma herpesvirus, and in particular with the virus. Epstein-Barr. Diseases associated with gamma herpesvirus infection include Burkitt's lymphoma, nasopharyngeal carcinoma, infectious mononucleosis and associated or post-transplant lymphoproliferative disorders. In particular, the invention relates to the therapeutic use of the compounds identified by the in vitro selection methods described above, for treating and / or preventing Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, infectious mononucleosis and / or lymphoproliferative disorders associated with or subsequent to transplantation. In addition to Doxorubicin, the Applicant has also identified daunorubicin, Epirubicin and Pirarubicin as compounds of interest.

EXAMPLES A. Materials and Methods A.1. Yeast strains and culture media All the yeast strains used are derived from the strain "W 303 WT" (Blondel et al., 2005): MATa, leu2-3,112 trp1-1 canl-100 ura3-1 ade2-1 his3 -11.15. The genotype of the ade2A strain is as follows: MATα, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 :: HIS5si''b '. The stem cells were cultured and used as described in Bach et al. (2003) and Bach et al. (2006). The culture media used for the growth of yeast cells were as follows: YPD [1% (w / w) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose], 1 / 2 YPD [0.5% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose]. The solid culture media contained 2% (w / v) agar. A.2. Construction of Plasmids All plasmids were constructed using standard procedures. Restriction enzymes T4 DNA ligase and intestinal alkaline phosphatase of calf were obtained from New England Biolabs (Ipswich, MA, USA). The purified synthetic oligonucleotides were obtained from Eurogentec. The conservation of the plasmids was carried out in the bacterial strains DH5a and TOP10.

The plasmid p416-pADH-HA-ADE2 was constructed as follows: the polynucleotide HA-ADE2 was amplified with (i) sense primer 5'-GCGCGAATTCATGTACCCATACGATGTTCCAGATTACGCTAGGGAT TCTAGAACAG-3 '(SEQ ID NO: 19) and ( ii) the 5'-GCGCGGTACCTTACTTGTTTTCTAGATAAGG-3 'antisense primer (SEQ ID NO: 20), which made it possible to introduce the EcoR1 and KpnI restriction sites; the amplified polynucleotide was then cloned into the p416-pADH vector. To construct the plasmid p416-pADH-HA-21GAr-ADE2, the polynucleotide HA-21GAr was amplified with (i) the sense primer 5'-GCGCGGATCCATGGGGTACCCATACGATGTT-3 '(SEQ ID NO: 21) and (ii) the 5'-GCGCGAATTCTGGTGAATTCAGGGCCCCTCC-3 'antisense primer (SEQ ID No. 22), which made it possible to introduce the BamH1 and EcoR1 restriction sites; the amplified polynucleotide was then cloned into the p416-pADH-HA-ADE2 vector upstream of the ADE2 gene. Plasmid p416-pADH-HA-43GAr-ADE2 was constructed in the same manner, using the same primers as for plasmid p416-pADH-HA-21GArADE2. Plasmid p416-pADH-HA-235GAr-ADE2 was constructed as follows: The HA-235GAr polynucleotide was amplified using (i) sense primer 5'-GCGCGGATCCATGTACCCCTACGACGTCCCCG-3 '(SEQ ID NO: 23) and (ii) antisense primer 5'-GCGCGAATTCCTCGAGGATATCACCTTCTTGG-3' (SEQ ID NO: 24), which made it possible to introduce the BamH1 and EcoR1 restriction sites; the amplified polynucleotide was then cloned into the p416-pADH-ADE2 vector upstream of the ADE2 gene. To construct the plasmid p416-pADH-HA-papio3OGAr-ADE2, the HA-papio3OGAr cassette was amplified using (i) sense primer 5'-GCGCGGATCCATGGCTTACCCCTACGACG-3 '(SEQ ID NO: 25) and (ii) the 5'-GCGCGGTGAATTCTCCTCCTGCTCC-3 'antisense primer (SEQ ID No. 26), which made it possible to introduce the BamH1 and EcoR1 restriction sites; the amplified polynucleotide was then cloned into the plasmid p416-pADH-ADE2 upstream of the ADE2 gene. To construct the p416-pTEF-HA-ADE2 plasmid, the p416-pADH-HA-ADE2 vector was digested with EcoR1 and KpnI and the HA-ADE2 cassette which was thus excised was inserted into the p416 vector. -pTEF. To construct the vectors p416-pTEFHA-21GAr-ADE2, p416-pTEF-HA43GAr-ADE2 and p416-pTEF-HA-235GAr-ADE2, the cassettes HA-21GAr-ADE2, HA-43GAr-ADE2 and HA-235GAr-ADE2 were obtained by subjecting the respective p416-pADH constructs with BamHI and KpnI; then the corresponding cassettes were inserted into the 416-p TEF vector. The pCDNA3-OVA, pCDNA3-235GAr-OVA, pCDNA3-EBNA 1 and pCDNA3-EBNA 1 AGAr constructs were obtained as described by Apcher et al. (2010) and Yin et al. (2003).

A3. Constructing Yeast Cells In order to achieve homogeneous expression in all cells, a single copy of HA-43GRA-ADE2 or HA-ADE2 under the control of the ADH promoter was first stably integrated into the genome of strain ade2A. The fragments pADH-HA-ADE2, pADH-HA-21GRA-ADE2, pADH-HA-43GRA-ADE2 and pTEF-HA235GAr-ADE2 were excised from the respective plasmids p416-pADH-HA-ADE2, p416 pADH-HA-21GAr-ADE2, p416-pADH-HA-43GRA-ADE2 and p416-pTEF-HA-235GRA-ADE2, using restriction enzymes SacI and KpnI; then the excised fragments were subcloned into the disintegrator plasmid pIS375 (Sadowski et al., 2007). The disintegrator plasmids make it possible to obtain the integration of a single copy of the constructs of interest at the junction of the deletion of the marker, as well as the complete elimination of the additional plasmid sequences (Sadowski et al., 2007). Because the built-in constructs do not contain any flanking sequence duplication, the integrations are highly stable. The yeast transformations were carried out according to the procedure using lithium acetate. The yeast strains obtained were respectively designated pADH-HA-ADE2, pADH-HA-21GAr-ADE2, pADH-HA-43GArADE2 and pTEF-HA-235GAr-ADE2.

A.4. Compound Libraries Approximately 2,000 compounds were screened from two chemical libraries, which includes (i) the Prestwick Chemical Library® library, which contains a collection of 1,200 compounds tested at least in Phase II. clinical trials and (ii) the BIOMOL's FDA Approved Drug Library (Enzo Life Sciences), a collection of 640 drug molecules approved by the US Drug Administration (FDA), which were selected to maximize chemical and pharmacological diversity. Compounds were provided (i) at a concentration of 10 mM in 96-well plate wells (Prestwick®) or (ii) as 2 mg / ml solutions in DMSO (BIOLMOL®). The compounds 5-Fluorouracil (5FU), doxorubicin and daunorubicin are marketed by Sigma Aldrich. The compounds Epirubicin, Idarubicin, Pirarubicin, Ectoposide and Tenoposide are marketed by the Santa Cruz Company. The compound Valrubicin is marketed by the company Chemos. A. 5. Yeast Screening Assay This assay was adapted from a test described by Bach et al. (2003) and Bach et al. (2006). An aliquot of an exponentially growing culture (200 μl of a culture at a D.0.600 of 0.55) of yeast pADH-HA-43GAr-ADE2 was homogeneously plated using sterile glass beads. (3 and 5 mm diameter) on square Petri dishes (12 cm x 12 cm) containing YPD solid medium. Sterile filters (Thermo-Fisher) were then placed on the surface of the agar. Then a volume of 2.11 of the test compounds was applied to each filter. In addition, the filter located in the upper left area received DMSO as a negative control. The cultures were incubated at 29 ° C for four to five days and then scanned using a scanner Scan1212 (Agfa Company).

A. 6. Culture of yeast cells To obtain yeast protein extract, an amount corresponding to a value of 6 D.0.660 of exponentially growing cells was collected by centrifugation, and the cell pellets were then delivered. suspended in lysis buffer (25 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.2% Triton X-100, antiprotease cocktail (Roche Company), 1 mM phenyl-methylsulfonyl fluoride). After addition of glass beads with a size of 425 μm-600 μm (Sigma Company), the cells were lysed by shaking on a Vortex device for six sequences of (i) 30 seconds of Vortex stirring and then (ii) seconds of cooling in the ice; then the resulting liquid was centrifuged for 30 minutes at 200 g at 4 ° C. Supernatant fractions were recovered and tested for protein content. After denaturation by heat for 3 minutes at 95 ° C., the protein extracts were analyzed by electrophoresis 10% SDSPAGE (precast NuPage, Invitrogen) and then transferred onto nitrocellulose membranes of 0.45 μm (Whatman). The membranes underwent a blocking step with 1X PBS / 0.1% Ipegal solution containing 5% skim milk and 0.5% BSA; then the membranes were incubated overnight at 4 ° C with the indicated primary antibodies (mouse anti-HA antibody, anti-actin IgM mouse antibody, Company Calbiochem). The membranes were then washed with a new solution of PBS 1X / 0.1% Ipegal and then incubated for 45 minutes with secondary antibodies (goat anti-mouse antibody, Biorad, goat anti-mouse IgM antibody, Calbiochem ) which were conjugated to horseradish peroxidase at the 1: 3000 dilution, then analyzed by Enhanced Chemiluminescence (ECL, Amersham Company), using an imager Vilber-Lourmat Photodocumentation Chemistart 5000. Serum mouse anti-HA was used at 1: 5000 dilution, as the anti-actin antibody. For mammalian protein analysis by Western blotting, the cells were recovered 48 hours after their transfection, and then lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40). containing protease inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured according to the Bradford test. The total cell extracts were fractionated by SDS-PAGE, transferred to BioTrace NT Nitrocellulose Blotting membranes (PALL), and then incubated with a polyclonal anti-OVA rabbit antibody (Sigma) or a mouse anti-EBNA1 monoclonal antibody. (OT1X), both at the 1: 1000 dilution, or with anti-p53 antibody (D01, 1: 1000), or with anti-p21 antibody (12D1, Cell Signaling, 1: 500 dilution). After incubation with appropriate secondary antibodies, the proteins were visualized by ECL (Amersham Biosciences).

A. 7. "Pulse-Chase" Test Yeast cells pADH-HA-ADE2 or pADH-HA-43GAr-ADE2 in exponential growth phase were labeled ("pulse labeling") with 90 p.Ci / OD600 ( EasyTag Express protein labeling mix [35S], PerkinElmer Life Sciences) for 15 minutes, after being cultured for 15 minutes in methionine-free medium; then the cells were subjected to a "chase" step in fresh medium containing 50 mM methionine and cysteine for the indicated times, before recovery. After centrifugation, the cell pellets were resuspended in lysis buffer (25 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.2% Triton X-100, cocktail antiprotease (Roche), 1 mM phenyl-methylsulfonyl fluoride ), and then treated as described previously. The cell lysates were pre-clarified with G-Sepharose beads for 45 minutes at 4 ° C, then immuno-precipitated with 1 μg of anti-HA monoclonal antibodies which were previously immobilized on G-Sepharose beads. Sepharose overnight at 4 ° C. The beads were then washed four times with 1X-0.2% Igepal PBS, and then scalded in SDS loading buffer. Immunoprecipitates were analyzed by SDS-PAGE using 10% precast NUPAGE gels (InVitrogen). The gel was dried and then analyzed using a Typhoon 9400 Phosphorimager (GE) type imager. A.B. Real-Time Quantitative PCR Total yeast cell RNA was extracted using the RNAeasy® and RNAase-free® kits marketed by Qiagen Company (USA). The cDNA sythesis was carried out using 1 μg of DNA-free RNA using the Quperscript kit marketed by the company InVitrogen (France), using the random hexamers marketed by the company Qiagen (United States of America). ). Duplicate cDNA samples were subjected to a quantitative PCR method using the QuantiTect SYBR Green PCR® kit marketed by Qiagen Company (USA) and the Abiprism 7000 Sequence Detection System commercialized by Applied Biosystems Company. (United States). The relative amount of the amplified mRNA was determined using actin as a standard for normalization. The primer pairs used for the PCR were the following: - forward primer ADE2: ATTGTGCAAATGCCTAGAGGTG (SEQ ID NO: 27) - ADE2 back primer: AATCATAAGCGCCAAGCAGTC (SEQ ID NO: 28) - forward primer Actin: ATGGTCGGTATGGGTCAAAAA (SEQ ID N 29) - reverse primer Actin: TTCCATATCGTCCCAGTTGGT (SEQ ID NO: 30) A.9. T-cell assay This assay was performed as described in Apcher et al. (2010). Briefly, HEK 293 T Kb cells were seeded in 6-well plates at a density of 200,000 cells per well. The next day, the cells were transfected with 1.1G of the OVA expression plasmid or the 235Gar-OVA expression plasmid with 3μl of Genejuice®, according to the manufacturer's recommendations (Merck Biosciences Company). The next day, the transfected cells were treated with increasing concentrations of candidate compounds to be tested. After 24 hours, the transfected and treated HEK 293 T Kb cells were seeded at a density of 50,000 cells per well in 96-well microplates, and then cultured in the presence of B3Z T cell hybridoma cells for 16 h. Cells were then recovered and washed twice in 1X cold PBS buffer. The cells were then lysed in a solution of 0.2% Triton X-100®, 0.5M KH2PO4 for 5 min on ice. The cell lysates were centrifuged for 5 minutes at 200 g. Then a 1.1.1 supernatant volume from each of the wells was transferred to Optiplate® 96-well count plates (Packard Bioscience, Randburg, SA) and tested for β-galactosidase activity using Luminescence Assay. Assay® (BD Biosciences, Clontech) with a Fluostar® reading device (BMG Labtech). The results were expressed in arbitrary units Gal and the data were normalized with regard to the presentation of the peptide SIINFEKL (SL8 -SEQ ID No. 5), corresponding to amino acids 257 to 264 of ovalbumin, marketed by the company Eurogentec (Seraing, Belgium).

Example 1: Test Model of Inhibition of Translation Via a GAr Region in Yeast Cells A test model of protein translation inhibition inhibition dependent on the presence of the GAr region in yeast cells was designed. This test is based on the use of the yeast-derived ADE2 reporter gene which encodes a phophoribosylaminoimidazole carboxylase, which is an enzyme involved in the adenine biosynthetic pathway. Yeast cells, in which the ADE2 gene has been deleted (ade2A strain), form red colonies in a rich medium, the red color being due to the accumulation of phosphoribosylimidazole (AIR), the substrate of the enzyme Ade2p, this substrate becoming red when oxidized by active respiration. In contrast, cells expressing a sufficient amount of the Ade2p enzyme form white colonies. Intermediate amounts of the enzyme Ade2p lead to the formation of pink colonies whose color intensity is proportional to the level of Ade2p expression. From the yeast strain ade2A, it was determined that the pADH promoter allowed a minimal expression of the ADE2 gene leading to the formation of white colonies, allowing the detection of any inhibitory effect of the translation of the ADE2 mRNA, even a weak inhibitory effect, by determining a color change of the colonies. Using the pADH promoter, the effect of fusing GAr domains of different lengths to the N-terminus of the Ade2p protein was tested. The various resulting constructs were fused at their N-terminus with a TAG tag peptide so as to allow their detection (Figure 1A). A decrease in Ade2p levels which is dependent on the length of the GAr domain that is present in the fusion constructs tested has been observed, as visualized by the determination of a colony gradient from white to red. . These results were confirmed by Western blot analysis, which shows increasing inhibition of fusion protein production that is proportional to the length of the GAr domain (Figure 1B). The inhibition effect is independent of the promoter since the presence of a full GAr domain (235Gar) also leads to the formation of pink, deep / red colonies and a sharp decrease in the level of Ade2p when expressed. under the control of the strongest pTEF promoter (Figures 2A, 2B). All of these results suggest that, as in mammalian cells, the GAr domain of the EBNA1 protein impairs the translation of mRNA in yeast in a manner dependent on the length of the GAr domain. To further compare the GAr-dependent effect on protein expression in yeast, we used the GAr domain of EBV-related simian virus, which has been shown to have no effect on cell translation. Mammal (Apcher et al., 2010, Apcher et al., 2009). The lymphocrypto-Papio virus infects Old World primates and expresses a homologue of the EBNA1 protein which comprises short sequences related to the GAr domain which have been shown to be incapable of preventing antigen presentation (Blake et al. al., 1997). The gRNA domain of the EBV virus consists of unique amino acid residues separated by one, two or three glycine residues, whereas the GAr domain of the Papio virus EBNA1 protein contains four unique serine residues inserted every seven residues of the repetition. In contrast to all EBV GAr domains that were tested, which includes the 21Gar short domain, a 30 amino acid sequence of the Papio GAr domain has no effect on mRNA translation or presentation. antigen (Apcher et al., 2010). The same 30 amino acid sequence of the Papio GAr domain was fused to Ade2p and this GAr domain was determined to have no effect on Ade2p levels, as compared to control cells expressing the ADE2 gene placed under control of the same ADH promoter (Figure 1C). All these results underline that the GAr-dependent effect on protein expression is operational in yeast and that the cellular mechanisms that contribute to it are conserved from yeast to humans. The protein level can be disrupted by altering (i) mRNA levels, (ii) mRNA translation, or (iii) protein stability, respectively. To determine whether the GAr domain affects the level of its own mRNA, the expression of several constructs in the ade2A strain was measured by quantitative real-time PCR (qRT-PCR). As shown in Figure 1E, ade2A mRNA levels obtained under the control of the pADH promoter were similar in the presence or absence of the GAr domain of 43 amino acids in length or with the Papi GAr domain. These results indicate that the GAr domain has no effect on the level of the ADE2 transcript. The effect of the GAr domain on the stability of the Ade2p protein was then determined. By using a cycloheximide chase test, Apde2p has been shown to be a very stable protein and the GAr domain does not alter its stability (Figure 1D). Taking into account the metabolic labeling results of the newly synthesized proteins as well as the results of pulse-chase tests (FIG. 3), all of these results show that the GAr domain inhibits the translation of its own mRNA in the yeast, in a manner dependent on the length of the GAr domain, as is the case in human cells. EXAMPLE 2 Test for Screening Anti-Viral Compounds The yeast inhibition test model described in Example 1 was used, and the ease of reading the test results by colony color analysis. , in order to develop a test for screening compounds for their ability to interfere with the effect of the GAr domain on protein translation.

The principle of the screening assay is based on the identification of compounds having the ability to alter cis inhibition of GAr mediated translation. First, this screening assay allows the selection of compounds having the ability to prevent cis inhibition of GAr-mediated translation and can prevent escape of EBV-infected cells from control by the immune system. these compounds may therefore constitute compounds of therapeutic interest. Secondly, this screening assay allows the selection of compounds of therapeutic interest that have the ability to increase the inhibitory effect of the GAr domain, since the EBNA1 protein is required for viral replication and that this protein also prevents the oncogenic and anti-apoptotic activities of the virus (Gruhne et al., 2009, Kennedy et al., 2003, Wilson et al., 1996). For the purposes of this screening assay, the fusion construct comprising the 43 GA domain was selected because this construct exerts an intermediate effect leading to the formation of pink colonies on a rich medium. The yeast cells were plated on a solid rich medium and were exposed to filters each impregnated with a test compound. Then (i) compounds that prevent the translation effect of the GAr domain can be selected at the same time, which result in a halo of whiter colonies around the filter impregnated with the compound of interest and (ii) compounds which increase the inhibitory effect of the GAr domain, which lead to the production of a halo of red or darker pink colonies on the rich medium. An advantage of the screening test which has been developed is the possibility of testing simultaneously and in a very simple manner, many candidate compounds, each in a wide range of concentrations, because of the diffusion of the candidate compounds in the solid medium. surrounding. In addition, this screening test has been found to be very sensitive, since many compounds are toxic when in high concentration. If so, the results of the screening test may lead to false positives when a candidate compound interferes with the mechanisms of oxidative phosphorylation, since active respiration is required for the oxidation of the AIR substrate to a red pigment. However, compounds leading to false positive results can be easily spotted, as these compounds will also prevent the growth of yeast cells on a non-fermentable substrate such as glycerol or ethanol. The effect of selected compounds on the expression of ADE2 was then determined using the qRT-PCR technique and Western blot analysis. Compounds which specifically affect the expression of 43Gra-ADE2 were then tested for their effect on the expression of GAr-OVA and EBNA1 in mammalian cells by Western blot analysis and by a "T-cell" test. Antigen presentation reporter restricted to C1 \ 41-1 Class I antigen.

Example 3 Identification of Compounds of Interest The compounds of interest were identified using the screening test described in Example 2. A screening of compounds of interest was carried out from two collections of compounds, respectively (i) the Prestwick® chemo library and (ii) the BIOLMOL® library, these chemical libraries each containing a collection of therapeutic compounds for which toxicity and bioavailability studies have already been carried out in humans. Consequently, the compounds selected at the end of the screening test are likely to enter directly into optimization programs. Of the 2,000 therapeutic compounds tested using the screening assay, only two compounds were selected for their ability to generate white halos of yeast colonies, corresponding to a sharp increase in Ade2p levels. These results show that the screening test that has been developed is both specific and reliable. The compounds identified by the screening assay are 5-fluorouracil (5FU) and doxorubicin (DXR), respectively. The results show that both the intensity of color and the diameter of the halo are proportional to the amount of therapeutic compound contained on the filter under consideration. It was then investigated whether the 5FU and DXR compounds affect the mRNA levels of the various constructs by the qRT-PCR method (Figures 4A and 4B). In order to determine whether the 5FU and DXR compounds interfere with the ability of GAr to inhibit translation, the effect of these compounds on the equilibrium state level of the 43GaR-Ade2p and Ade2p proteins was determined (FIGS. 4D). Both the 5FU and DXR compounds led to a significant increase in the 43GAr-Ade2p protein levels, which corroborates their ability to induce a halo of whiter colonies in the yeast cell assay. However, only the DXR compound exerted a GAr-dependent effect because DXR did not exert an effect on the level of the Ade2p protein, unlike 5FU which also led to an increase in the level of the Ade2p protein. These results suggest that DXR specifically affects the translation inhibition that depends on the GAr domain, whereas 5FU could have a general effect on translation. Example 4: DXR interferes with suppression of antigen presentation and inhibition of GAr mediated translation. After identifying and validating the DXR and 5FU compounds with the yeast cell screening assay, the ability of these two compounds to interfere with the suppression of GAr-mediated antigen presentation was assessed using a T cell base described by Apcher et al. (2010) and by Karttunen et al. (1992). For this purpose, a construct resulting from the fusion (i) of GAr domain 235 amino acids in length (235GAr) was used with (ii) the N-terminus of chicken ovalbumin (Ova). Ova contains the antigenic peptide SIINFEKL (SL8 - SEQ ID NO: 5) which is detected by the specific reporter CD8 + T cells (B3Z cells), when the SL8 antigenic peptide is presented in association with murine class I CMH molecules ( Karttunen et al., 1992). Ova alone was used as a control. HEK 293 T cells that stably express the Class I Kb molecule (HEK 293T Kb, reference) were transfected by the cDNAs encoding these polypeptides into. The transfected HEK 293T Kb cells were then treated or not with various concentrations of DXR or 5FU for 24 hours. The compounds were then removed and the HEK 293T Kb cells mixed with an equal number of B3Z cells and incubated overnight. The amount of SL8 peptide displayed on the surface of HERK 293T Kb cells was then determined indirectly by measuring the (3-Gal) activity in B3Z cells, which is proportional to the activation of SL8 peptide-specific T cell receptors. This method made it possible to monitor the effect of the compounds on the presentation of the antigen from the Ova and 235GAr-Ova constructs.The compounds DXR (Figure 5B) and 5FU (Figure 5C) induced a significant increase in However, as observed in the yeast cell-based assay, this effect is only specific for GAr for the DXR compound because this therapeutic compound does not have the antigen presentation from the 235GAr-Ova construct. does not increase the presentation of Ova alone, whereas 5FU induces an increase in Ova presentation to a similar degree to the 235GAr-Ova construct.Ova and 2 protein levels were also determined. 35GAr-Ova in HEK 393T Kb cells and the DXR compound was shown to lead to a significant, dose-dependent increase in DXR on the GAr-Ova equilibrium level, whereas DXR did not significant effect on Ova alone (Figure 5A), as had also been observed in the yeast cell-based assay. All of these results show that the DXR compound affects the expression and antigenic presentation of the GAr-Ova construct, in a manner dependent on the presence of the GAr domain. These results validate the test of screening of therapeutic compounds with yeast cells which is described in the previous examples.

Example 5: DXR leads to an increase in the expression level of EBNA1 dependent on the presence of GAr. The effect of the DXR compound on the level of expression of the EBNA1 protein was determined. HEK 293T Kb cells expressing either EBNA1 or EBNA14GAr were treated or not with increasing concentrations of DXR for 24 hours. As shown in Figure 5D, the DXR compound leads to a dose-dependent increase in equilibrium levels of the EBNA1 protein, whereas DXR did not exert any effect on EBNA14GAr levels. The effect of DXR on endogenous EBNA1 expression was determined using Raji cells, which consist of Burkitt lymphoma cells carrying the EBV virus. The endogenous EBNA1 protein in Raji cells migrates more rapidly than the exogenous EBNA1 protein due to its shorter GAr sequence. Endogenous EBNA1 and exogenous EBNA1 levels expressed in the same cells increased after 8 hours treatment with 400 nM DXR (Figure 5E).

All of these results show that, beyond its effect on the expression of GAr-Ova and on the antigen-dependent presentation of GAr, DXR also leads to a GAr-dependent increase in the equilibrium level of the protein. EBNA1 complete ("full length"), whether EBNA1 is overexpressed or expressed endogenously in EBV-infected cells. Example 6: The effect of DXR on 5 GAr-mediated translation control is independent of DNA damage Based on the observation that DXR, but not 5FU, specifically overcomes the effect of GAr on translation, DXR commercial analogues, whose chemical structures are illustrated in Figure 6A, were tested in the yeast cell screening assay described in the previous examples. The purpose of these tests is to validate the selection of DXR as a compound of interest, and to determine whether there is a link between the known genotoxic activity of DXR and its effect on the inhibition of translation by the domain. Gar. As shown in Figure 6B, daunorubicin, epirubicin and pirarubicin are also active in this yeast cell-based assay, although to a lesser extent than DXR. However, idarubicin and valrubicin, which are also close to DXR, were inactive, indicating a yet unknown specific effect of DXR and its analogs on GAr-dependent translation. DXR and its five analogues have all been described as causing damage to DNA, due to their ability to target topoisomerase II / DNA complexes (Nitiss, 2009). In keeping with this genotoxic effect, these compounds have been shown to activate the p53 tumor suppressor pathway, as evidenced by their ability to induce the expression of both p53 and p21 genes. (Figure 6C). It can be noted that both etoposide and teniposide, which represent a distinct class of compounds targeting topoisomerase II, have no effect on the control of GAr-dependent translation in the yeast cell-based assay. while these compounds activate the p53 pathway as well as DXR and its analogs (Figure 6B & 6C). These results indicate that the effect of DXR on GAr mediated translation inhibition is not coupled with its already known genotoxic effect. To further validate the structure-activity relationship (SAR) of DXR, the effect of a few DXR chemical analogues was tested in the mammalian T cell-based assay. In agreement with its effect in yeast, treatment with daunorubicin resulted in a significant increase in antigen presentation from the 235GRA-Ova construct (Figure 7A). In contrast, and as shown with the yeast cell assay, both valrubicin (Figure 7B) and etoposide (Figure 7C) have no effect. Epirubicin, which is moderately active in the yeast cell assay, exerts a modest effect in the T cell-based assay (Figure 7D). All of these results suggest that the test in yeast cells can be used to perform structure-activity relationship (SAR) studies for compounds that interfere with the effect of the GAr domain on translation. In addition, based on the comparison of effects on DNA damage and a GAr-dependent translation suppression effect, these results show that these two effects are not coupled.

SEQUENCE LISTING SEQ ID No. 1 MSDEGPGTGPGNGLGEKGDT SGPEGSGGSGPQRRGGDNHGRGRGRGRGRGGGRP GAP GGS GS GPRHRDGVRRPQKRP S CIGCKGTHGGTGAGAGAGGAGAGGAGAGG GAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGA GAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGA GGAGGAGAGGAGAGGGAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGG AGGAGAGGAGGAGAGGGAGGAGAGGGAGGAGAGGAGGAGAGGAGGAGAGG AGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRGGSGGRGRGGSGGRRGRGRE RARGGSRERARGRGRGRGEKRPRSP SSQSSS SGSPPRRPPPGRRPFFHPVGEADYFE YHQEGGPDGEPDVPPGAIEQGPADDPGEGP ST GPRGQ GD GGRRKKGGWF GKHRG QGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKT SLYNLRRGTA LAIPQCRLTPL SRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDL VMTKPAPTCNIRVTVC SFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDE GEEGQE SEO ID No. 2 GAGAGAGGAGAGGAGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGA GGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGG AGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGGGAGGAGGAGAGGAGAGG AGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAGGGAGGAGA GGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGG SEO ID ° 3 GAGCAGGAGCAGGAGCGGGAGGGGCAGG AGCAGGAGGGGCAGGAGCAGGAG GAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGGGGCAGGAGGGG CAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAG GGGC AGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGC AGGAGGGGC AG GAGCAGGAGGAGGGGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGG CAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAG GGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAG GGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAG GGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAG GAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAG GGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAG GGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAG GGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGG CAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGTGGAG CCG SEO ID No. 4 MG SIGAA SMEF CFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKD S TRT QIN KVVRFDKLPGF GD SIEAQC GT SVNVHS SLRDILNQITKPND VYSF SLASRLYAEER YPILPEYLQCVKELYRGGLEPINF QTAADQARELINSWVES QTNGIIRNVLQP S SVD SQTAMVLVNAIVFKGLWEKTFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVAS MA SEKMKILELPF AS GTM SML VLLPDEV SG LEQLE SIINFEKL TEW TS SNVMEERKI KVYLPRMKMEEKYNLT SVLMAMGITDVF SS SANL SIMS SAESLKISQAVHAAHAE NEAGREVVGS AEAGVD AA SV SEEF RADHPFLF CIKHIATNAVLFF RCMP V SP SEO ID NO: 5 SIINFEKL SEO ID NO: 6 MAPPGMRLRSGRSTGAPLTRGSCRKRNRSPERCDLGDDLHLQPRRKHVAD S VD G RECGPHTLPIPGSPTVF TS GLPAF VS SPTLPVAPIPSPAPATPLPPPALLPPVTTS S SPIP P SHPVSP GT TD THSP SPALPPTQ SPE SQRPPL S S S SPTGRPD STPMRPPPSQQ TTPPHS PTTPPPEPPSKS SPD SLAP STLRSLRKRRL S SPQGP STLNPICQ SPPVSPPRCDFANRS VYPPWATESPIYVGS S SD GD TPPRQPP T S SP SPI SIGS SEGSWGDDTAMLVLLAEIAE EASKNEKEC SENNQAGEDNGDNEISKESQVDKDDNDNKDDEEEQETDEEDEEDD EEDDEEDDEEDDEEDDEEDDEEDDEEEDEEEDEEEDEEEDEEEEEDEEDDDDEDN EDEEDDEEEDKKEDEEDGGDGNKTL SIQ S SQQQQEPQQQEPQQQEPQQQEPQQQE PQQQEPQQQEPQQQEPQQREPQQREPQQREPQQREPQQREPQQREPQQREPQQRE PQQREPQQREPQQREPQQREPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQE PQQQEPQQQEPQQQEPQQQEPQQQEPQQQDEQQQDEQQQDEQQQDEQQQDEQQ QDEQQQDEQQQDEQEQQDEQQQDEQQQQDEQEQQEEQEQQEEQQQDEQQQDEQ QQDEQQQDEQEQQDEQQQDEQQQQDEQEQQEEQEQQEEQEQQEEQEQQEEQEQ ELEEQEQELEEQEQELEEQEQELEEQEQE LEEQEQELEEQEQELEEQEQELEEQEQE LEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQEQELEEVEEQEQEQE EQELEEVEEQEQEQEEQEEQELEEVEEQEEQELEEVEEQEEQELEEVEEQEQQGVE QQEQETVEEPIILHGS S SEDEMEVDYPVVSTHEQIAS SPPGDNTPDDDPQPGP SREY RYVLRT SPPHRPGVRMRRVPVTHPKKPHPRYQ QPPVPYRQIDD CPAKARP QHIF YR RFLGKDGRRDPKCQWKFAVIFWGNDPYGLKKL SQAFQFGGVKAGPVSCLPHPGP DQ SPITYCVYVYCQNKDT SKKVQMARLAWEASHPLAGNLQ S SIVKFKKPLPLTQP GENQGPGD SPQEMT SEO ID NO: 7 MAPPGMRLRSGRSTGAPLTRGSCRKRNRSPERCDLGDDLHLQPRRKHVAD S ID GR ECGPHTLPIPGSPTVFT S GLPAF VS SPTLPVAPIP SPAPATPLPPPALLPPVTT SS SPIPP SHPVSPGTTDTHSP SPALPPTQ SPE S SQRPPLS SPTGRPD S STPMRPPP SQQTTPPHSP TTPPPEPP SKS SPD SLAPSTLRSLRKRRL S SPQGP STLNPICQ SPPV SPPRCDF ANRS V YPPWATESPIYVGS S SD GD TPPRQPP SPI T S S SP SEGSWGDDTAMLVLLAEIAEE IGS AS KNEKEC SENNQAGEDNGDNEISKESQVDKDDNDNKDDEEEQETDEEDEEDDE EDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEEDEEEDEEEDEEEE DEEDDDDEDNEDEEDDEEEDKKEDEEDGGDGNKTL SIQ S SQQQQEPQQQEPQQQE PQQQEPLQEPQQQEPQQQEPQQQEPLQEPQQQEPQQQEPLQEPQQQEPQQQEPQQ QEPQQQEPQQQEPQQQEPQQQE PQQQEPQQQEPQQQEPQQREPQQREPQQREPQQ REPQQREPQQREPQQREPQQREPQQREPQQQDEQQQDEQQQDEQQQDEQQQDEQ QQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQ DEQQQDEQQQDEQQQDEQQQDEQQQDEQEQQDEQEQQDEQEQQDEQQQDEQQ QQDEQQQQDEQQQQDEQQQQDEQQQQDEQEQQEEQEQQEEQEQELEEQEQELE DQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELE EQEQELEEQEVEEQEQEVEEQEQEQEEQELEEVEEQEQEQEEQEEQELEEVEEQEE QELEEVEEQEEQELEEVEEQEQQELEEVEEQEQQGVEQQEQETVEEPIILHGS S SED EMEVDYPVVSTHEQIAS SPPGDNTPDDDPQPGP SREYRYVLRTSPPHRPGVRMRR VPVTHPKKPHPRYQQPPVPYRQIDDCPAKARPQHIFYRRFLGKDGRRDPKCQWKF AVIFWGNDPYGLKKL SQAF QFGGVKAGPVSCLPHPGPDQ SPITYCVYVYCQNKDT SKKVQMARLAWEASHPLAGNLQ SS IVKFKKPLPL scooter GENQ GP GD SPQEMT SEO ID No. 8 MAPPGMRLRSGRSTGAPLTRGSCRKRNRSPERCHLGDDLHLQPRRKHVADSVDG RECGPHTLPIPGSPTVF TS GLPAF VS SPTLPVAPIP SPAPATPLPPPALLPPVTTS S SPIP P SHPVSP GT TD THSP SPALPPTQ SPE S SQRPPL S SPTGRPD S STPMRPPP SQQ TTPPHS PTTPPPEPP SKS SPD SLAP STLRSLRKRRL S SPQGP STLNPICQ SPPVSPPRCDFANRS VYPPWATESPIYVGS S SD GD TPPRQPP T SPI SIGS S SP SEGSWGDDTAMLVLLAE IAE EASKNEKEC SENNQAGEDNGDNEISKESQVDKDDNDNKDDEEEQETDEEDEEDD EEDDEEDDEEDDEEDDEEDDEEDDEEDDEEEDEEEDEEEEDEEEEEEDEEDDDDE DNEDEEEDKKEDEEDGGDGNKTL SIQ SSQQQQEPQQQEPQQQEPQQQEPQQQEPL QEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQDEQQQDEQQQDEQQQDEQ QQDEQEQQDEQQQDEQQQDEQQQQDEQEQQEEQEQQEEQEQQEEQEQELEEQE QELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQE QELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQE QELEEQEQELEEQEQELEEQEQEQELEEVEEQEQEQEEQELEEVEEQEQEQEEQEE QELEEVEEQEEQELEEVEEQEEQELEEVEEQEQQGVEQQEQETVEEPIILHGS S SED EMEVDYPVVSTHEQIAS SPPGDNTPDDDPQPGP SREYRYVLRTSPPHRPGVRMRR VPVTHPKKPHPRYQQPPVPYRQIDDCPAKARPQHIFYRRFLGKDGRRDPKCQWKF AVIFWGNDPYGLKKL SQAF QFGGVKAGPVSCLPHPGPDQ SPITYCVYVYCQNKDT SKKVQMARLAWEASHPLAGNLQ S S IVKFKKPLPL scooter GENQ GP GD SPQEMT SEO ID No. 9 ATGGCGCCCCCGGGAATGCGCCTGAGGTCGGGACGGAGCACCGGCGCGCCCTT AACGAGAGGAAGTTGTAGGAAACGAAACAGGTCTCCGGAAAGATGTGACCTT GGCGATGACCTACATCTACAACCGCGAAGGAAGCATGTCGCCGACTCCATCGA CGGCCGGGAATGTGGACCCCACACCTTGCCTATACCTGGAAGTCCCACAGTGT TCACATCCGGGCTGCCAGCATTTGTG TCTAGTCCTACTTTACCGGTGGCTCCCA TTCCTTCACCCGCTCCCGCAACACCTTTACCTCCACCGGCACTCTTACCCCCCG TAACCACGTCTTCCTCCCCAATCCCTCCATCCCATCCTGTGTCTCCGGGGACCA CGGATACTCATTCTCCATCTCCTGCATTGCCACCCACGCAGTCTCCAGAGTCTT CTCAAAGGCCACCGCTTTCAAGTCCTACAGGAAGGCCAGACTCTTCAACACCT ATGCGTCCGCCACCCTCGCAGCAGACTACACCTCCACACTCACCCACGACTCC TCCACCCGAGCCTCCCTCCAAGTCGTCACCAGACTCTTTAGCTCCGTCTACCCT GCGTAGCCTGAGAAAAAGAAGGCTATCGTCCCCCCAAGGTCCCTCTACACTAA ACCCAATATGTCAGTCGCCCCCAGTCTCTCCCCCTAGATGTGACTTCGCCAACC GTAGTGTGTACCCCCCATGGGCCACAGAGTCCCCGATCTACGTGGGATCATCC AGCGATGGCGATACTCCGCCACGCCAACCGCCTACATCTCCCATCTCCATAGG ATCATCATCCCCGTCTGAGGGATCCTGGGGTGATGACACAGCCATGTTGGTGC TCCTTGCGGAGATTGCAGAAGAAGCATCCAAGAATGAAAAAGAATGTTCCGA AAATAATCAGGCTGGCGAGGATAATGGGGACAACGAGATTAGCAAGGAAAGT CAGGTTGACAAGGATGACAATGACAATAAGGATGATGAGGAGGAGCAGGAGA CAGATGAGGAGGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACG AGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGG AGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGG AGGACGAGGAGGAGGACGAGGAGGAGGACGAGGAGGAGGAGGACGAGGAGG AT GA C GAT GATGAGGACAAT GAGGAC GAGGAGGATGAC GAGGAGGAGGAC A AGAAGGAGGAC GAGGAGGAC GGGGGC GAT GGAAACAAAAC GT TGAGCAT CC AAAGT TC ACAAC AGCAGCAGGAGC CACAACAGCAGGAGC C ACAGC AGCAGGA GCCACAGCAGCAGGAGCC CCTGCAGGAGC CAC AACAGC AGGAGC CACAGCAG CAGGAGC CAC AGCAGC AGGAGCC CCTGCAGGAGC CAC AAC AGCAGGAGC CAC AGCAGC AGGAGCC CCTGCAGGAGC CAC AACAGC AGGAGC CACAACAGC AGGA GC C ACAGC AGCAGGAGC CAC CAC AGCAGC AGGAGC AGCAGCAGGAGC CACAG CGIC AGGAGC C ACAGC AGCAGGAGC C AC AGCAGC AGGAGC CAC CAC AGCAGCAG GAGCCACAGCAGCAGGAGCCACAGCAGCGGGAGCCACAGCAGCGGGAGCCCC AGCAGCGGGAGCCACAGCAGCGGGAGCCACAGCAGCGGGAGCCACAGCAGC GGGAGCCACAGCAGCGGGAGCCACAGCAGCGGGAGCCACAGCAGCGGGAGC AGCAGC AGGATGAGCAGCAGCAGGATGAGCAGCAGC AGGATGAGCAGC A GCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGAT GAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGC AGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGG AT GAGCAGC AGCAGGAT GAGCAGCAGC AGGAT GAGCAGC AGC AGGAT GAGC A GCAGCAGGAT GAGCAGC AGC AGGATGAGC AGCAGCAGGAT GAGC AGCAGC AG GATGAGCAGGAGCAGCAGGAT GAGCAGG AGC AGC AGGAT GAGC AGGAGC AG CAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGCAGGATGAGCAGCAGCAGC AGGAT GAGCAGCAGC AGCAGGAT GAGC AGCAGC AGCAGGAT GAGCAGCAGC A GCAGGATGAACAGGAGCAGCAGGAGGAGCAGGAGCAGCAGGAGGAGCAGGA GCAGGAGT TAGAGGAGCAGGAGCAGGAGTTAGAGGAT CAGGAGCAGGAGT TA GAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAG GAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAG TTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGC AGGAGCAGGAGTTAGAGGAGCAGGAGGTGGAAGAGCAAGAGCAGGAGGTGG AAGAGCAAGAGCAGGAGCAGGAAGAGCAGGAATTAGAGGAGGTGGAGGAGC AAGAGCAGGAGCAGGAGGAGCAGGAGGAGCAGGAGTTAGAGGAGGTGGAAG AGCAGGAAGAGCAGGAGTTAGAGGAGGTGGAAGAGCAGGAAGAGCAGGAGT TAGAGGAGGTGGAAGAGCAGGAGCAGCAGGAGTTAGAGGAGGTGGAAGAGC AGGAGC AGCAGGGGGT GGAACAGCAGGAGC AGGAGAC GGT GGAAGAGC C CA TAATC TT CCGA GGGT C GT CAT CC GAGGAC GAAAT GGAAGT GGAT TAC CC TGT T GT TAGCACACAT GAACAAAT T GC C AGTAGC CC AC CAGGAGATAATACAC CAGA CGATGACCCACAACCTGGCCCATCTCGCGAATACCGCTATGTACTCAGAACAT CAC CAC CCCACAGAC CTGGAGTTC GTATGAGGC GCGTTC CAGTTACCCACC CA AAAAAGC CAC ATC CAAGATA CC AACAAC CAC C GGTC CTC TACAGAC AGATAG ATGATTGTCCTGCGAAAGCTAGGCCACAACACATCTTTTATAGACGCTTTTTGG GAAAGGAT GGAAGAC Gagat CC AAAGTGTCAATGGAAGT TT GCAGT GATT TT TT GGGGCAAT GAC C CATAC GGAC T TAAAAAAT TAT CT CAGGC C TT CC AGT TT GG AGGAGTAAAGGCAGGCCCCGTGTCCTGCTTGCCCCACCCTGGACCAGACCAGT C GC CC ATAAC T TAT TGTGTATATGT GTATT GT CAGAAC AAAGACAC AAGTAAG AAAGTACAAATGGCCCGC CTAGCC TGGGAAGC tagt CAC CC CCTGGCAGGAA ACCTACAATCTTCCATAGTTAAGTTTAAAAAGCCCCTGCCATTAACCCAGCCAG GGGAAAAC CAAGGT GGGGAC CTC TC TC C ACAGGAAAT AT GAC AA SEQ ID N ° 10 ATGGCGCCCCCGGGAATGCGCCTGAGGTCGGGACGGAGCACCGGCGCGCCCTT AACGAGAGGAAGTTGTAGGAAACGAAACAGGTCTCCGGAAAGATGTCACCTT GGCGATGACCTACATCTACAACCGCGAAGGAAGCATGTCGCCGACTCCGTCGA CGGCCGGGAATGTGGACCCCACACCTTGCCTATACCAGGAAGTCCCACAGTGT TCACATCCGGGCTGCCAGCATTTGTGTCTAGTCCTACTTTACCGGTGGCTCCCA TTCCTTCACCCGCTCCCGCAACACCTTTACCTCCACCGGCACTCTTACCCCCCG TAACCACGTCTTCCTCCCCAATCCCTCCATCCCATCCTGTGTCTCCGGGGACCA CGGATACTCATTCTCCATCTCCTGCATTGCCACCCACGCAGTCTCCAGAGTCTT CTCAAAGGCCACCGCTTTCAAGTCCT ACAGGAAGGCCAGACTCTTCAACACCT ATGCGTCCGCCACCCTCGCAGCAGACTACACCTCCACACTCACCCACGACTCC TCCACCCGAGCCTCCCTCCAAGTCGTCACCAGACTCTTTAGCTCCGTCTACCCT GCGTAGCCTGAGAAAAAGAAGGCTATCGTCCCCCCAAGGTCCCTCTACACTAA ACCCAATATGTCAGTCGCCCCCAGTCTCTCCCCCTAGATGTGACTTCGCCAACC GTAGTGTGTACCCCCCATGGGCCACAGAGTCCCCGATCTACGTGGGATCATCC AGCGATGGCGATACTCCGCCACGCCAACCGCCTACATCTCCCATCTCCATAGG ATCATCATCCCCGTCTGAGGGATCCTGGGGTGATGACACAGCCATGTTGGTGC TCCTTGCGGAGATTGCAGAAGAAGCATCCAAGAATGAAAAAGAATGTTCCGA AAATAATCAGGCTGGCGAGGATAATGGGGACAACGAGATTAGCAAGGAAAGT CAGGTTGACAAGGATGACAATGACAATAAGGATGATGAGGAGGAGCAGGAGA CAGATGAGGAGGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACG AGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGG AGGATGACGAGGAGGAGGACGAGGAGGAGGACGAGGAGGAGGAGGACGAGG AGGAGGAGGAGGAGGACGAGGAGGATGACGATGATGAGGACAATGAGGACG AGGAGGAGGACAAGAAGGAGGACGAGGAGGACGGGGGCGATGGAAACAAAA CGTTGAGCATCCAAAGTTCACAACAGCAGCAGGAGCCACAGCAGCAGGAGCC ACAACAGCAGGAGCCACAGCAGCAGGAGCCACAGCAGCAGGAGCCCCTGCAG GAGCCACAGCAGCAGGAGCCACAACAGCAGGAGCCACAACAGCAGGAGCCAC AACAGCAGGAGCCA CAACAGCAGGAGCCACAACAGCAGGAGCCACAGCAGC AGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGA GCAGCAGCAGGATGAGCAGGAGCAGCAGGATGAGCAGCAGCAGGATGAGCA GCAGCAGGATGAGCAGCAGCAGCAGGATGAACAGGAGCAGCAGGAGGAGCA GGAGCAGCAGGAGGAGCAGGAGCAGCAGGAGGAGCAGGAGCAGGAGTTAGA GGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGA GCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTA GAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAG GAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAG TTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGC AGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGG AGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGG AGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGC AGGAGCAGGAGTTAGAGGAGGTGGAAGAGCAAGAGCAGGAGCAGGAAGAGC AGGAATTAGAGGAGGTGGAGGAGCAAGAGCAGGAGCAGGAGGAGCAGGAGG AGCAGGAGTTAGAGGAGGTGGAAGAGCAGGAAGAGCAGGAGTTAGAGGAGG TGGAAGAGCAGGAAGAGCAGGAGTTAGAGGAGGTGGAAGAGCAGGAGCAGC AGGGGGTGGAACAGCAGGAGCAGGAGACGGTGGAAGAGCCCATAATCTTGCA CGGGTCGTCATCCGAGGACGAAATGGAAGTGGATTACCCTGTTGTTAGCACAC ATGAACAAATTGCCAGTAGC CCACCAGGAGATAATACACCAGACGATGACCC ACAACCTGGCCCATCTCGCGAATACCGCTATGTACTCAGAACATCACCACCCC ACAGAC CT GGAGT TC GTATGAGGC GC GT TC C AGTTAC C CAC C CAAAAAAGC CA CAT CC AAGATAC CAACAAC C AC C GGTCCCTTACAGACAGATAGATGATTGTCC TGCGAAAGCTAGGCCACAACACATC TT TTATAGAC GC TT T TT GGGAAAGGATG 5 GAAGAC GAGATC CAAAGT GT CAAT GGAAGT TT GC AGT GATT T TT T GGGGCAAT GACCCATACGGAC TTAAAAAAT TAT CT CAGGC CT CT C AGT T TGGAGGAGTAAA GGCAGGCCCCGTGTCCTGCTTGCCCCACCCTGGACCAGACCAGTCGCCCATAA CTTATTGTGTATATGTGTATTGTCAGAACAAAGACACAAGTAAGAAAGTACAA AT GGCC CGCC TAGCCTGGGAAGCTAGTCACCCCCTGGCAGGAAACCTACAATC 10 TTCCATAGTTAAGTTTAAAAAGCCCCTGCCATTAACCCAGCCAGGGGAAAACC AAGGTCC TGGGGAC TC TC CACAGGAAAT GACATAA SEO ID NO: 11 GAGGAGGAGAGGAGGAGAGGA 15 SEO ID NO: 12 GGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGA GCAGGAGGGGCC 20 SEQ ID NO: 13 GAGGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGA SEO ID NO: 14 GGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGA AGGAGGAGC AGGAGGAGGAGGAGCAGG AGGAGGGGC AGGAGGAGGAGGAGCAGG AGGGGCC SEO ID No. 15 30 GAGAGAGGAGAGGAGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGA GGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGG AGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGGGAGGAGGAGAGGAGAGG AGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAGGGAGGAGA GGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGA 35 SEQ ID NO: 16 GGAGCAGGAGCAGGAGCGGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGA GGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGGGGCAGGAGGG GCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGA GGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCA 40 GGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGG GCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGA GGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGA GGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGA GGGGC AGGAGGGGCAGGAGC AGGAGGGGC AGGAGC AGGAGGGGC AGGAGC 45 GGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGA GGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGA GGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGA GGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGG GCAGGAGCAGGAGGAGGGGC AGGAGC AGGA GGGGC AGGAGC A SEO ID NO: 17 DDEEEQETDEEDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEEDDEED DEEEDEEEDEEEDEEEEDEEDDDDEDNEDEEDDEEEDKKEDEEDGGDGNKTL SIQ SSQQQQEPQQQEPQQQEPQQQEPLQEPQQQEPQQQEPQQQEPLQEPQQQEPQQQE PLQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEPQQQEP QQREPQQREPQQREPQQREPQQREPQQREPQQREPQQREPQQREPQQQDEQQQDE QQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQ QDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQQQDEQEQQDEQEQ QDEQEQQDEQQQDEQQQQDEQQQQDEQQQQDEQQQQDEQQQQDEQEQQEEQE QQEEQEQELEEQEQELEDQEQELEEQEQELEEQEQELEEQEQELEEQEQELEEQEQ ELEEQEQELEEQEQELEEQEQELEEQEVEEQEQEVEEQEQEQEEQELEEVEEQEQE QEEQEEQELEEVEEQEEQELEEVEEQEEQELEEVEEQEQQELEEVEEQEQQGVEQQ EQE SEO ID NO: 18 GATGATGAGGAGGAGCAGGAGACAGATGAGGAGGACGAGGAGGATGAC GAG GAG GAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAG GATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGATGACGAGGAGGAT GACGAGGAGGATGACGAGGAGGAGGACGAGGAGGAGGACGAGGAGGAGGAC GAGGAGGAGGAGGACGAGGAGGATGACGATGATGAGGACAATGAGGAC GAGGATGACGAGGAGGAGGACAAGAAGGAGGACGAGGAGGACGGGGGCGAT GGAAAC AAAAC C CAAA GT TGAGCAT GTT CAC AACAGCAGC AGGAGC CACAAC AGCAGGAGCCACAGCAGCAGGAGCCACAGCAGCAGGAGCCCCTGCAGGAGCC ACAACAGCAGGAGC CAC AGCAGC AGGAGC C ACAGC AGCAGGAGC CCC TGC AG GAGC CACAAC AGCAGGAGC CAC AGCAGC AGGAGCC CCTGCAGGAGC CAC AAC AGCAGGAGCCACAACAGCAGGAGC CACAGC AGCAGGAGC CAC AGCAGC AGG AGC CACAGCAGC AGGAGC C ACAGC AGCAGGAGC C ACAGCAGCAGGAGC CAC A GCAGCAGGAGC CACAGC AGCAGGAGC CAC AGCAGCAGGAGC CACAGCAGC G GGAGC ACC AGCAGC GGGAGCC CCAGCAGC GGGAGC C ACAGC AGC GGGAGC CA CGIC AGC GGGAGC CAC AGCAGC GGGAGCCACAGCAGCGGGAGC CACAGCAGC GGGAGC CACAGCAGC GGGAGC CAC AGCAGC AGGATGAGCAGCAGCAGGAT G AGCAGC AGCAGGAT GAGC AGCAGC AGGAT GAGCAGCAGCAGGATGAGCAGC A GCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGAT GAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGC AGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGGATGAGCAGCAGCAGG AT GAGCAGC AGCAGGATGAGC AGCAGC AGGAT GAGCAGC AGCAGGATGAGC A GCAGCAGGATGAGCAGCAGCAGGATGAGCAGGAGCAGCAGGATGAGCAGGA GCAGCAGGAT GAGC AGGAGCAGCAGGAT GAGC AGCAGC AGGAT GAGCAGC A GCAGCAGGAT GAGCAGC AGCAGC AGGATGAGCAGCAGCAGCAGG ATGAGC AG CAGCAGCAGGATGAGCAGCAGCAGCAGGATGAACAGGAGCAGCAGGAGGAG CAGGAGCAGCAGGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAG TTAGAGGATCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGC AGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGG AGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGG AGCAGGAGCAGGAGTTAGAGGAGCAGGAGCAGGAGTTAGAGGAGCAGGAGG TGGAAGAGCAAGAGCAGGAGGTGGAAGAGCAAGAGCAGGAGCAGGAAGAGC AGGAATTAGAGGAGGTGGAGGAGCAAGAGCAGGAGCAGGAGGAGCAGGAGG AGCAGGAGTTAGAGGAGGTGGAAGAGCAGGAAGAGCAGGAGTTAGAGGAGG TGGAAGAGCAGGAAGAGCAGGAGTTAGAGGAGGTGGAAGAGCAGGAGCAGC AGGAGTTAGAGGAGGTGGAAGAGCAGGAGCAGCAGGGGGTGGAACAGCAGG AGCAGGAG SEO ID NO: 19 GCGCGAATTCATGTACCCATACGATGTTCCAGATTACGCTAGGGATTCTAGAA 10 CAG SEO ID NO: 20 GCGCGGTACCTTACTTGTTTTCTAGATAAGG 15 SEO ID NO: 21 GCGCGGATCCATGGGGTACCCATACGATGTT SEO ID NO: 22 GCGCGAATTCTGGTGAATTCAGGGCCCCTCC-3 '20 SEO ID NO: 23 GCGCGGATCCATGTACCCCTACGACGTCCCCG SEO ID # 24 25 GCGCGAATTCCTCGAGGATATCACCTTCTTGG SEO ID # 25 GCGCGGATCCATGGCTTACCCCTACGACG 30 SEO ID # 26 GCGCGGTGAATTCTCCTCCTGCTCC SEO ID NO. 27 ATTGTGCAAATGCCTAGAGGTG 35 SEO ID # 28 AATCATAAGCGCCAAGCAGTC SEO ID # 29 40 ATGGTCGGTATGGGTCAAAAA SEO ID # 30 TTCCATATCGTCCCAGTTGGT 45

Claims (15)

REVENDICATIONS1. An in vitro method for selecting active compounds against infection with a gamma herpesvirus, comprising the steps of: a) culturing yeast cells in the genome of which at least one copy of a nucleic acid has been inserted rendering said cells capable of expressing a recombinant protein comprising at least a [GMP] moiety and at least a [REPORTER] moiety, wherein: [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells, and the yeast cells being cultured in the presence of a candidate compound, b ) measuring the level of production of said recombinant protein by the cells cultured in step a), and c) comparing the level of production of said recombinant protein measured in step b) with a value of reference. 2. Method according to claim 1, wherein said recombinant protein is a recombinant protein of formula (I): NH2- [TAG1] - [GMP] - [REPORTER] - [TAG2] y-COOH (I), in which: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1, - [GMP] signifies a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells. 3. Method according to one of claims 1 and 2, the recombinant protein of formula (I) comprising a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus. 4. Method according to one of claims 1 to 3, the recombinant protein comprising a portion [REPORTER] consisting of the enzyme Ade2p Saccharomyces cerevisiae. 5. Method according to one of claims 1 to 4, the yeast cells being Saccharomyces cerevisiae cells, preferably Saccharomyces cerevisiae cells of genotype MATA leu2-3, 112 trp1-1 canl-100 ura3-1 ade2- 1 :: his5 S. pombe An in vitro method for selecting compounds active against Epstein-Barr virus infection comprising the steps of: a) culturing mammalian cells having the following characteristics: (i) said cells express a class I MHC antigen, and (ii) said cells express a recombinant protein comprising at least a portion [GMP] and at least a portion [REPORTER], wherein: [GMP] means a polypeptide comprising all or part of the amino acid sequence of an Epstein-Barr virus stability protein, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells, the mammalian cells being cultured in the presence of a candidate compound, b) measuring the level of expression of the class I MHC-associated recombinant protein on the surface of the cells cultured in step a), and c) comparing the level of expression of the recombinant protein ante measured in step b) with a reference value. The method according to claim 6, wherein said recombinant protein is a recombinant protein of the following formula (I): NH2- [TAG1] - [GMP] - [REPORTER] - [TAG2] y-COOH (I), in which: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1, - [GMP] signifies a polypeptide comprising all or part of the amino acid sequence of an Epstein-Barr virus genome stability protein, and - [REPORTER] means a detectable polypeptide, not expressed as such by said mammalian cells. 8. Process according to claim 6 or 7, the candidate compound being selected by carrying out the method according to one of claims 1 to 5. 9. Method according to one of claims 6 to 8, wherein step b) is carried out according to the following steps: b1) cultivating the mammalian cells obtained at the end of step a) in the presence of recognizable CD8 + T lymphocyte cells specifically the peptide of sequence SEQ ID No. 5, and b2) measure the level of proliferation of CD8 + T lymphocyte cells cultured in step 1) 1). 10. The method of claim 9, the CD8 + T lymphocyte cells cultured in step b1) expressing a detectable recombinant protein. 11. Method according to one of claims 6 to 10, the mammalian cells being selected from the group comprising the cells of the line HEK293T kb and Raji cells. A yeast cell in the genome from which at least one copy of a nucleic acid has been inserted, making these cells capable of expressing a recombinant protein comprising at least one [GMP] part and at least one [REPORTER] part, in which - [GMP] means a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cell. A yeast cell according to claim 12, wherein said recombinant protein is a recombinant protein of the following formula (I): NH 2 - [TAG 1] - [GMP] - [REPORTER] - [TAG 2] y -COOH (I ), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to a ligand, - x and y, identical or different, represent an integer equal to 0 or 1, - [GMP] signifies a polypeptide comprising all or part of the amino acid sequence of a genome stability protein of a gamma herpes virus, and - [REPORTER] means a detectable polypeptide, not expressed as such by said yeast cells. A mammalian cell having the following characteristics: (i) said cell expresses a MHC class I antigen, and (ii) said cell expresses a recombinant protein comprising at least a portion [GAr] and at least a portion [REPORTER] , wherein: [GAr] means a peptide comprising all or part of the continuous amino acid sequence of the GAr domain of EBNA1 protein of Epstein-Barr virus, and - [REPORTER] means a detectable, non-expressed polypeptide as such by said mammalian cell. The mammalian cell of claim 14, having the following characteristics: (i) said cell expresses a MHC Class I antigen, and (ii) said cell expresses a recombinant protein of the following formula (I): NH2- [TAG1 ] ,, - [GAr] - [REPORTER] - [TAG2] y -COOH (I), wherein: [TAG1] and [TAG2] signify a peptide capable of binding to an identical ligand, - x and y or different, represent an integer equal to 0 or 1, - [GAr] means a peptide comprising from 8 to 239 continuous amino acids of the GAr domain of the EBNA1 protein of the Epstein-Barr virus, and - [REPORTER] signifies a polypeptide detectable, not expressed as such by said mammalian cell.