WO2004067560A2 - Nouveaux polypeptides liant la proteine vp16 herpes simplex - Google Patents

Nouveaux polypeptides liant la proteine vp16 herpes simplex Download PDF

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WO2004067560A2
WO2004067560A2 PCT/EP2004/000681 EP2004000681W WO2004067560A2 WO 2004067560 A2 WO2004067560 A2 WO 2004067560A2 EP 2004000681 W EP2004000681 W EP 2004000681W WO 2004067560 A2 WO2004067560 A2 WO 2004067560A2
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polypeptide
polynucleotide
vacid
sequence
protein
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PCT/EP2004/000681
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WO2004067560A3 (fr
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Michael Meisterernst
Gerhard Mittler
Ulf Schaberg
Thomas STÜHLER
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Gsf - Forschungszentrum Für Umwelt Und Gesundheit, Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to polypeptides which are involved in the regulation of gene expression, i.e. are capable of binding to the transcription activating domain HI of Herpes simplex protein VP16. More specifically, the present invention relates to the novel polypeptides hVaCID, mVaCID, DoCID and ACID. This invention also relates to genes and polynucleotides encoding said polypeptides, as well as vectors, host cells, antibodies directed to said polypeptides, and the recombinant methods for producing the same.
  • the invention further relates to screening methods for identifying agonists and antagonists of said polypeptides.
  • the nervous system plays a central role in the pathogenesis of herpes simplex virus (HSV) infections.
  • HSV-1, HSV-2 herpes simplex virus
  • Both serotypes of the virus are capable of life-long persistence with sensory peripheral ganglia in a nonreplicative latent state.
  • both serotypes can reactivate to produce recurrent epithelial eruptions and virus shedding.
  • the central nervous system is a major target for HSV-related morbidity and mortality resulting from encephalitis .
  • VP16 a phosphoprotein component of the virion tegument, activates transcription of the viral immediate-early (IE or ) genes.
  • IE or immediate-early
  • VP16 mediates transinduetion through specific promoter elements containing TAATGA-purine-AT and GCGGAA sequences. VP16 does not bind directly to either of these conserved motifs, although it makes minimal contact with the DNA.
  • a domain of VP16 located between amino acids 1 to 400 forms a multicomponent DNA-binding complex with cellular protein Oct-1 as the major determinant of DNA binding and with HCF which facilitates the interaction between VP16 and DNA-bound Oct-1.
  • VPl ⁇ contributes to this complex a potent transcriptional activation domain, located between 410 and 490, that interacts with targets within the basal transcription machinery.
  • VP16 is essential for propagation of the virus: E.g., a VPl ⁇ -null mutant requires for propagation VP16 in trans . Truncation of the VP16 gene at codon 422 (deleting most of the transcriptional activation domain) resulted in an HSV with reduced IE gene activation.
  • Recent studies with HSV-1 mutant with deletions of various portions of the activation domain of VP16 show that these mutations result in a significantly reduced infectivity.
  • the present invention relates to novel Herpes simplex VP16 protein binding polypeptides and the encoding polynucleotides of said polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting a susceptibility to a Herpes simplex infection and therapeutic methods for treating a Herpes simplex infection or disorders associated therewith. The present invention further relates to screening methods for identifying binding partners, activators/agonists and inhibitors/antagonists of said polypeptides.
  • a protein having an apparent molecular weight of 103 kDa binds to VP16-H1.
  • M marker; KE, HeLa nuclear extract; unbound proteins are marked by an asterisk (*); cf. the gel of (C);
  • A Domains and sequence motifs derived from computer predictions. At the N-terminus of the protein a longer sequence can be found (aa 16-145), which is homologous to the VWA-domain (cf. (C) ) of Von-Willebrand-Factor . Two proline reach regions (P) can be found, aa237 to aa275 and aa301 to 387. Further in-silico-analyses revealed the presence of an evolutionary conserved domain, CID ("conserved interesting domain”) (small arrows; aa397 to 541) which has so far not been described and characterized, respectively.
  • CID conserved interesting domain
  • the arrows symbolize potential sequence repeats within the CID domain (small arrows) and between CID-domains (large arrow, found in proteins having two CID domains) , respectively.
  • the CID domain is followed by a short Q reach sequence region (Q) (aa546 to 563) which is followed by a prolin- and tryptophane reach region (P/W) at the C-terminus of the protein.
  • Q Q reach sequence region
  • P/W prolin- and tryptophane reach region
  • the N-terminal region (aal to 290) which had been used for the generation of the mAb VCI9C2 as GST fusion protein expressed in E. coli is underlined.
  • Amino acids showing a high degree of conservation in all four polypeptides are lined dark grey. Amino acids showing a high degree of conservation in three of the four polypeptides are lined grey. Amino acids showing a low degree of conservation are lined light grey. Drosophila melanogaster , ("drome”); Homo sapiens, ("human”); Mus musculus, (“murine”), Xenopus laevis, (“xlaevis”).
  • Drosophila VaCID (dArc92, XP_081452), "human” VaCID (TCBAP0758, NP_112235) , “murine” VaCID (AAH21333), Xenopus VaCID (AW635548 & BJ074617; incomplete) .
  • the VWA- and CID domains are underlined.
  • (A) + (B) MTNTM blots with 12 different human tissue specific poly(A) + -RNA preparations.
  • (C) + (D) MTNTM blots with 6 different human tissue specific poly(A) + -RNA preparations.
  • hVaCID human VaCID (ESTs and gene found and verified by sequencing; cDNA from mouse and man obtained and sequenced; VP16 interaction and dominant negative effect demonstrated. ) ;
  • FIG 7 Transient analysis of VaCID effects on transcriptional activation by a GA 4 (aa 1-147) -VP16 fusion protein (HI: aa 411-452, H2 : aa 453-490; VP16: aa 411-490) in human Swl3 cells (for details: see Example 4)
  • Figure 8 EBNA2 and NTD in BL41-P3HR1
  • NTD V-ACID (aa 1-290)
  • NTD V-ACID (aa 1-290)
  • pSRE reporter being a serum response element
  • the technical problem of the invention is to provide means for treatment of such infections .
  • a 103 kDa protein (pl03) could be isolated which binds to VP16:H1 and its N-terminal amino acid sequence (Meth-Val-Pro-Gly- Ser-Glu-Gly-Pro-Ala-.. ) could be dermined.
  • the pl03 encoding gene could be localized on chromosome 19ql3.3 (cosmid clones R31181 (accession number AC006942) and F23669 (accession number AC018766, NCBI data base) .
  • TCBAP0758 (theoretically) predicted protein TCBAP0758 (NCBI accession number NP_112235) and is identical to a cloned DNA sequence obtained from human testis mRNA (NCBI accession number A 136746) .
  • the nucleotide sequence and deduced amino acid sequence are shown in Figure 6A.
  • a fragment of pl03 was used as an immunogen for obtaining a specific anti-pl03-antibody.
  • VWA domain has an average length of about 180 amino acids with the first 112 amino acid residues (62%) being conserved in pl03.
  • pl03 is a nuclear protein. The high percentage of proline residues is reflected by three proline reach regions.
  • pl03 /VaCID directly interacts with the Hi domain of VP16.
  • pl03/VaCID seems to interact (directly or indirectly) with VP16:H2 and it is plausible to speculate that pl03/VaCID is part of a protein complex binding to both subregions (Hi and H2) of the VP16 transactivation domain.
  • the binding behavior of pl03/VaCID to VP16 is critical for the binding behavior of human Mediator, a cofactor of transcription.
  • VP16 Since binding of pl03/VaCID to the Herpes simplex transactivator protein VP16 seems to be an essential requirement for the activation of Herpes simplex promoters required for viral replication it can be expected that inhibition of the function of pl03/VaCID and/or the binding of pl03/VaCID (or the related polypeptides of the invention) to VP16 is useful for the treatment of Herpes simplex infections.
  • the present invention relates to an isolated or substantially pure form of a polypeptide having a biological activity of pl03/VaCID, i.e., being capable of binding to the transcription activating domain HI of Herpes simplex protein VP16, or an immunologically and/or biologically active fragment thereof, which comprises an amino acid sequence encodable by a polynucleotide selected from the group consisting of
  • polynucleotides encoding a polypeptide derived from the polypeptide encoded by a polynucleotide of (a) or
  • polynucleotides comprising the complementary strand which hybridizes with a polynucleotide of any one of (a) to (c) ;
  • polynucleotides encoding a polypeptide the sequence of which has an identity of 60% or more to the amino acid sequence of the polypeptide encoded by a polynucleotide of any one of (a) to (d) ;
  • polynucleotides encoding a polypeptide capable of binding to VP16:H1 comprising a fragment or an epitope- bearing portion of a polypeptide encoded by a polynucleotide of any one of (a) to (e) ;
  • polynucleotides the nucleotide sequence of which is degenerate as a result of the genetic code to a nucleotide sequence of a polynucleotide of any of (a) to
  • treatment covers any treatment of a disease in a mammal, preferably a human, and includes: (a) preventing the disease, e.g. infection, from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • the present invention is directed towards treating patients with medical conditions relating to a Herpes simplex infection. Accordingly, a treatment of the invention would involve preventing, inhibiting or relieving any medical condition related to Herpes simplex infections.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring) , and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • polypeptides of the invention comprise recombinantly produced polypeptides and include isolated naturally occurring polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods . Means for preparing such polypeptides or related polypeptides are well understood in the art. These polypeptides are preferably in a substantially purified form.
  • the polypeptide pl03/VaCID isolated or produced in accordance with the present invention typically has an approximate molecular weight of about 100 to 110 kDa, more preferably about 103 kDa, as measured on SDS-PAGE, particularly on a 10% gel at pH 8.6 in TRIS-Glycine SDS buffer under reducing conditions.
  • the polypeptide is generally glycosylated, however, the present invention also relates to unglycosylated versions of said polypeptide.
  • the present invention also describes the characterization and cloning of genes that are candidates for the above- described polypeptides; see the Examples, below.
  • the amino acid and nucleotide sequences of pl03/VaCID, mVaCID, DoCID and ACID are set out in Figures 6A, 6B, 6C and 6D.
  • the polypeptides of the present invention comprise the amino acid sequences of Figure 6A, 6B, 6C and 6D, respectively, optionally including mutations or deletions which do not substantially affect the activity thereof.
  • Such mutations include substitution of one or more amino acids, particularly by homologues thereof, as well as additions of one or more amino acids, especially at the N- or C-termini.
  • Deletions include deletions from the N- or C- termini . Substitutions by both naturally-occurring and synthetic amino acids are possible. Also included are polypeptides modified by chemical modification or enzymatic modification. Further, fragment peptides, whether chemically synthesized or produced by a biological method, whether modified or unmodified, are included within the scope of this invention.
  • the present invention not only provides the pl03/VaCID, mVaCID, DoCID and ACID encoding polynucleotide sequences shown in Figures 6A, 6B, 6C and 6D, but also related polynucleotide sequences encoding a polypeptide having a biological activity of pl03/VaCID, i.e., being capable of binding to the transcription activating domain Hi of Herpes simplex protein VP16, or an immunologically and/or biologically active fragment thereof. Said polynucleotides are described in more detail, below.
  • the polynucleotides of the invention can be both DNA and RNA molecules. Suitable DNA molecules are, for example, genomic or cDNA molecules. It is understood that all nucleic acid molecules encoding all or a portion of pl03/VaCID, mVaCID, DoCID and ACID are also included, as long as they encode a polypeptide with biological activity.
  • the polynucleotides of the invention an be isolated from natural sources or can be synthesized according to know methods.
  • polynucleotides of present invention also comprise polynucleotides encoding a polypeptide the amino acid sequence of which shows an identity of at least 40%, in particular an identity of at least 60%, preferably of at least 80% and, particularly preferred, of at least 90% to the amino acid sequence of pl03/VaCID.
  • polynucleotides are characterized by deletion, substitution and/or insertion of amino acid residue (s) compared to the amino acid sequences shown in Figures 6A, 6B, 6C and 6D or are the result of recombination. They can be naturally occurring variations, for example sequences from other organisms, or mutations that can either occur naturally or that have been introduced by specific mutagenesis.
  • nucleic acid molecules can be used, for example, that have exactly or basically the nucleotide sequence as depicted in Figures 6A, 6B, 6C and 6D, respectively, or parts of these sequences.
  • the fragments used as hybridization probe can be synthetic fragments that were produced by means of conventional synthetic methods and the sequence of which basically corresponds to the sequence of a polynucleotide of the invention.
  • the term answeringhybridization extract has the meaning of hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook et al . , Molecular Cloning, A Laboratory Manual 2 nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • a hybridizing polynucleotide can also be detected at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency), salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC) .
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • the polynucleotides of the present invention also include molecules which differ from the nucleic acid molecules with sequences shown in Figures 6A, 6B, 6C and 6D due to the degeneracy of the genetic code .
  • the present invention also provides polynucleotides which comprise fragments of the polynucleotides described above encoding a polypeptide of the invention.
  • IndeedFragments are understood to be parts of the nucleic acid molecules that are long enough to encode one of the described polypeptids .
  • These fragments also comprise nucleic acid molecules specifically hybridizing to transcripts of the polynucleotides of the invention.
  • fragments can be used, for example, as probes or primers in the diagnostic assay described below and, preferably, are oligonucleotides having a length of at least 13, in particular of at least 20 and particularly preferred of at least 50 nucleotides.
  • the nucleic acid molecules and oligonucleotides of the invention can also be used, for example, as primers for a PCR reaction.
  • nucleic acid molecules of the invention by means of conventional molecular biological processes it is possible (see, e.g., Sambrook et al . , supra) to introduce different mutations into the nucleic acid molecules of the invention.
  • polypeptides of the invention or related polypeptides with possibly modified biological properties are synthesized.
  • One possibility is the production of deletion mutants in which nucleic acid molecules are produced by continuous deletions from the 5 ' - or 3 '-terminal of the coding DNA sequence and that lead to the synthesis of polypeptids that are shortened accordingly.
  • Another possibility is the introduction of single-point mutation at positions where a modification of the amino acid sequence influences, e.g., the VP16:H1 binding properties.
  • muteins can be produced, for example, that possess a modified K m -value or that are no longer subject to the regulation mechanisms that normally exist in the cell, e.g. with regard to allosteric regulation or covalent modification. Such muteins might also be valuable as therapeutically useful antagonists of pl03 /VaCID.
  • the polynucleotides of the invention or parts of these molecules can be introduced into plasmids allowing a mutagenesis or a modification of a sequence by recombination of DNA sequences.
  • bases can be exchanged and natural or synthetic sequences can be added.
  • each other adapters or linkers can be added to the fragments. Furthermore, manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be performed. As analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.
  • polypeptids encoded by the various variants of the polynucleotidesd of the invention show certain common characteristics, such as VP16:H1 binding, molecular weight, immunological reactivity or conformation or physical properties like the electrophoretical mobilty, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum.
  • the invention furthermore relates to vectors containing the polynucleotides of the invention.
  • they are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells.
  • a polynucleotide of the invention is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an mRNA in prokryotic and/or eukaryotic cells that can be translated.
  • the nucleotide sequence to be transcribed can be operably linked to a promoter like a T7, metallothionein I or polyhedrin promoter.
  • the present invention relates to recombinant host cells transiently or stably containing the polynucleotides or vectors or the invention.
  • a host cell is understood to be an organism that is capable to take up in vi tro recombinant DNA and, if the case may be, to synthesize the polypeptids encoded by the polypeptides of the invention.
  • these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells.
  • the host cells of the invention are preferably characterized by the fact that the introduced polynucleotide of the invention either is heterologous with regard to the transformed cell, i.e. that it does not naturally occur in these cells, or is localized at a place in the genome different from that of the corresponding naturally occurring sequence.
  • a further embodiment of the invention relates to a method for the production of the VP16 binding polypeptides of the invention, whereby, e.g., a host cell of the invention is cultivated under conditions allowing the synthesis of the polypeptide and the polypeptide is subsequently isolated from the cultivated cells and/or the culture medium. Isolation and purification of the recombinantly produced polypeptide may be carried out by conventional means including preparative chromatography and affinity and immunological separations using, e.g., an anti-pl03/VaCID- antibody or, e.g., can be substantially purified by the one- step method described in Smith and Johnson, Gene 67; 31-40 (1988) .
  • the VP16 binding polypeptide of the invention is capable of mediating the transcription activating function of VP16. This activity can be assayed by the assays described below.
  • the present invention also relates to an antibody that binds specifically to pl03/VaCID, mVaCID, DoCID or ACID or a related polypeptide as defined above.
  • the term tauantibody preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specifities, as well as distinct monoclonal antibody preparations .
  • Monoclonal antibodies are made from an antigen containing fragments of the polypeptides of the invention by methods well known to those skilled in the art (see, e.g., K ⁇ hler et al . , Nature 256 (1975), 495).
  • the term is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to protein.
  • Fab and f(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less nonspecific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library.
  • antibodies of the present invention include chimerical, single chain, and humanized antibodies.
  • the antibody of the present invention can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.
  • the present invention also relates to a nucleic acid molecule of at least 13, preferably of at least 20 and, more preferably of at least 50 nucleotides in length hybridizing specifically with a polynucleotide of the invention as defined above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences encoding no or substantially different proteins. Such nucleic acid molecules may be used as probes in diagnosis and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length.
  • nucleic acids of up to 100 and more nucleotides in length may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of nucleic polynucleotides according to the invention or for detecting mutations within said nucleic acid molecules. Another application is the use as a hybridization probe to identify polynucleotides hybridizing to the polynucleotides of the invention by homology screening of genomic DNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a polynucleotide as described above may also be used for repression of expression of a gene, e.g., inhibition of translation of mRNA, comprising such a polynucleotide, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-Bl 0 291 533, EP-Al 0 321 201, EP- A2 0 360 257) which specifically cleave the (pre) -mRNA of a gene comprising a polynucleotide of the invention.
  • nucleic acid molecules may be chemically synthesized or transcribed by an appropriate vector containing a chi eric gene which allows for the transcription of said nucleic acid molecule in the cell. Such nucleic acid molecules may further contain ribozyme sequences as described above.
  • the antisense RNA and ribozyme of the invention are complementary to the coding region of the mRNA, e.g. to the 5' part of the coding region.
  • the person skilled in the art provided with the sequences of polynucleotides of the present invention will be in a position to produce and utilize the above described antisense RNAs or ribozymes.
  • the invention also relates to a transgenic non-human animal such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish such as torpedo fish comprising a polynucleotide or vector of the invention, preferably wherein said polynucleotide or vector is stably integrated into the genome of said non-human animal, preferably such that the presence of said polynucleotide or vector leads to the expression of the pl03/VaCID, mVaCID, DoCID or ACID polypeptide (or related polypeptide) of the invention.
  • a transgenic non-human animal such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish such as torpedo fish
  • a transgenic non-human animal such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish
  • Said animal may have one or several copies of the same or different polynucleotides encoding one or several forms of pl03/VaCID, mVaCID, DoCID or ACID polypeptide or mutant forms thereof.
  • This animal has numerous utilities, including as a research model for Herpes simplex promoter activation and, therefore, presents a novel and valuable animal in the development of therapies, treatment, etc. for Herpes simplex infections and diseases caused by such infection.
  • the non-human mammal is preferably a laboratory animal such as a mouse or rat.
  • the transgenic non-human animal of the invention further comprises at least one inactivated wild type allele of the corresponding pl03/VaCID, mVaCID, DoCID or ACID encoding or related gene.
  • This embodiment allows for example the study of the interaction of various mutant forms of pl03 /VaCID, mVaCID, DoCID and ACID polypeptides with VP16. All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes . It might be also desirable to inactivate pl03/VaCID, mVaCID, DoCID or ACID protein expression or function at a certain stage of development and/or life of the transgenic animal.
  • tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of, e.g., an antisense or ribozyme directed against the RNA transcript encoding the pl03/VaCID, mVaCID, DoCID or ACID encoding mRNA; see also supra.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al . (Trends Biotech. 12 (1994), 58- 62). Similar, the expression of a mutant pl03/VaCID, mVaCID, DoCID or ACID polypeptide may be controlled by such regulatory elements.
  • polynucleotides according to the invention opens up the possibility to produce transgenic non-human animals with a reduced level of the pi03/VaCID, mVaCID, DoCID and ACID protein as described above and, thus, with a defect in Herpes simplex promoter activation.
  • Techniques how to achieve this are well known to the person skilled in the art. These include, for example, the expression of antisense-RNA, ribozymes, of molecules which combine antisense and ribozyme functions and/or of molecules which provide for a co-suppression effect.
  • the nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the animal species used for transformation.
  • nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding a pl03 /VaCID, mVaCID, DoCID and ACID protein are also possible.
  • the homology is preferably higher than 80%, particularly higher than 90% and still more preferably higher than 95%.
  • the reduction of the synthesis of a polypeptide according to the invention in the transgenic mammalian cells can result in an reduction of promoter activation. In transgenic animals comprising such cells this can lead to various physiological, developmental and/or morphological changes .
  • the transgenic non-human animals of the present invention may show, for example, a deficiency in HSV promoter activation compared to wild type animals due to the stable or transient presence of a foreign DNA resulting in at least one of the following features:
  • transgenic non-human animal of the present invention preferably transgenic mouse
  • methods for the production of a transgenic non-human animal of the present invention are well known to the person skilled in the art.
  • Such methods e.g., comprise the introduction of a polynucleotide or vector of the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom.
  • the non- human animal can be used in accordance with a screening method of the invention described herein and may be a non- transgenic healthy animal, or may have a disorder, preferably a disorder caused by at least one mutation in the pl03 /VaCID protein.
  • transgenic animals are well suited for, e.g., pharmacological studies of drugs in connection with mutant forms of the above described pl03 /VaCID, mVaCID, DoCID or ACID polypeptide.
  • Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • the DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra.
  • the present invention relates to a method for identifying a binding partner to a pl03 /VaCID, mVaCID, DoCID or ACID polypeptide (or related polypeptide) of the invention comprising:
  • pl03 /VaCID, mVaCID, DoCID or ACID polypeptides may be used to screen for proteins or other compounds that bind to pl03/VaCID, mVaCID, DoCID or ACID or for proteins or other compounds to which pl03/VaCID, mVaCID, DoCID or ACID binds.
  • the binding of pl03/VaCID, mVaCID, DoCID or ACID and the molecule may activate (agonist) , increase, inhibit (antagonist) , or decrease activity of pl03 /VaCID, mVaCID, DoCID or ACID.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to a natural ligand of pl03/VaCID, mVaCID, DoCID or ACID, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic; see, e.g., Coligan, Current Protocols in Immunology 1(2) (1991); Chapter 5.
  • the molecule can be closely related to VP16 to which pl03/VaCID, mVaCID, DoCID or ACID might bind, or at least, a fragment of VP16, e.g. the Hi region, capable of being bound by pl03/VaCID, mVaCID, DoCID or ACID. In either case, the molecule can be rationally designed using known techniques .
  • the screening for these molecules involves producing appropriate cells which express pl03/VaCID, mVaCID, DoCID or ACID, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
  • Cells expressing pi03/VaCID, mVaCID, DoCID or ACID (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of pl03/VaCID, mVaCID, DoCID or ACID.
  • the assay may simply test binding of a candidate compound to pl03/VaCID, mVaCID, DoCID or ACID, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to pl03/VaCID, mVaCID, DoCID or ACID.
  • the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
  • the assay may also simply comprise the steps of mixing a candidate compound with a solution containing pl03/VaCID, mVaCID, DoCID or ACID, measuring pl03/VaCID-, mVaCID-, DoCID- or ACID/molecule activity or binding, and comparing the pl03/VaCID-, mVaCID-, DoCID- or ACID /molecule activity or binding to a standard.
  • an ELISA assay can measure pl03/VaCID, mVaCID, DoCID or ACID level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody of the invention.
  • the antibody can measure pl03/VaCID, mVaCID, DoCID or ACID level or activity by either binding, directly or indirectly, to pl03/VaCID, mVaCID, DoCID or ACID or by competing with pl03/VaCID, mVaCID, DoCID or ACID for a substrate. All of these above assays can also be used as diagnostic or prognostic markers.
  • the molecules discovered using these assays can be used to treat infection or to bring about a particular result in a patient, e.g., by inhibiting the pl03/VaCID, mVaCID, DoCID or ACID molecule.
  • the assays can discover agents which may inhibit or enhance the production of pl03/VaCID, mVaCID, DoCID or ACID from suitably manipulated cells or tissues.
  • the invention also includes a method of identifying compounds which bind to a pl03/VaCID, mVaCID, DoCID or ACID polypeptide or related polypeptide comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention, e.g., pl03/VaCID; and (b) determining if binding has occurred.
  • EBV causes Pfeiffer's disease and is possibly a cofactor of lymphomas .
  • the central transactivator of EBV is a protein called EBNA2 which is necessary for the cellular transformation.
  • EBNA2 can be inhibited by the amino terminal mediator binding domain (aa 1-290 of V-ACID; called NTD) as mentioned above. This means that EBV reacts in the same manner as herpes simplex VP16, in particular in its binding to ACID. This function has been shown by transfection assays with several luciferase reporters (Fig. 8) . Overexpressed EBNA2 and the activation domain of EBNA2 fused to GAL4 are inhibited in their transcription effect.
  • LANA is the central transactivator of this virus. LANA (like VP16 or EBNA) is also inhibited in its transactivation by overexpressing the amino terminal 1-290 amino acids of V-ACID (NTD called) (Fig. 9) .
  • the present invention relates to method of identifying and obtaining a drug candidate (e.g., an inhibitor/antagonist of a polypeptide of the invention) for therapy of a Herpes simplex infection, EBV infection or Karposi Sarcoma Herpes infection or a disorder associated therewith comprising the steps of
  • a polypeptide of the present invention e.g., pl03/VaCID, or a cell expressing said polypeptide in the presence of components capable of providing a detectable signal in response to promoter VP16, EBNA or LANA mediated promoter activation, with said drug candidate to be screened under conditions to permit such activation, and
  • the assay described in Example 4 can be used to measure promoter activation.
  • the drug candidate may be a single compound or a plurality of compounds.
  • the term "plurality of compounds" in a method of the invention is to be understood as a plurality of substances which may or may not be identical.
  • Said compound or plurality of compounds may be chemically synthesized or microbiologically produced and/or comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compound (s) may be known in the art but hitherto not known to be capable of suppressing or activating pl03/VaCID, mVaCID, DoCID and ACID polypeptides, i.e. their capability to bind to and, preferably, activate V16, EBNA2 or LANA.
  • the reaction mixture may be a cell free extract or may comprise a cell or tissue culture.
  • Suitable set ups for the method of the invention are known to the person skilled in the art and are, for example, generally described in Alberts et al . , Molecular Biology of the Cell, third edition (1994) .
  • the plurality of compounds may be, e.g., added to the reaction mixture, culture medium, injected into a cell or otherwise applied to a transgenic animal of the invention.
  • the cell or tissue that may be employed in the method of the invention preferably is a host cell, mammalian cell or non-human transgenic animal of the invention described in the embodiments hereinbefore .
  • a sample containing a compound or a plurality of compounds is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound capable of suppressing or activating pl03/VaCID, mVaCID, DoCID or ACID, or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample.
  • the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance (s) .
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical .
  • the immobilized polymers are contacted with a labeled receptor and scanned for label to identify polymers binding to the receptor.
  • the synthesis and screening of peptide libraries on continuous cellulose membrane supports that can be used for identifying binding ligands of the polypeptide of the invention and thus possible inhibitors and activators is described, for example, in Kramer, Methods Mol. Biol. 87 (1998), 25-39. This method can also be used, for example, for determining the binding sites and the recognition motifs in the polypeptide of the invention.
  • the substrate specificity of the DnaK chaperon was determined and the contact sites between human interleukin-6 and its receptor; see Rudiger, EMBO J.
  • WO 98/25146 described further methods for screening libraries of complexes for compounds having a desired property, especially, the capacity to agonize, bind to, or antagonize a polypeptide or its cellular receptor.
  • the complexes in such libraries comprise a compound under test, a tag recording at least one step in synthesis of the compound, and a tether susceptible to modification by a reporter molecule. Modification of the tether is used to signify that a complex contains a compound having a desired property.
  • the tag can be decoded to reveal at least one step in the synthesis of such a compound.
  • All these methods can be used in accordance with the present invention to identify activators/agonists and, preferably, inhibitors/antagonists of a pl03 /VaCID, mVaCID, DoCID or ACID polypeptide or related polypeptide of the invention.
  • Mimetic analogs of the polypeptide of the invention or biologically active fragments thereof can be generated by, for example, substituting the amino acids that are expected to be essential for the biological activity with, e.g., stereoisomers, i.e. D- amino acids; see e.g., Tsukida, J. Med. Chem. 40 (1997), 3534- 3541.
  • pro-mimetic components can be incorporated into a peptide to reestablish at least some of the conformational properties that may have been lost upon removal of part of the original polypeptide; see, e.g., Nachman, Regul. Pept. 57 (1995), 359-370.
  • the pl03/VaCID, mVaCID, DoCID or ACID polypeptide of the invention can be used to identify synthetic chemical peptide mimetics that bind to or can function as a ligand, substrate, binding partner or the receptor of the polypeptide of the invention as effectively as does the natural polypeptide; see, e.g., Engle an, J. Clin. Invest.
  • folding simulations and computer redesign of structural motifs of the polypeptide of the invention can be performed using appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput . Appl. Biosci . 11 (1995), 675-679).
  • Computer modeling of protein folding can be used for the conformational and energetic analysis of detailed peptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).
  • the appropriate programs can be used for the identification of interactive sites of the pl03/VaCID, mVaCID, DoCID and ACID polypeptide and VP16, EBNA2 , LANA or other interacting proteins by computer assistant searches for complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114-120. Further appropriate computer systems for the design of protein and peptides are described in the prior art, for example in Berry, Biochem. Soc. Trans. 22 (1994), 1033- 1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991.
  • results obtained from the above-described computer analysis can be used for, e.g., the preparation of peptide mimetics of the protein of the invention or fragments thereof.
  • pseudopeptide analogues of the natural amino acid sequence of the protein may very efficiently mimic the parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).
  • incorporation of easily available achiral ⁇ -amino acid residues into a protein of the invention or a fragment thereof results in the substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptide mimetic (Banerjee, Biopolymers 39 (1996) , 769-777) .
  • polypeptide of the invention can be used for the design of peptide mimetic inhibitors of the biological activity of the polypeptide of the invention (Rose, Biochemistry 35 (1996) , 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545- 1558) .
  • the polynucleotide of the invention can also serve as a target for activators and, preferably, inhibitors.
  • Activators may comprise, for example, proteins that bind to the mRNA of a gene encoding a pl03/VaCID, mVaCID, DoCID or ACID polypeptide of the invention, thereby stabilizing the native conformation of the mRNA and facilitating transcription and/or translation, e.g., in like manner as Tat protein acts on HIV-RNA.
  • nucleic acid molecules such as an RNA fragment that mimics the structure of a defined or undefined target RNA molecule to which a compound binds inside of a cell resulting in retardation of cell growth or cell death; see, e.g., WO 98/18947 and references cited therein.
  • These nucleic acid molecules can be used for identifying unknown compounds of pharmaceutical interest, and for identifying unknown RNA targets for use in treating an infection.
  • These methods and compositions can be used in screening for novel antibiotics, bacteriostatics, or modifications thereof or for identifying compounds useful to alter expression levels of proteins encoded by a nucleic acid molecule.
  • Still other methods are, for example, the drug design methods as described in WO 95/35367, US-A- 5,322,933, where the crystal structure of the RNA fragment can be deduced and computer programs are utilized to design novel binding compounds which can act as antibiotics .
  • the compounds which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra) .
  • expression libraries e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra) .
  • genes encoding a putative regulator of pi03/VaCID polypeptide and/or which exert their effects up- or downstream the pl03/VaCID polypeptide of the invention may be identified using, for example, insertion mutagenesis using, for example, gene targeting vectors known in the art.
  • insertion mutagenesis using, for example, gene targeting vectors known in the art.
  • standard native gel-shift analyses can be carried out.
  • the protein or regulatory sequence can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
  • nucleic acid molecules which encode polypeptides which interact with pl03/VaCID, mVaCID, DoCID or ACID polypeptides described above can also be achieved, for example, as described in Scofield (Science 274 (1996) , 2063- 2065) by use of the so-called yeast "two-hybrid system".
  • yeast two-hybrid system
  • the polypeptide encoded by a nucleic acid molecule according to the invention or a smaller part thereof is linked to the DNA-binding domain of the GAL4 transcription factor.
  • the complex is able to direct expression of the reporter gene.
  • the polynucleotides according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with pl03/VaCID, mVaCID, DoCID or ACID.
  • Activation or repression of pl03/VaCID, mVaCID, DoCID or ACID could then be achieved in animals by applying the transacting factor (or its inhibitor) or the gene encoding it, e.g. in an expression vector.
  • the transacting factor or its inhibitor
  • the active form of the transacting factor is a dimer
  • dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
  • further components in the pathway leading to activation e.g. signal transduction
  • repression of a gene involved in the control of pl03/VaCID, mVaCID, DoCID or ACID then can be identified.
  • the present invention also relates to the use of the two-hybrid system as defined above for the identification of pl03/VaCID, mVaCID, DoCID or ACID activators or, preferably, inhibitors of pl03/VaCID, mVaCID, DoCID or ACID.
  • the compounds isolated by the above methods also serve as lead compounds for the development of analog compounds .
  • the analogs should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to pl03/VaCID, mVaCID, DoCID or ACID or VP16 in substantially the same way as the lead compound.
  • the analog compounds have spatial electronic properties which are comparable to the binding region, but can be smaller molecules than the lead compound, frequently having a molecular weight below about 2 kD and preferably below about 1 kD.
  • Identification of analog compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis.
  • SCF self-consistent field
  • CI configuration interaction
  • normal mode dynamics analysis normal mode dynamics analysis.
  • the present invention also relates to a method for treating a Herpes simplex infection, EBV infection, Karposi sarcoma Herpes Virus infection or diseases associated therewith which comprises administering to a mammalian subject a therapeutically effective amount of one of the compounds of the invention, i.e., a polypeptide, polynucleotide, vector, antibody or inhibitor/antagonist of the invention.
  • the present invention also relates to a composition
  • a composition comprising the above compounds, e.g., a pharmaceutical composition containing said compounds and, further, a pharmaceutically acceptable excipient, diluent or carrier.
  • suitable pharmaceutical carriers etc. are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the route of administration depends, inter alia, on the kind of compound contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the attending physician and other clinical factors.
  • dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of infection or disease, general health and other drugs being administered concurrently.
  • the delivery of the polynucleotides, nucleic acid molecules, antisense RNAs, ribozymes etc. of the invention can be achieved by direct application or, preferably, by using a recombinant expression vector such as a chimeric virus containing these compounds or a colloidal dispersion system.
  • Direct application to the target site can be performed, e.g., by ballistic delivery, as a colloidal dispersion system or by catheter to a site in artery.
  • the colloidal dispersion systems which can be used for delivery of the above nucleic acid molecules include macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions (mixed) , micelles, liposomes and lipoplexes,
  • the preferred colloidal system is a liposome.
  • the composition of the liposome is usually a combination of phospholipids and steroids, especially cholesterol.
  • the skilled person is in a position to select such liposomes which are suitable for the delivery of the desired nucleic acid molecule.
  • Organ-specific or cell- specific liposomes can be used in order to achieve delivery only to the infected organ.
  • the targeting of liposomes can be carried out by the person skilled in the art by applying commonly known methods.
  • This targeting includes passive targeting (utilizing the natural tendency of the liposomes to distribute to cells of the RES in organs which contain sinusoidal capillaries) or active targeting (for example by coupling the liposome to a specific ligand, e.g., an antibody, a receptor, sugar, glycolipid, protein etc., by well known methods) .
  • a specific ligand e.g., an antibody, a receptor, sugar, glycolipid, protein etc.
  • monoclonal antibodies are preferably used to target liposomes to specific tissues via specific cell-surface ligands.
  • Preferred recombinant vectors useful for gene therapy are viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a Retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus. Examples of such retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) and Rous sarcoma virus (RSV) .
  • a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV) , providing a broader host range compared to murine vectors .
  • GaLV gibbon ape leukemia virus
  • Such assistance can be provided, e.g., by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art.
  • Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified.
  • the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody.
  • a polynucleotide encoding a sugar, a glycolipid, or a protein, preferably an antibody.
  • Those skilled in the art know additional methods for generating target specific vectors.
  • Further suitable vectors and methods for in vitro- or in vivo-gene therapy are described in the literature and are known to the persons skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
  • the nucleic acid molecules of the present invention can be linked to a tissue specific promoter and used for gene therapy.
  • tissue specific promoters are well known to those skilled in the art (see e.g. Zimmermann et al . , (1994) Neuron 12, 11-24; Vidal et al . ; (1990) EMBO J. 9, 833-840; Mayford et al . , (1995), Cell 81, 891-904; Pinkert et al . , (1987) Genes & Dev. 1, 268-76) .
  • pl03 /VaCID or a related protein can result in an increased activation of Herpes simplex VP16, EBV EBNA2 or Karposi Sarcoma herpes virus LANA and thus, will promote replication and infectivity of the virus.
  • the present invention also relates to a method of diagnosing a susceptibility to a pathological condition in a subject related to a Herpes simplex, EBV or Karposi Sarcoma Herpes Virus infection comprising:
  • the present invention also relates to a method of diagnosing a susceptibility to a pathological condition in a subject related to a Herpes simplex, EBV or Karposi Sarcoma Herpes Virus infection comprising:
  • an increased concentration or activity of pl03/VaCID, mVaCID, DoCID or ACID (compared to the concentration or activity in control tissue) or, alternatively, an increased level of pl03 /VaCID, mVaCID, DoCID or ACID encoding mRNA is indicative for a susceptibility to a pathological condition associated with a Herpes simplex, EBV or Karposi Sarcoma Herpes Virus infection.
  • the pl03 /VaCID, mVaCID, DoCID or ACID polypeptide or the corresponding mRNA, e.g. in biological fluids or tissues, may be detected directly in situ, e.g. by in situ hybridization or it may be isolated from other cell components by common methods known to those skilled in the art before contacting with a probe. Detection methods include Northern Blot analysis (e.g., according to the examples, below), RNase protection, in situ methods, e.g.
  • RNA-transcription/amplification PCR, LCR, QRNA replicase or RNA-transcription/amplification (TAS, 3SR) , reverse dot blot disclosed in EP-Bl 0 237 362)
  • PCR in vitro amplification methods
  • TAS QRNA replicase or RNA-transcription/amplification
  • TAS reverse dot blot
  • Western Blot Western Blot and other detection assays that are known to those skilled in the art.
  • the probe e.g. a specific antibody or specific oligonucleotide
  • said probe is an anti-pl03/VaCID-, anti-mVaCID-, anti-DoCID- or anti-ACID-antibody and allows said diagnosis, e.g., by ELISA and the antibody is bound to a solid support, for example, a polystyrene microtiter dish or nitrocellulose paper, using techniques known in the art.
  • said diagnosis is based on a RIA and uses said antibody marked with a radioactive isotope.
  • antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 1 5 I, 121 I), carbon ( 14 C) , sulfur ( 35 S) , tritium ( 3 H) , indium ( 112 In) , and technetium rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 1 5 I, 121 I), carbon ( 14 C) , sulfur ( 35 S) , tritium ( 3 H) , indium ( 112 In) , and technetium rhodamine, and biotin.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc .
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al . , whilImmunopharmacokinetics of Radiolabeled Antibodies and Their Fragments". (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes, eds .
  • pl03/VaCID, mVaCID, DoCID or ACID can be determined by using (modified) assays described above for the screening of compounds capable of interacting with or binding to pl03/VaCID, mVaCID, DoCID or ACID, or a related polypeptide.
  • VP16 depends on PC6 and that this is in turn disturbed by point mutation in Hi. It was postulated that PC6 is an interaction partner necessary for the transactivation by
  • affinity chromatography was carried out as a purification strategy for the PC6 activity.
  • proteins binding non- specifically to the column material and to the H2 and/or HI mutant were depleted from the low salt fractions of the Pll chromatography of a HeLa nuclear extract.
  • affinity chromatography steps were taken via a GST, a GST-H2 and a GST-Hlmut glutathione sepharose column, the column flow being further used in each case.
  • the specifically Hl- binding proteins of the last flow fraction were immobilized on a GST-HI gluthatione sepharose column and eluted at 500 mM salt.
  • one of the murine sequences was used as a probe in the screening of a HeLa cDNA phage library.
  • a clone was identified whose sequence was previously known as a cDNA sequence and whose genomic locus was disposed about 30 kB below (positioned 3') the identified exons of the pl03 gene on chromosome 19. It was initially given the laboratory name of 3IIR and was considered the 3' end of the pl03 gene. However, it was later found that 3IIR is a gene of its own.
  • chromosome 19ql3.3 has meanwhile been fully sequenced in the form of two partially overlapping cosmids (cosmid R31181, Ace. AC006942; cosmid F23669, Ace. AC018766) .
  • some cDNA sequences existed which, however, did not represent a constant sequence of the pl03 gene and in particular did not contain the sequence of the 3IIR clone considered to be the 3' end (Ace. AW504573; Ace. AF283769) .
  • the gene of pl03 should be amplified by PCR with gene- specific primers of the cDNA.
  • genomic sequence information for determining the primers, a statement on the exon-intron structure of the gene had to be made beforehand.
  • a prediction regarding splicing sites is possible by means of the "GENSCAN" program (Burge and Karlin, J.Mol.Biol. 268 (1997), 78-94). Using this program, an analysis of the entire known gene locus was carried out to be able to make a statement on the quality of the prediction and on possible splicing and/or termination variants by means of possibly adjacent genes.
  • the structure of the formerly unknown gene of pl03 is predicted as evident in Figures 2 and 3.
  • the predicted exon sequences correspond with the previously sequenced, known cDNA sequences from databases.
  • the sequence of clone AW504573 comprises the first four predicted exons .
  • the sequence of clone AF283769 comprises exons 6 to 17 and only differs from the GENSCAN prediction as regards the terminal exon 18. Here, it is, however, necessary to point out that two completely different terminal exons are selected in the clone sequence and in the GENSCAN prediction.
  • a search for known protein motifs in the protein sequence of CIDl by means of the ScanProsite program showed that amino acids 17 to 184 have some similarity with the Willebrandt factor type A domain (vWA) .
  • the homology comprises approximately the first two thirds of the vWA domain, i.e. a complete vWA domain is not concerned.
  • the part present in CIDl however, has a significant similarity with the vWA domain over the entire 167 amino acid residues.
  • ScanProsite also determined other significant structural motifs in the sequence of CIDl.
  • the region of amino acids 546 to 612 is regarded as a region rich in glutamine(Q) .
  • Drosophila CIDl protein consists of a C terminus highly rich in Q altogether, which distinguishes itself by several poly-Q sequences. Later, a human cDNA clone was identified which contains the entire predicted sequence of the CIDl gene (Ace. AL136746) .
  • CID motifs In other BLAST searches for CID motifs, the homologous regions of the already identified CID protein motifs of murine CIDl and CID2 , bovine CIDl, rat CIDl, Drosophila CIDl and CID2 were found and considered for the analyses .
  • CID domain sequence is highly conserved in all of the listed mammalian species .
  • the Drosophila sequence differs more strongly but the conservation can still be detected clearly.
  • the homology in the second one of the CID2 domains (CID2-II) strongly decreases after one third of the sequence.
  • the domain is highly rich in leucine (17%) .
  • the isoelectric point of the domain was determined by computer analyses; the entire domain is basic (pi 10.07).
  • MTNTM blots (“multiple tissue northern blots") of Clontech, Palo Alto (Heidelberg, Germany) were hybridized with radioactively labeled gene-specific probes.
  • Radioactively labeled PCR products (primer pl03-INup, pl03-Indw, pl031N_up 5 ' -TTTGTGATTGAGGGTACGGCCAACC-3 ' , pi03lN_down 5 ' - GCTGGTGGGAGCGTGACATTGTACG-3 ' ) served for the CIDl gene as a probe.
  • the RNA amounts of each lane were standardized by the manufacturer. Nevertheless, the RNA amount was checked in a second experiment by decoration with a radioactively labeled ⁇ -actin probe (2 kB, Clontech) .
  • two signals, in the peripheral leukocytes three signals, are constantly generated by the CIDl probe ( Figure 5A and C) .
  • the band of about 2,8 kB length corresponds to the length of about 2,8 kB of the hCIDl-iriRNA, expected on account of the genomic sequence.
  • the signal at a length of about 4 kB can be produced by a longer splicing variant or represent a non-specific cross- reaction, a definite statement on this, however, cannot be made.
  • the 2.8 kB signal is available in all of the investigated tissues.
  • the 2,8 kB mRNA is strongly expressed.
  • the heart, skeletal muscle, kidney, placenta and fetal liver have the strongest expression level.
  • the 4 kB signal is also detected in all of the studied tissues, however, it is altogether weaker. For example, almost no signals become visible in the large intestine, thymus, liver, small intestine, peripheral leukocytes and lymph nodes.
  • the signals are somewhat more intense in the brain, placenta and bone marrow but again they are most intense in the heart, skeletal muscle, kidney and fetal liver.
  • RNA amounts in each lane of the MTNTM blots are almost identical.
  • the expected position of the signals at a length of about 2 kB meets the expectations.
  • an about 1.6 to 1.8 kB long isoform of the ⁇ - actin-mRNA is detected with priority in the heart tissue and in the muscle tissue.
  • the respectively applied RNA amounts of these tissues are also slightly raised over the other lanes. This difference was considered in the interpretation of the expression data of the hCIDl-mRNA.
  • Example 4 Deletion mutants of pl03/VaCID ( ⁇ aal-290) show a dominant negative effect
  • a transient analysis of VaCID effects on transcriptional activation by a GAL4 (aa 1-147) -VP16 fusion protein (HI: aa 411-452, H2: aa 453-490; VP16: aa 411-490) in human Swl3 cells was carried out.
  • a promoter carrying 5 ' -upstream GAL4 binding sites was cotransfected with an expression vector of the indicated GAL-VP16 proteins and the indicated amounts of a standard CMV expression vector carrying the amino-terminal 290 aa of mVaCID (NTD) .
  • Similar results were obtained in various cell lines (HeLa, Jurkat) upon over-expression of mouse and human VaCID (aa 1-290) proteins. The results are shown in Figure 7.
  • nucleotide sequences of mVaCID and DoCID as well as the deduced amino acid sequences are shown in Figures 6B and 6C, respectively.

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Abstract

L'invention concerne de nouveaux polypeptides capable de lier le domaine H1 d'une protéine VP16 Herpes simplex, et des polynucléotides isolés codant ces polypeptides. La présente invention porte également sur des vecteurs, des cellules hôtes, des anticorps, et sur des procédés de recombinaison pour obtenir ces polypeptides. Cette invention concerne aussi des méthodes de diagnostic et des thérapies utiles pour traiter des infections dues au virus Herpes simplex, EBV ou Karposi Sarcoma ainsi que des troubles associés à ces infections.
PCT/EP2004/000681 2003-01-27 2004-01-27 Nouveaux polypeptides liant la proteine vp16 herpes simplex WO2004067560A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072633A1 (fr) * 2001-03-14 2002-09-19 Consejo Superior De Investigaciones Científicas Nouvelle proteine surexprimee dans le cancer de la prostate
WO2002103028A2 (fr) * 2001-05-30 2002-12-27 Biomedical Center Criblage in silico de sequences exprimees associees a un phenotype

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072633A1 (fr) * 2001-03-14 2002-09-19 Consejo Superior De Investigaciones Científicas Nouvelle proteine surexprimee dans le cancer de la prostate
WO2002103028A2 (fr) * 2001-05-30 2002-12-27 Biomedical Center Criblage in silico de sequences exprimees associees a un phenotype

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL 1 May 2000 (2000-05-01), ADAMS, D. ET AL.: "CG12254-PA (LD07688p)" XP002294846 Database accession no. Q9VDR1 *
DATABASE EMBL 11 January 2001 (2001-01-11), NCI-CGAP: "nae02a10.x1 NCI_CGAP_Ov18 Homo sapiens cDNA clone IMAGE: 3434011 3' similar to TR: Q9VDR1 Q9VDR1 CG12254 PROTEIN, mRNA sequence" XP002312321 Database accession no. BF732211 *
DATABASE EMBL 14 October 2000 (2000-10-14), MARRA, M. ET AL.: "uy57f10.y1 McCarrey Eddy round spermatid Mus musculus cDNA clone IMAGE: 3663691 5' similar to TR: Q9VDR1 Q9VDR1 CG12254 PROTEIN, mRNA sequence" XP002294845 Database accession no. BF021756 *
DATABASE EMBL 15 July 1998 (1998-07-15), ECKNER, R. ET AL.: "E1A-associated protein p300" XP002312014 Database accession no. Q09472 *
DATABASE EMBL 23 October 2001 (2001-10-23), MELTON, D. ET AL.: "id21g12.x1 Melton Normalized Mixed Mouse Pancreas 1 N1-MMS1 Mus musculus cDNA clone IMAGE: 5663831 3' similar to TR: Q9UGR7 Q9UGR7 DJ85F18.1, mRNA sequence" XP002312013 Database accession no. BI901314 *
DATABASE EMBL 30 November 2002 (2002-11-30), MELTON, D. ET AL.: "io43d02.x1 Human insulinoma Homo sapiens cDNA clone IMAGE: 6129074 3' similar to TR: Q9VDR1 Q9VDR1 CG12254 PROTEIN, mRNA sequence" XP002312320 Database accession no. BU789592 *
DATABASE EMBL 4 February 2001 (2001-02-04), NCI-CGAP: "mab90c03.y1 NCI_CGAP_SP2 Mus musculus cDNA clone IMAGE: 3977476 5' similar to TR: Q9VDR1 Q9VDR1 CG12254 PROTEIN, mRNA sequence" XP002294844 Database accession no. BG146318 *
IKEDA, K. ET AL.: "The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms" GENES TO CELLS, vol. 7, no. 1, January 2002 (2002-01), pages 49-58, XP002294841 *
MEISTERERNST, M.: "Transcription Regulation in Eukaryotes: Structure and function of proteins that modulate RNA polymerase II transcription" 1999, HUMAN FRONTIER SCIENCE PROGRAM , XP008034481 pages 130-138 positive cofactor PC6 page 130, line 1 - line 24 page 131, line 31 - page 133, line 21; figure 50 *
MITTLER, G. ET AL.: "A novel docking site on Mediator is critical for activation by VP16 in mammalian cells" EMBO JOURNAL, vol. 22, no. 24, 15 December 2003 (2003-12-15), pages 6494-6504, XP002294843 *
N[[R, A.M. ET AL.: "Composite co-activator ARC mediates chromatin-directed transcriptional activation" NATURE, vol. 398, no. 6730, 29 April 1999 (1999-04-29), pages 828-832, XP002294840 *
WANG, C. ET AL.: "A prostate-derived cDNA that is mapped to human chromosome 19 encodes a novel protein" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 296, no. 2, 16 August 2002 (2002-08-16), pages 281-287, XP002294842 *

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