WO2004007536A2 - Nouvelles interactions de la proteine du virus d'epstein-barr ebna1, compositions et methodes associees - Google Patents

Nouvelles interactions de la proteine du virus d'epstein-barr ebna1, compositions et methodes associees Download PDF

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WO2004007536A2
WO2004007536A2 PCT/CA2003/001019 CA0301019W WO2004007536A2 WO 2004007536 A2 WO2004007536 A2 WO 2004007536A2 CA 0301019 W CA0301019 W CA 0301019W WO 2004007536 A2 WO2004007536 A2 WO 2004007536A2
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ebnal
complex
protein
polypeptide
usp7
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PCT/CA2003/001019
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WO2004007536A3 (fr
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Lori Frappier
Melissa Holowaty
Jack Greenblatt
Mahel Zeghouf
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Affinium Pharmaceuticals, Inc.
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Publication of WO2004007536A3 publication Critical patent/WO2004007536A3/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Epstein-Barr Virus causes primary viral infections, such as mononucleosis, and persistent, latent EBN infections are associated with a variety of cancers.
  • EBV-based vectors are being investigated for use in the stable maintenance of gene therapy vectors.
  • Epstein-Barr Virus Nuclear Antigen 1 (EBNAl) is the only EBV protein that is expressed in all known EBV-associated cancers.
  • EBNAl is a DNA binding protein with activity in maintenance of the extrachromosomal EBV genome (episome). EBNAl also acts as a transactivator for expression of certain genes.
  • EBNAl is resistant to ubiquitin/proteasome protein degradation, and this appears to prevent EBNAl fragments from being displayed on the surface of the infected cell, and therefore prevents the immune system from recognizing and eliminating EBNAl -expressing cells.
  • a crystal structure of EBNAl bound to DNA has been solved.
  • EBNAl is included in various EBV-based gene therapy vectors.
  • EBV-associated cancers The interaction of EBNAl with various cellular molecules may be important for the immortalization of human cells by EBV, which appears to be an essential step in the development of EBV-associated cancers. Such interactions and the complexes of molecules resulting therefrom may present drug targets to prevent or treat EBV-associated cancers, including, for example, lymphomas associated with immunosupression (observed in AIDS and organ transplant patients), Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • lymphomas associated with immunosupression observed in AIDS and organ transplant patients
  • Burkitt's lymphoma Burkitt's lymphoma
  • nasopharyngeal carcinoma SUMMARY OF THE INVENTION
  • One aspect of the invention is based upon the identification of proteins which possess the ability to interact with any mammalian EBNAl protein or a protein complex comprising EBNAl .
  • EBNAl as a "bait" protein
  • EBNAl as a "bait" protein
  • mammalian proteins that form complexes with EBNAl under physiologic conditions, including USP7, NAP 1, TAF I ⁇ , CK2, PRMT5, karyopherin ⁇ 2, karyopherin ⁇ 3, importin ⁇ , and pp32.
  • the various interactions of these proteins may reveal new information regarding the biochemical nature and cellular and physiological consequences of EBNAl interactions.
  • Such interactions and complexes of polypeptides resulting therefrom may present drug targets for the prevention and treatment of virus-associated diseases and disorders, such as, for example, an EBV-associated disease or disorder, including, lymphomas associated with immunosupression (observed in AIDS and organ transplant patients), Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • virus-associated diseases and disorders such as, for example, an EBV-associated disease or disorder, including, lymphomas associated with immunosupression (observed in AIDS and organ transplant patients), Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • the invention provides an EBNAl complex comprising (a) EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (b) EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (c) a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; or (d) a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the EBNAl complex comprises EBNAl, or a fragment of EBNAl, and USP7, or a fragment of USP7.
  • the EBNAl complex comprises a fragment of EBNAl comprising residues 395-450 and USP7.
  • the EBNAl complex comprises a fragment of USP7 comprising residues 67-161 of USP7.
  • the EBNAl complex comprises EBNAl and PRMT5.
  • the EBNAl complex comprises EBNAl and CK2.
  • the EBNAl complexes are provided in a variety of forms.
  • the EBNAl complexes may be prepared by mixing the polypeptides or fragments comprising the EBNAl complexes in vitro under conditions which promote complex formation. In certain embodiments, these polypeptides or fragments are recombinantly produced.
  • the EBNAl complexes are isolated from a cell. In certain embodiments, the EBNAl complexes are at least about 75% pure by weight as compared to the weight of the total protein in the sample. In other embodiments, the EBNAl complexes are at least about 85% pure by weight as compared to the weight of the total protein in the sample.
  • the EBNAl complexes are at least about 95% pure by weight as compared to the weight of the total protein in the sample. In still other embodiments, the EBNAl complexes are crystallized; e.g. the invention provides a crystal comprising an EBNAl complex.
  • the EBNAl complexes of the present invention may be comprised of a variety of polypeptide compositions.
  • at least one EBNAl complex polypeptide or polypeptide fragment is a fusion protein.
  • at least one EBNAl complex polypeptide or polypeptide fragment is labeled.
  • at least one EBNAl complex polypeptide or polypeptide fragment is recombinantly produced.
  • all of the EBNAl complex polypeptides or polypeptide fragments are recombinantly produced.
  • the EBNAl complexes may additionally comprise an antibody.
  • at least two of the polypeptides in an EBNAl complex may be covalently linked to each other.
  • a fusion polypeptide comprising all or a portion of the amino acid sequence of an EBNAl complex comprising (a) EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (b) EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (c) a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; or (d) a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the present invention provides a molecule comprising all or a portion of the amino acid sequence of an EBNAl complex comprising (a) EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (b) EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (c) a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5 ; or (d) a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the present invention further provides polypeptide fragments comprising all or a portion of the amino acid sequence of an EBNAl complex polypeptide selected from the group consisting of EBNAl, USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • an isolated polypeptide comprising the amino acid sequence of residues 325- 376, 330-619, 330-641, 452-641, 395-641, 41-376, or 395-450 of EBNAl is provided.
  • an isolated polypeptide comprising the amino acid sequence of residues 1-324 and 377-641, 1-40 and 377-641, or 1-394 and 451-641 of EBNAl is provided.
  • an isolated polypeptide comprising the amino acid sequence of residues 67-745, 12-543, 67-543, 67-355, 81-322, 67-312, 67-301, 67-335, 67- 322, 67-254, 67-161, or 81-161 of USP7 is provided.
  • a polypeptide fragment having at least 3, 5, 7, 10, 20, 25, 30, 40, 50, or more contiguous amino acids of said isolated polypeptides is provided.
  • Such isolated polypeptides and polypeptide fragments may be produced by a variety of methods, including but not limited to recombinant methods and chemical synthesis.
  • the invention further includes peptidomimetics based on the EBNAl complex polypeptide fragments of the invention, for example, an isolated polypeptide comprising at least 5 consecutive residues of the amino acid sequence of 395-450 of EBNAl or 67-161 of USP7.
  • the present invention provides an isolated antibody that has a higher binding affinity for a EBNAl complex than for the individual EBNAl complex polypeptides.
  • the present invention provides an isolated antibody that binds to an interaction site on an EBNAl complex polypeptide selected from the group consisting of EBNAl, USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the isolated antibodies of the invention disrupt or stabilize an EBNAl complex.
  • the present invention provides an isolated antibody that binds to an EBNAl complex polypeptide comprising the amino acid sequence of residues 395 to 450 of EBNAl.
  • the present invention further provides compositions related to producing, isolating, detecting, or characterizing the above EBNAl complex and EBNAl complex polypeptide compositions, such as nucleic acids, vectors, host cells, and the like.
  • a host cell comprising at least one recombinant nucleic acid encoding at least one of the EBNAl complex polypeptides is provided.
  • the recombinant nucleic acid in the host cell comprises a reporter gene that is reflective of the activity of an EBNAl complex.
  • the invention further provides, in another of its aspects, various methods of exploiting the EBNAl complexes, as well as the individual EBNAl complex polypeptides.
  • These various permutations of EBNAl complexes, by virtue of these interactions, are implicated in the modulation of various functional activities of EBNAl.
  • These functional activities may include, but are not limited to: (i) physiological processes (e.g., cell cycle control, cellular differentiation and apoptosis); (ii) response to viral infection; (iii) intracellular signal transduction; (iv) transcriptional regulation; and (v) pathophysiological processes (e.g., hyperproliferative disorders including tumorigenesis and tumor spread, degenerative disorders including neurodegenerative disorders, virus infection).
  • methods for identifying modulators of the EBNAl complexes and methods for identifying modulators of EBV-mediated diseases or disorders using the EBNAl complexes are provided.
  • a method for identifying modulators of EBNAl complexes comprising: (i) forming a reaction mixture including an EBNAl complex comprising (a)
  • a method for identifying a compound that modulates an EBV-mediated disease or disorder comprising:
  • a modulation in the extent of said EBV-mediated disease or disorder in the presence of said test compound indicates that the test compound may be a candidate therapeutic for said EBV-mediated disease or disorder.
  • a method for identifying a compound that modulates an EBV-mediated disease or disorder comprising:
  • a method for identifying a compound that modulates an EBV-mediated disease or disorder comprising:
  • Compounds for use with the above-described methods may be include, for example, lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides.
  • the compound is a polynucleotide.
  • said polynucleotide is an antisense nucleic acid.
  • said polynucleotide is an siRNA.
  • the compound may be a member of a library of compounds.
  • the invention provides pharmaceutical compositions comprised of compounds identified through the above-described methods that modulate the EBNAl complexes or an EBV-mediated disease or disorder.
  • the invention provides methods of treating EBV-mediated diseases or disorders using the subject pharmaceutical compositions.
  • the invention provides reagents comprising the EBNAl complexes and EBNAl complex polypeptide compositions described above.
  • the invention provides diagnostic assays based on the above-mentioned methods for detecting the EBNAl complexes. Kits comprising the pharmaceutical compositions or reagents of the present invention are also within the scope of the invention.
  • FIGURES Figure 1 Flowchart of the procedure used to isolate EBNAl complex polypeptides from human cell extracts via an EBNAl affinity column.
  • Figure 2 Depiction of a gel electrophoresis profile of cellular polypeptides that bound to empty (“none”), EBNAl (left) and TBP (right) affinity columns.
  • Figure 3 Depiction of a gel electrophoresis profile of cellular polypeptides that bound to an EBNAl affinity column after a first pass of human cell extract (lane 1) and a second pass of the EBNAl affinity column eluate visualized in lane 1 (lane 2) through the column.
  • Figure 4. Results of immunoprecipitation of EBNAl from insect cells in which EBNAl or an EBNAl mutant and USP7 were co-expressed.
  • the top panel depicts a diagram of the EBNAl polypeptide in which the position of the USP7 binding site, as determined from the immunoprecipitation results, is indicated.
  • the bottom panel depicts autoradiograph visualization of the EBNAl immunoprecipitation results.
  • the position of USP7 is indicated with an asterisk.
  • Each lane is labeled with the polypeptides that were co- expressed in the insect cells.
  • FIG. 5 Flowchart of the tandem affinity purification (TAP) tagging method used to isolate EBNAl complex polypeptides from human cells.
  • Figure 6. Depiction of a gel electrophoresis profile of cellular polypeptides that bound to TAP-tagged beta-galactosidase (MW 116 kDa, lane " ⁇ -gal”), EBNAl (lane "EBNAl”), and EBNAl mutant (lane “ ⁇ 395-450”) as isolated by the TAP tagging method.
  • TAP tandem affinity purification
  • FIG. 7 Profiling of cellular protein interactions by EBNAl affinity chromatography.
  • A HeLa cell lysate was applied to either an EBNAl column, a TBP column, or a column lacking a protein ligand (None), and retained proteins were eluted in 1 M NaCl. Eluted proteins were separated by SDS-PAGE, visualized by silver staining and identified by MALDI-ToF mass spectrometry as indicated.
  • B Cellular proteins eluted from EBNAl affinity columns (lane 1) were dialyzed then reapplied to a second EBNA 1 column. The 1 M NaCl elution from the second EBNA 1 column is shown in lane 2. The dominant NAP 1 band in this experiment was partially proteoly sed resulting in a slightly faster migration than in part A.
  • Figure 8 Comparison of EBNAl and ⁇ 325-376 affinity column profiles. HeLa cell lysate was applied to columns containing either EBNAl, ⁇ 325-376 or no protein (None). Retained proteins were eluted and analyzed as described for Figure 7 A.
  • Figure 9. Mapping of the USP7 binding region of EBNAl by coimmunoprecipitation.
  • A Schematic representation of EBNAl and EBNAl mutants and summary of their ability to bind USP7 in the co-immunoprecipitation assay.
  • B Schematic representation of EBNAl and EBNAl mutants and summary of their ability to bind USP7 in the co-immunoprecipitation assay.
  • FIG. 10 Isolation of TAP-tagged EBNAl and associated cellular proteins.
  • TAP-tagged proteins were expressed in 293 T cells and sequentially purified on IgG and calmodulin columns. Purified proteins were separated by SDS-PAGE, visualized by silver staining and identified by MALDI-ToF mass spectrometry. Positions of molecular weight markers are indicated beside each gel in KDa. Bands that are not labeled were not identified by mass spectrometry.
  • the band at 116 KDa in the ⁇ -gal lane is TAP-tagged ⁇ -gal.
  • FIG. 11 Comparison of turnover rates of EBNAl and EBNAl ⁇ 395-450. 293T cells expressing either EBNAl or ⁇ 395-450 were blocked with cycloheximide and cells were collected and lysed 0, 1, 5, 9, 25 and 32 hours post transfection as indicated. Equal amounts of protein from each cell lysate were analysed by Western blotting using anti-
  • FIG. 12 Effect of the EBNAl ⁇ 395-450 mutation on the DNA replication and plasmid maintenance abilities of EBNAl.
  • C33A cells were transfected with pc3oriP plasmids expressing either EBNAl, EBNAl ⁇ 395-450 or no EBNAl protein (None) as indicated.
  • Transfected cells were either grown for 2 weeks to assess plasmid maintenance ability (A) or grown for 72 hours to measure DNA replication activity (B). Plasmids were then harvested, linearized, digested with Dpn I and analyzed by Southern blotting.
  • the bracket marks the position of the linearized pc3oriP plasmids.
  • a 100 pg marker of pc3oriP is shown in A.
  • FIG. 13 Log-phase cultures of C33A cells expressing EBNAl from a transfected plasmid or Raji cells were stained for EBNAl using mouse monoclonal antibody, stained for USP7 using rabbit antiserum, and counterstained with 4,6-diamidino-2-phenylindole
  • DAPI DAPI
  • B Time course of cleavage of GST-Ub52 by purified USP7.
  • Purified USP7 was incubated with purified GST-Ub52 at a 1:1000 molar ratio and aliquots were withdrawn and quenched at the indicated times.
  • Cleaved (bottom band) and uncleaved (top band) substrates were separated by SDS-PAGE, visualized by staining with S YPRO- Orange and scanning on a STORM860 laser scanner, and quantified with ImageQuant software.
  • C EBNAl was incubated in the ubiquitination reaction for increasing amounts of time up to 1 hour (lanes 2-5).
  • Figure 15 Effect of salt, pH and protease inhibitors on USP7 cleavage of GST- Ub52.
  • the cleavage of purified GST-Ub52 by purified USP7 was monitored and quantified as in Figure 14B.
  • Each data point plotted is the average value from 3 to 8 experiments.
  • B Reactions were performed in 20 mM Tris-HCl pH 7.5, 35 mM NaCl, 10 mM DTT and either 5 mM CaCl 2 , 5 mM MnCl 2 , 5mM MgCl 2 or 1 mM EDTA.
  • Reactions were performed in either 20 mM MES pH 6.2, 20 mM sodium phosphate pH 7.0, 20 mM HEPES pH 7.5 or 20 mM sodium carbonate pH 9.5, plus 10 mM DTT. USP7 was dialyzed against these reaction buffers prior to inclusion in these reactions. D. Reactions were performed in 20 mM Tris-HCl pH 7.5, 35 mM NaCl, 10 mM DTT with or without 1 mM ALLN or PMSF. For reactions containing 1 mM NEM the DTT was omitted.
  • FIG. 16 Retention of USP7 domains on EBNAl affinity columns.
  • Purified USP7 was partially digested with chymotrypsin (load) and analysed on an EBNAl affinity column or a control column as in A. The bands marked by the asterisk in the SDS lane are EBNAl (top) and an EBNAl proteolysis product (bottom).
  • FIG. 18 Retention of USP7 domains on ICPO affinity columns.
  • M molecular weight markers
  • the bands marked by the asterisks in the eluate lanes are from the GST or GST-ICPO preparation; the top band is GST-ICPO and the bottom band is GST.
  • B A summary of all of the USP7 fragments generated by partial tryptic digestion and analysed for binding to ICPO as in part A from 3 separate experiments. Fragments that bound and did not bind ICPO are marked by the solid and broken lines, respectively.
  • FIG. 19 Analysis of EBNAl and p53 peptide binding to the USP7 N-terminal domain by tryptophan fluorescence.
  • the USP7 N-terminal fragment containing amino acids 1-205 was incubated with increasing amount of EBNAl peptide containing amino acids 395-450 (A) or p53 peptides containing either amino, acids 355-393 or 311-393 (B). Binding was assessed by measuring the total tryptophan fluorescence at 350 rim, using an excitation wavelength of 295 nm. The change in fluorescence signal, as compared to the USP7 1-205 fragment alone, was plotted as a function of target peptide concentration.
  • FIG. 20A-L Polypeptide sequences of EBNAl complex polypeptides along with their respective GenBank Accession numbers.
  • F CK2 alpha' subunit (SEQ ID NO: 6
  • G CK2 beta subunit (SEQ ID NO: 7), H: NAPl (SEQ ID NO: 8)
  • pp32 SEQ ID NO: 12
  • EBNAl plays several roles in latent EBV infection, including the replication and segregation of the viral genomes, transcriptional activation of other viral genes and escape of host immune detection due to the failure of EBNAl to be processed by the proteasome. All of these functions likely involve specific interactions with cellular proteins.
  • the present invention relates to the discovery of novel protein complexes of EBNAl. Such complexes of molecules may present drug targets to prevent or treat virus-associated diseases and disorders, such as, for example, EBV-associated diseases or disorders, including, for example, lymphomas associated with immunosupression (observed in AIDS and organ transplant patients), Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • Two methods have been used to identify cellular protein targets that interact with EBNAl using a recombinant EBNAl lacking most of the Gly Ala repeat region.
  • One approach for detection of EBNAl complex proteins involved profiling human proteins that bound an EBNAl affinity column. After eliminating those interactions that occur through nucleic acid and non-specific charge interactions, specific interactions were revealed between EBNAl and the nuclear ubiquitin-specific protease, USP7 (also called HAUSP), and between EBNAl and casein kinase II.
  • EBNAl -USP7 interaction suggests that EBNAl might avoid targeting to the proteasome due to the removal of conjugated ubiquitin from EBNAl, and that EBNAl might affect the turnover of cellular proteins by sequestering USP7.
  • Another approach taken to identify cellular targets of EBNAl involved the purification of TAP-tagged EBNAl and associated proteins from human cells ' . This approach confirmed the interaction with USP7 and casein kinase II, and revealed an interaction with a protein arginine-methyltransferase that was not detected by the EBNAl affinity column methods. Thus, at least three novel EBNAl complexes have been identified as described herein.
  • the EBNAl complex polypeptides include USP7 (also called HAUSP), PRMT5, and casein kinase II.
  • USP7 is a protein affiliated with the ubiquitin-mediated protein degradation system. USP7 localizes to nuclear bodies (ND10 or PML bodies) and is known to associate with a herpes virus protein. USP7 deubiquitinates and stabilizes p53. ND10 structures are thought to have pro-apoptotic antiviral activities, and proteins from EBV, HSV and CMV are all known to disrupt ND10 structures.
  • PRMT5 JBPl is an arginine methyltransferase and a member of a complex termed the methylosome that methylates RNA splicing proteins (Sm proteins). Sm proteins are targeted for assembly into the spliceosome. PRMT5 may interact with hepatitis C virus protein NS3 helicase. Casein kinase II (CK2) phosphorylates a host of different viral proteins in vitro, including the EBV proteins EBNA-LP, EBNA-SM and ZEBRA. Otherwise, CK2 is a ubiquitous protein kinase that has been implicated in numerous signal transduction pathways. 2. Definitions
  • Biological activities include, but are not limited to, binding to polypeptides, binding to other proteins or molecules, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, enzymatic activity, methyl transferase activity, phosphorylase or kinase activity, conformational changes, changes in intracellular localization, changes in the transcription level of the gene encoding the peptide, changes in second messenger levels, etc.
  • An activity may be modulated by directly affecting the subject polypeptide.
  • a bioactivity may be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.
  • antibody refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody may be monoclonal or polyclonal.
  • the antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • antibodies used with the methods and compositions described herein are derivatives of the IgG class.
  • antibody fragment refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFv, Fv, dsFv diabody, and Fd fragments.
  • the antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced.
  • the antibody fragment may optionally be a single chain antibody fragment.
  • the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages.
  • the fragment may also optionally be a multimolecular complex.
  • a functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being mcluded in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • the names of the natural amino acids are abbreviated herein in accordance with the recommendations of IUPAC-IUB.
  • antisense nucleic acid refers to oligonucleotides which specifically hybridize (e.g., bind) under cellular conditions with a gene sequence, such as at the cellular mRNA and/or genomic DNA level, so as to inhibit expression of that gene, e.g., by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a polypeptide of the invention and a binding partner, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen- bond interactions under physiological conditions.
  • exemplary interactions include protein- protein, protein-nucleic acid, protein-small molecule, and small molecule-nucleic acid interactions.
  • biological sample when used in reference to a diagnostic assay is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • casein kinase II or "CK2" as used herein refers to a polypeptide comprising one or more of the ⁇ , ⁇ ' and ⁇ subunits of casein kinase II.
  • CK2 is a tetramer comprising an ⁇ , an ⁇ ', and two ⁇ subunits.
  • the ⁇ and ⁇ ' subunits contain the catalytic site, thus, in certain embodiments a CK2 polypeptide comprising the ⁇ and ⁇ ' subunits may be used.
  • a CK2 polypeptide may comprise one or more ⁇ subunits, for example, to analyze the interaction of those subunits with another polypeptide.
  • CK2 refers to a polypeptide comprising one or more polypeptides comprising (a) an amino acid sequence set forth in SEQ ID NOs: 5, 6 and/or 7, (b) an amino acid sequence set forth in SEQ ID NOs: 5, 6 and/or 7 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, identical to the sequence set forth in SEQ ID NOs: 5, 6 and/or 7.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of CK2.
  • complex refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another.
  • moieties e.g. chemical or biochemical
  • complexes include associations between antigen/antibodies, lectin/avidin, target polynucleotide/probe oligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligand and the like.
  • Member of a complex refers to one moiety of the complex, such as an antigen or ligand.
  • Protein complex or “polypeptide complex” refers to a complex comprising at least one polypeptide.
  • a complex refers to an "EBNAl complex" comprising (a) EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (b) EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (c) a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; or (d) a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the complex comprises EBNAl and USP7. In another embodiment, the complex comprises a fragment of EBNAl and a fragment of USP7. In another embodiment, the complex comprises a fragment of EBNAl comprising residues 395-450 and USP7. In another embodiment, the complex comprises a fragment of USP7 comprising residues 67-161. In still another embodiment, the complex comprises EBNAl and PRMT5. In yet another embodiment, the complex comprises
  • the complex comprises EBNAl and NAPl. In still another embodiment, the complex comprises EBNAl and TAF I ⁇ .
  • EBNAl complex polypeptide refers to an individual polypeptide that may be present in an EBNAl complex, including EBNAl and polypeptides that may interact with EBNAl either directly or indirectly.
  • an EBNAl complex polypeptide refers to EBNAl, USP7, NAP 1, TAF I ⁇ , CK2, or PRMT5.
  • an EBNAl complex polypeptide refers to a fusion protein comprising all or a portion of one or more EBNAl complex polypeptides such as EBNAl, USP7, NAP 1, TAF I ⁇ , CK2, and/or PRMT5.
  • EBNAl or "EBNAl polypeptide”, with reference to a polypeptide, refers to a polypeptide comprising (a) an amino acid sequence set forth in SEQ ID NOs: 1 or 2, (b) an amino acid sequence set forth in SEQ ID NOs: 1 or 2 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, identical to the sequence set forth in SEQ ID NOs: 1 or 2.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of EBNAl.
  • an EBNAl fragment refers to an EBNAl polypeptide in which amino acid residues are deleted as compared to the full length polypeptide.
  • an EBNAl fragment refers to a polypeptide comprising amino acid residues 325-376, 330- 619, 330-641, 452-641, 395-641, 41-376, or 395-450 of SEQ ID NO: 1.
  • an EBNAl fragment refers to a polypeptide comrpisng amino acid residues 1- 324 and 377-641 of SEQ ID NO: 1.
  • an EBNAl fragment refers to a polypeptide comrpisng amino acid residues 1-40 and 377-641 of SEQ ID NO: 1. In yet another embodiment, an EBNAl fragment refers to a polypeptide comrpisng amino acid residues 1-394 and 451-641 of SEQ ID NO: 1. In an exemplary embodiment, an EBNAl fragment refers to a polypeptide comprising at least 5, 10, 15, 20, 25, 30, 40, 50, or more, consecutive residues of SEQ ID NO: 1 or 2. In another embodiment, an EBNAl fragment refers to a polypeptide comprising at least 5, 10, 15, 20, 25, 30, 40, 50, or more, consecutive residues from the region of SEQ ID NO: 1 having amino acid residues 395-450.
  • an EBNAl fragment retains at least one biological activity of an EBNAl polypeptide, such as, for example, the ability to bind to another EBNAl complex polypeptide, such as, for example, USP7, NAP 1, TAF I ⁇ , CK2 (subunits ⁇ , ⁇ ', and or ⁇ ), and/or PRMT5.
  • an EBNAl polypeptide such as, for example, the ability to bind to another EBNAl complex polypeptide, such as, for example, USP7, NAP 1, TAF I ⁇ , CK2 (subunits ⁇ , ⁇ ', and or ⁇ ), and/or PRMT5.
  • EBV-mediated disease or disorder refers to any disease or disorder that is induced in a subject by the presence of or infection by EBV in said subject.
  • EBV-mediated diseases or disorders include, but are not limited to lymphomas associated with immunosupression (observed in AIDS and organ transplant patients), Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • a “fusion protein” or “fusion polypeptide” refers to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences may be linked in frame.
  • a fusion protein may include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion expressed by different kinds of organisms.
  • the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide.
  • the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences.
  • the fusion polypeptides may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide.
  • Exemplary fusion proteins include polypeptides comprising a glutathione S-transferase tag (GST-tag), histidine tag (His-tag), an immunoglobulin domain or an immunoglobulin binding domain.
  • fusion proteins of the invention include polypeptides comprising all or a portion of a least two EBNAl complex polypeptides, including EBNAl, USP7, NAP 1, TAF I ⁇ , CK2, or PRMT5.
  • gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide having exon sequences and optionally intron sequences.
  • intron refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • the "level of expression of a gene in a cell” or “gene expression level” refers to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s) and degradation products, encoded by the gene in the cell.
  • Gene construct refers to a vector, plasmid, viral genome or the like which includes a "coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may transfect cells, in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct.
  • the gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, poly adenylation sites, origins of replication, marker genes, etc.
  • “Host cell” refers to a cell transduced with a specified transfer vector.
  • the cell is optionally selected from in vitro cells such as those derived from cell culture, ex vivo cells, such as those derived from an organism, and in vivo cells, such as those in an organism.
  • “Host cells” or “recombinant host cells” or “heterologous cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.
  • isolated polypeptide refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • isolated nucleic acid refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid” is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
  • label refers to incorporation or attachment, optionally covalently or non-covalently, of a detectable marker into a molecule, such as a polypeptide.
  • a detectable marker such as a polypeptide.
  • Various methods of labeling polypeptides are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes, fluorescent labels, heavy atoms, enzymatic labels or reporter genes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Examples and use of such labels are described in more detail below.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • linker is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides.
  • the linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and the library member by a specific distance.
  • Non-limiting examples of linkers for use in the present invention are described in section 3.
  • the term "mammal” is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).
  • modulation when used in reference to a functional property or biological activity or process (e.g., enzyme activity or receptor binding), refers to the capacity to either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a quality of such property, activity or process.
  • up regulate e.g., activate or stimulate
  • down regulate e.g., inhibit or suppress
  • regulation may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
  • modulator refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species or the like (naturally-occurring or non-naturally-occurring), or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that may be capable of causing modulation.
  • Modulators may be evaluated for potential activity as inhibitors or activators (directly or indirectly) of a functional property, biological activity or process, or combination of them, (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, anti-microbial agents, inhibitors of microbial infection or proliferation, and the like) by inclusion in assays. In such assays, many modulators may be screened at one time.
  • NAPl refers to a polypeptide comprising (a) an amino acid sequence set forth in SEQ ID NO: 8, (b) an amino acid sequence set forth in SEQ ID NO: 8 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, identical to the sequence set forth in SEQ ID NO: 8.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of NAPl.
  • nucleic acid refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • a “patient”, “subject” or “host” to be treated by the subject method may mean either a human or non-human animal.
  • a “peptide nucleic acid” or “PNA” refers to an analogue of a nucleic acid in which the backbone of the molecule is not sugar-phosphate, but rather a peptide or peptidomimetic. A detailed description of PNAs may be found in Nielsen, et al. Curr. Issues Mol. Biol. (1999) 1 :89-104.
  • Peptidomimetic refers to a compound containing peptide-like structural elements that is capable of mimicking the biological action (s) of a natural parent polypeptide.
  • “Pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds.
  • “Pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the supplement and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene gly col; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene gly col; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum
  • PRMT5 or “an PRMT5 polypeptide”, with reference to a polypeptide refers to a polypeptide comprising (a) an amino acid sequence set forth in SEQ ID NO: 4, (b) an amino acid sequence set forth in SEQ ID NO: 4 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, identical to the sequence set forth in SEQ ID NO: 4.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of PRMT5.
  • Protein Protein
  • polypeptide and “peptide” are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence.
  • gene product it is meant a molecule that is produced as a result of transcription of a gene.
  • Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts.
  • Exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • a protein may comprise two or more polypeptide chains that are associated through covalent or non-covalent interactions.
  • polypeptide fragment when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and/or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.
  • reaction site refers to a region on a polypeptide that facilitates the interaction of the polypeptide with a second molecule, such as another polypeptide or a nucleic acid or small molecule.
  • purified refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a “purified fraction” is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present.
  • the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species (including the one of interest) dissolved or dispersed are taken into account.
  • a purified composition will have one species that comprises more than about 85 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present.
  • the object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.
  • a skilled artisan may purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide may be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis and mass- spectrometry analysis.
  • Recombinant protein "heterologous protein” and “exogenous protein” are used interchangeably to refer to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.
  • reporter gene refers to a nucleic acid comprising a nucleotide sequence encoding a protein that is readily detectable either by its presence or activity, including, but not limited to, luciferase, fluorescent protein (e.g., green fluorescent protein), chloramphenicol acetyl transferase, ⁇ -galactosidase, secreted placental alkaline phosphatase, ⁇ -lactamase, human growth hormone, and other secreted enzyme reporters.
  • fluorescent protein e.g., green fluorescent protein
  • chloramphenicol acetyl transferase e.g., chloramphenicol acetyl transferase
  • ⁇ -galactosidase e.g., secreted placental alkaline phosphatase
  • ⁇ -lactamase ⁇ -lactamase
  • human growth hormone and other secreted enzyme reporters.
  • a reporter gene encodes a polypeptide not otherwise produced by the host cell, which is detectable by analysis of the cell(s), e.g., by the direct fluorometric, radioisotopic or spectrophotometric analysis of the cell(s) and preferably without the need to kill the cells for signal analysis.
  • a reporter gene encodes an enzyme, which produces a change in fluorometric properties of the host cell, which is detectable by qualitative, quantitative or semiquantitative function or transcriptional activation.
  • sample includes material obtained from a subject or an object of interest.
  • samples may be obtained from a human or animal subject, a plant, a cell culture or an environmental location, such as a water or air sample.
  • the sample also includes materials that have been processed or mixed with other materials.
  • a blood sample may be processed to obtain serum, red blood cells, etc., each of which may be considered a sample.
  • Small molecule refers to a composition, which has a molecular weight of less than about 1000 kDa .
  • Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules.
  • libraries of chemical and/or biological extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.
  • Temporal activating factor I beta or "TAF I ⁇ " or “TAF I ⁇ polypeptide” refers to a polypeptide comprising (a) an amino acid sequence set forth in SEQ ID NO: 9, (b) an amino acid sequence set forth in SEQ ID NO: 9 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, identical to the sequence set forth in SEQ ID NO: 9.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of TAF I ⁇ .
  • test compound refers to a molecule to be tested by one or more screening method(s) as a putative modulator of a polypeptide of the invention or other biological entity or process.
  • a test compound is usually not known to bind to a target of interest.
  • test compound is meant to include polypeptides, polynucleotides, carbohydrtaes, lipids, and small molecules, or mixtures thereof.
  • USP7 refers to a polypeptide comprising (a) an amino acid sequence set forth in SEQ ID NO: 3, (b) an amino acid sequence set forth in SEQ ID NO: 3 with 1 to about 20 conservative amino acid substitutions, or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%), or greater, identical to the sequence set forth in SEQ ID NO: 3.
  • the term is also meant to include homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of USP7.
  • USP7 fragment or “USP7 polypeptide fragment"
  • USP7 polypeptide refers to a USP7 polypeptide in which amino acid residues are deleted as compared to the full length polypeptide.
  • a USP7 fragment refers to a polypeptide comprising amino acid residues 67-745, 12-543, 67-543, 67-355, 81- 322, 67-312, 67-301, 67-335, 67-322, 67-254, 67-161, or 81-161 of SEQ ID NO: 3.
  • a USP7 fragment refers to a polypeptide comprising at least 5, 10, 15, 20, 25, 30, 40, 50, or more, consecutive residues of SEQ ID NO: 3.
  • a USP7 fragment refers to a polypeptide comprising at least 5, 10, 15, 20, 25, 30, 40, 50, or more, consecutive residues from the region of SEQ ID NO: 3 having amino acid residues 67-161.
  • a USP7 fragment retains at least one biological activity of USP7, such as, for example, the ability to bind to EBNAl .
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops, which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • Exemplary Compositions Relating to the Novel Interactions 3. a. Polypeptide and Nucleic Acid Compositions
  • the application provides an EBNAl complex comprising EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; or a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the EBNAl complex comprises EBNAl , or a fragment thereof, and USP7, or a fragment thereof.
  • the EBNAl complex comprises a fragment of EBNAl copmprising residues 395-450 of SEQ ID NO: 1 and USP7.
  • the EBNAl complex comprises a fragment of USP7 comprising residues 67-161 of USP7.
  • the EBNAl complex comprises a fragment of EBNAl comprising residues 395-450 and a fragment of USP7 comprising residues 67-161 of USP7.
  • the EBNAl complex comprises EBNAl and PRMT5.
  • the EBNAl complex comprises EBNAl and CK2. In another embodiment, the EBNAl complex comprises EBNAl and NAP 1. In another embodiment, the EBNAl complex comprises EBNAl and TAF I ⁇ . The sequences of the full-length EBNAl complex polypeptides comprising the EBNAl complexes are shown in Figure 20.
  • the present invention makes available in a variety of embodiments soluble, purified and/or isolated forms of the EBNAl complexes or the EBNAl complex polypeptides, or fragments thereof.
  • an EBNAl complex polypeptide may comprise (a) a full-length EBNAl complex polypeptide amino acid sequence, (b) a full-length EBNAl complex polypeptide amino acid sequence with 1 to about 20 conservative amino acid substitutions, (c) a polypeptide amino acid sequence that is at least 80% identical to an EBNAl complex polypeptide sequence of interest, or (d) a fragment of an EBNAl complex polypeptide as indicated in (a), (b), or (c).
  • the present invention contemplates a composition comprising an isolated EBNAl complex or EBNAl complex polypeptide and less than about 25%, or alternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides.
  • an EBNAl complex polypeptide is a fusion protein containing a domain which increases its solubility and/or facilitates its purification, identification, detection, and/or structural characterization.
  • Exemplary domains include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His- Asp or FLAG fusion proteins and tags.
  • Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system- targeting peptides, transcytosis domains, nuclear localization signals, etc.
  • an EBNAl complex polypeptide may comprise one or more heterologous fusions.
  • Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains.
  • the fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide. It is also within the scope of the invention to include linker sequences between an EBNAl complex polypeptide and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein.
  • the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and EBNAl complex polypeptide in order to remove the tag after protein expression or thereafter.
  • suitable endoproteases include, for example, Factor Xa and TEV proteases.
  • a fusion polypeptide is provided, comprising all or a portion of one or more EBNAl complex polypeptide amino acid sequences.
  • a fusion polypeptide may comprise (a) EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (b) EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; (c) a fragment of EBNAl and at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5; or (d) a fragment of EBNAl and a fragment of at least one polypeptide selected from the group consisting of USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • Such fusion polypeptides may mimic the EBNAl complexes or a portion thereof.
  • Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed to generate a chimeric gene sequence
  • Linkers also known as “linker molecules” or “cross- linkers” or “spacers” may be used to conjugate EBNAl complex polypeptides, or to conjugate EBNAl complex polypeptides once they have formed an EBNAl complex.
  • Linkers are chemicals able to react with a defined chemical group of several, usually two, molecules and thus conjugate them. The majority of known cross-linkers react with amine, carboxyl, and sulfliydryl groups. The choice of target chemical group is crucial if the group may be involved in the biological activity of the polypeptides to be conjugated.
  • Linkers may be homofunctional (containing reactive groups of the same type), heterofunctional (containing different reactive groups), or photoreactive (containing groups that become reactive on illumination).
  • Linker molecules may be responsible for different properties of the conjugated compositions. The length of the linker should be considered in light of molecular flexibility during the conjugation step, and the availability of the conjugated molecule for its target (cell surface molecules and the like.) Longer linkers may thus improve the biological activity of the compositions of the present invention, as well as the ease of preparation of them.
  • the geometry of the linker may be used to orient a molecule for optimal reaction with a target.
  • a linker with flexible geometry may allow the cross-linked polypeptides to conformationally adapt as the bind other polypeptides.
  • the nature of the linker may be altered for other various purposes. For example, the aryl-structure of MBuS was found less immunogenic than the aromatic spacer of MBS.
  • the hydrophobicity and functionality of the linker molecules may be controlled by the physical properties of component molecules.
  • the hydrophobicity of a polymeric linker may be controlled by the order of monomeric units along the polymer, e.g. a block polymer in which there is a block of hydrophobic monomers interspersed with a block of hydrophilic monomers.
  • At least one EBNAl complex or EBNAl complex polypeptide thereof is labeled.
  • an EBNAl complex or EBNAl complex polypeptide is labeled with a fluorescent or chromogenic label to facilitate detection, purification, or structural characterization.
  • an EBNAl complex or EBNAl complex polypeptide is fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal, including, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • Renilla reniformis green fluorescent protein GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • EYFP enhanced cyan fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • an EBNAl complex or EBNAl complex polypeptide is labeled with an isotopic label to facilitate its detection and or structural characterization using nuclear magnetic resonance or another applicable technique.
  • isotopic labels include radioisotopic labels such as, for example, potassium-40 ( °K), carbon- 14 ( C), tritium ( H), sulphur-35 ( S), phosphorus-32 ( P), and the like, or isotopic labels comprising an atom with non zero nuclear spin, including, for example, hydrogen- 1 (1H), hydrogen-2 ( 2 H), hydrogen-3 ( 3 H), phosphorous-31 ( 31 P), sodium-23 ( 23 Na), nitrogen- 14 ( 14 N), nitrogen- 15 ( 15 N), carbon- 13 ( 13 C) and fluorine- 19 ( 19 F).
  • the EBNAl complex or EBNAl complex polypeptide is labeled to facilitate structural characterization using x-ray crystallography or another applicable technique.
  • exemplary labels include heavy atom labels such as, for example, cobalt, selenium, krypton, bromine, strontium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, and the like.
  • the EBNAl complex or EBNAl complex polypeptide is labeled with seleno-methionine.
  • the label is located in one or more specific locations within the EBNAl complex or EBNAl complex polypeptide, for example, the label may be specifically incorporated into one or more of the leucine residues of the polypeptide. In another example, the label may be specifically conjugated into one or more of the lysine residues of the polypeptide.
  • the invention also encompasses the embodiment wherein a single polypeptide comprises two, three or more different labels, for example, the EBNAl complex or EBNAl complex polypeptide comprises both N and C labeling, or comprises acceptor and donor fluorescent labels suitable for use in FRET.
  • an expression vector comprising a nucleic acid encoding a polypeptide is introduced into a host cell, and the host cell is cultured in a cell culture medium in the presence of a source of the label, thereby generating a labeled polypeptide.
  • the expression vector comprises a nucleic acid encoding an EBNAl complex polypeptide fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal.
  • an EBNAl complex or EBNAl complex polypeptide is reacted with a label containing a moiety that reacts with an amino acid residue of the EBNAl complex or EBNAl complex polypeptide, such as, for example, reacting a polypeptide with a succinimidyl ester of a fluorophore.
  • the invention relates to an EBNAl complex or EBNAl complex polypeptide contained within a vessel useful for manipulation of the sample.
  • the EBNAl complexes or EBNAl complex polypeptides may be contained within a microtiter plate to facilitate detection, screening or purification.
  • the EBNAl complexes or EBNAl complex polypeptides may also be contained within a syringe as a container suitable for administering them to a subject in order to generate antibodies or as part of a vaccination regimen.
  • the EBNAl complexes or EBNAl complex polypeptides may also be contained within an NMR tube in order to enable characterization by nuclear magnetic resonance techniques.
  • the EBNAl complexes or EBNAl complex polypeptides may either be in solution (e.g. dissolved into a solvent) or in a dry form (e.g. lyophilized).
  • the invention relates to a crystallized form of an EBNAl complex or EBNAl complex polypeptide described herein and crystallized EBNAl complexes or EBNAl complex polypeptides which have been mounted for examination by x-ray crystallography as described further below.
  • the present invention contemplates a crystallized EBNAl complex or EBNAl complex polypeptide including an EBNAl complex or EBNAl complex polypeptide and one or more of the following: a co- factor (such as a salt, metal, nucleotide, oligonucleotide or polypeptide), a modulator, or a small molecule.
  • a co- factor such as a salt, metal, nucleotide, oligonucleotide or polypeptide
  • a modulator or a small molecule.
  • the present invention contemplates a crystallized EBNAl complex or EBNAl complex polypeptide including any other molecule or atom (such as a metal ion) that associates with the complex in vivo.
  • the EBNAl complexes or EBNAl complex polypeptides may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell.
  • Chemical synthesis of EBNAl complexes or EBNAl complex polypeptides may be carried out using a variety of art recognized methods, including stepwise solid phase synthesis, semi-synthesis through the conformationally-assisted re-ligation of peptide fragments, enzymatic ligation of cloned or synthetic peptide segments, and chemical ligation.
  • Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate.
  • the transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site.
  • Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules, (see e.g., U.S. Patent Nos. 6,184,344 and 6,174,530; and T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420; M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H.
  • homologs may function in a limited capacity as a modulator to promote or inhibit a subset of the biological activities of the naturally-occurring form of the EBNAl complex or EBNAl complex polypeptide.
  • specific biological effects may be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with modulators which are directed to all of the biological activities of an EBNAl complex or EBNAl complex polypeptide.
  • homologs may be generated which interfere with the ability of the wild-type EBNAl complex polypeptide of the invention to associate with certain proteins, but which do not substantially interfere with the formation of complexes between the native polypeptide and other cellular proteins.
  • Another aspect of the invention relates to an EBNAl complex polypeptide that is derived from a full-length EBNAl complex polypeptide.
  • Isolated peptidyl portions of the EBNAl complex polypeptides may be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such polypeptides.
  • fragments may be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • proteins may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or may be divided into overlapping fragments of a desired length.
  • the fragments may be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments having a desired property, for example, the capability of functioning as a modulator of an EBNAl complex polypeptide.
  • peptidyl portions of an EBNAl complex polypeptide may be tested for binding activity, as well as inhibitory ability, by expression as, for example, thioredoxin fusion proteins, each of which contains a discrete fragment of the EBNAl complex polypeptide (see, for example, U.S. Patents 5,270,181 and 5,292,646; and PCT publication WO94/ 02502).
  • modified polypeptides when designed to retain at least one activity of the naturally- occurring form of the protein, are considered "functional equivalents" of the polypeptides described in more detail herein.
  • modified polypeptides may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.
  • This invention further contemplates a method of generating sets of combinatorial mutants of an EBNAl complex polypeptide, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs).
  • the purpose of screening such combinatorial libraries is to generate, for example, homologs which may modulate the activity of an EBNAl complex or EBNAl complex polypeptide, or alternatively, which possess novel activities altogether.
  • Combinatorially-derived homologs may be generated which have a selective potency relative to a naturally-occurring protein. Such homologs may be used in the development of therapeutics.
  • mutagenesis may give rise to homologs which have intracellular half- lives dramatically different than the corresponding wild-type protein.
  • the altered protein may be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of the protein.
  • homologs, and the genes which encode them may be utilized to alter protein expression by modulating the half-life of the protein.
  • proteins may be used for the development of therapeutics or treatment.
  • the library of potential homologs may be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence may be carried out in an automatic DNA synthesizer, and the synthetic genes may then be ligated into an appropriate vector for expression.
  • One purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential protein sequences.
  • the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al, (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed.
  • mutagenesis may be utilized to generate a combinatorial library.
  • protein homologs may be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al, (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597- 601; Nagashima et al., (1993) J Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of protein homologs.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.
  • candidate combinatorial gene products are displayed on the surface of a cell and the ability of particular cells or viral particles to bind to the combinatorial gene product is detected in a "panning assay”.
  • the gene library may be cloned into the gene for a surface membrane protein of a bacterial cell (Ladner et al, WO 88/06630; Fuchs et al, (1991) Bio/Technology 9:1370- 1371; and Goward et al., (1992) TIBS 18:136-140), and the resulting fusion protein detected by panning, e.g. using a fluorescently labeled molecule which binds the cell surface protein, e.g. FITC-substrate, to score for potentially functional homologs.
  • Cells may be visually inspected and separated under a fluorescence microscope, or, when the morphology of the cell permits, separated by a fluorescence-activated cell sorter. This method may be used to identify substrates or other polypeptides that can interact with an EBNAl complex or EBNAl complex polypeptide.
  • the gene library may be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences may be expressed on the surface of infectious phage, thereby conferring two benefits.
  • coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins may be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al., PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al., (1991) Nature 352:624-628; and Barbas et al, (1992) PNAS USA 89:4457-4461). Other phage coat proteins may be used as appropriate.
  • the invention also provides for reduction of an EBNAl complex polypeptide to generate mimetics, e.g. peptide or non-peptide agents, which are able to mimic binding of the authentic protein to another cellular partner.
  • mimetics e.g. peptide or non-peptide agents
  • Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of a protein which participates in a protein-protein interaction with another protein.
  • the critical residues of a protein which are involved in molecular recognition of a substrate protein may be determined and used to generate peptidomimetics that may bind to the substrate protein.
  • the peptidomimetic may then be used as an inhibitor of the wild-type protein by binding to the substrate and covering up the critical residues needed for interaction with the wild-type protein, thereby preventing interaction of the protein and the substrate.
  • peptidomimetic compounds may be generated which mimic those residues in binding to the substrate.
  • non-hydrolyzable peptide analogs of such residues may be generated using benzodiazepine (e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G.R.
  • the present invention further provides compositions related to producing, detecting, or characterizing an EBNAl complex or EBNAl complex polypeptide, such as nucleic acids, vectors, host cells, and the like. Such compositions may serve as compounds that modulate an EBNAl complex or EBNAl complex polypeptide, such as antisense nucleic acids.
  • the nucleic acid of the invention encodes an EBNAl complex polypeptide.
  • Preferred nucleic acids encode a polypeptide at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homologous with an EBNAl complex polypeptide.
  • Isolated nucleic acids which differ from the nucleotide sequences encoding an EBNAl complex polypeptide due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations that do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the EBNAl complex polypeptides will exist among mammalian cells.
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
  • the present invention also pertains to nucleic acids encoding mutational variants of the EBNAl complex polypeptides which are derived, for example, by combinatorial mutagenesis. Fragments of the nucleic acid encoding a biologically active portion of an EBNAl complex polypeptide are also within the scope of the invention.
  • a fragment of the nucleic acid encoding an active portion of an EBNAl complex polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, the polypeptides in Figure 20, and which encodes a polypeptide which retains at least a portion of the biological activity of the full-length protein, or alternatively, which is functional as an modulator of the biological activity of the full-length protein.
  • such fragments include, as appropriate to the full-length protein from which they are derived, a polypeptide containing a domain mediating the interaction of the EBNAl with another protein such as USP7, NAP 1, TAF I ⁇ , CK2, and/or PRMT5.
  • Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant polypeptides.
  • antisense therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding one of the subject EBNAl complexes so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a member of an EBNAl complex.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding a member of an EBNAl complex.
  • oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.
  • exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775).
  • the invention provides double stranded small interfering RNAs
  • siRNAs decrease or block gene expression. While not wishing to be bound by theory, it is generally thought that siRNAs inhibit gene expression by mediating sequence specific mRNA degradation.
  • RNA interference is the process of sequence-specific, post-transcriptional gene silencing, particularly in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene (Elbashir et al. Nature 2001; 411(6836): 494- 8).
  • siRNAs and long dsRNAs may be used to inhibit the expression of a nucleic acid encoding an EBNAl complex polypeptide, and particularly when the polynucleotide is expressed in a mammalian or plant cell.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an EBNAl complex polypeptide and operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
  • the vector's copy number the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered.
  • the subject nucleic acids may be used to cause expression and over-expression of an EBNAl complex polypeptide in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins or polypeptides.
  • This invention pertains to a host cell transfected with a recombinant gene in order to express an EBNAl complex polypeptide.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an EBNAl complex polypeptide may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject.
  • Other suitable host cells are known to those skilled in the art.
  • the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide will be known to those in the art.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • An EBNAl complex polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, an EBNAl complex polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • An EBNAl complex polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffmity purification with antibodies specific for particular epitopes of an EBNAl complex polypeptide.
  • a nucleotide sequence encoding all or a selected portion of an EBNAl complex polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes.
  • Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant EBNAl complex polypeptides by microbial means or tissue-culture technology in accord with the subject invention.
  • Expression vehicles for production of a recombinant protein include plasmids and other vectors.
  • suitable vectors for the expression of an EBNAl complex polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83).
  • These vectors may replicate in E. coli due the presence of the ⁇ BR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • drug resistance markers such as ampicillin may be used.
  • mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • in vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein.
  • An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F).
  • CBP cap-binding protein
  • eIF4F eukaryotic initiation factor 4F
  • a variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts.
  • Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagens, La Jolla, Calif.; Amersham, Arlington Heights, III; and GIBCO/BRL, Grand Island, N. Y.
  • In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes.
  • an in vitro transcription system may be used.
  • Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors.
  • In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.
  • a carboxy terminal fragment of a polypeptide When expression of a carboxy terminal fragment of a polypeptide is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed.
  • ATG start codon
  • a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP).
  • MAP methionine aminopeptidase
  • the present invention provides an isolated antibody that has a higher binding affinity for an ⁇ BNAl complex than for the individual ⁇ BNAl complex polypeptides.
  • the present invention provides an isolated antibody that binds to an interaction site on an ⁇ BNAl complex polypeptide selected from the group consisting of ⁇ BNAl, USP7, NAP 1, TAF I ⁇ , CK2, and PRMT5.
  • the isolated antibodies of the invention disrupt or stabilize an ⁇ BNAl complex.
  • the present invention provides an isolated antibody that binds to an EBNAl complex polypeptide comprising the amino acid sequence of residues 395 to 450 of EBNAl. Another aspect of the invention pertains to antibodies specifically reactive with an EBNAl complex polypeptide.
  • Antibodies may be elicited by methods known in the art.
  • a mammal such as a mouse, a hamster or rabbit may be immunized with an immunogenic form of an EBNAl complex or EBNAl complex polypeptide of the invention (e.g., an antigenic fragment which is capable of eliciting an antibody response).
  • immunization may occur by using a nucleic acid of the acid, which presumably in vivo expresses an EBNAl complex polypeptide giving rise to the immunogenic response observed.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • a peptidyl portion of an EBNAl complex polypeptide may be administered in the presence of adjuvant.
  • the progress of immunization may be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays may be used with the immunogen as antigen to assess the levels of antibodies.
  • antibody producing cells may be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an EBNAl complex or EBNAl complex polypeptide and the monoclonal antibodies isolated.
  • Antibodies directed against an EBNAl complex or EBNAl complex polypeptide can be used to selectively block the action of an EBNAl complex or EBNAl complex polypeptide.
  • Antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
  • the EBNAl complex polypeptides may be modified so as to increase their immunogenicity.
  • a polypeptide, such as an antigenically or immunologically equivalent derivative may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of
  • the antibodies of the invention, or variants thereof are modified to make them less immunogenic when administered to a subject.
  • the antibody may be "humanized"; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al. (1991) Biotechnology 9, 266-273.
  • transgenic mice, or other mammals may be used to express humanized antibodies. Such humanization may be partial or complete.
  • the invention provides an antibody fragment. Preparation of antibody fragments may be accomplished by any number of well-known methods.
  • phage display technology may be used to generate antibody fragment selectivity components that are specific for a desired target molecule, including, for example, Fab fragments, Fv's with an engineered intermolecular disulfide bond to stabilize the VH - VL pair, scFvs, or diabody fragments.
  • Fab fragments fragments
  • Fv's with an engineered intermolecular disulfide bond to stabilize the VH - VL pair
  • scFvs or diabody fragments.
  • an immune response to a selected immunogen is elicited in an animal (such as a mouse, rabbit, goat or other animal) and the response is boosted to expand the immunogen-specific B-cell population.
  • Messenger RNA is isolated from those B-cells, or optionally a monoclonal or polyclonal hybridoma population.
  • the mRNA is reverse- transcribed by known methods using either a poly-A primer or murine immunoglobulin- specif ⁇ c primer(s), typically specific to sequences adjacent to the desired V H and VL chains, to yield cDNA.
  • the desired VH and V chains are amplified by polymerase chain reaction (PCR) typically using V H and V specific primer sets, and are ligated together, separated by a linker.
  • VH and VL specific primer sets are commercially available, for instance from Stratagene, Inc. of La Jolla, California. Assembled V H -linker-NL product (encoding an scFv fragment) is selected for and amplified by PCR. Restriction sites are introduced into the ends of the V ⁇ -linker-V L product by PCR with primers including restriction sites and the scFv fragment is inserted into a suitable expression vector (typically a plasmid) for phage display. Other fragments, such as an Fab' fragment, may be cloned into phage display vectors for surface expression on phage particles.
  • the phage may be any phage, such as lambda, but typically is a filamentous phage, such as fd and Ml 3, typically Ml 3.
  • phage display systems In phage display vectors, the V ⁇ -linker-V L sequence is cloned into a phage surface protein (for Ml 3, the surface proteins g3p (pill) or g8p, most typically g3p).
  • Phage display systems also include phagemid systems, which are based on a phagemid plasmid vector containing the phage surface protein genes (for example, g3p and g8p of Ml 3) and the phage origin of replication. To produce phage particles, cells containing the phagemid are rescued with helper phage providing the remaining proteins needed for the generation of phage.
  • Phagemid packaging systems for production of antibodies are commercially available.
  • One example of a commercially available phagemid packaging system that also permits production of soluble ScFv fragments in bacteria cells is the Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida.
  • RPAS Recombinant Phage Antibody System
  • Phage display systems, their construction and screening methods are described in detail in, among others, United States Patent Nos. 5,702,892, 5,750,373, 5,821,047 and 6,127, 132, each of which are incorporated herein by reference in their entirety.
  • epitope-specific phage are selected by their affinity for the desired immunogen and, optionally, their lack of affinity to compounds containing certain other structural features.
  • a variety of methods may be used for physically separating immunogen-binding phage from non-binding phage.
  • the immunogen is fixed to a surface and the phage are contacted with the surface. Non-binding phage are washed away while binding phage remain bound. Bound phage are later eluted and are used to re-infect cells to amplify the selected species.
  • a number of rounds of affinity selection typically are used, often increasingly higher stringency washes, to amplify immunogen-binding phage of increasing affinity.
  • Negative selection techniques also may be used to select for lack of binding to a desired target. In that case, un-bound (washed) phage are amplified.
  • EBNAl complexes may be produced by a variety of methods.
  • EBNAl complexes may be naturally-occurring, for instance in a cell infected with EBV, or produced in a host cell comprising nucleic acids encoding EBNAl complex polypeptides, or produced in vitro in a solution comprising EBNAl complexes or EBNAl or EBNAl complex polypeptides.
  • a variety of materials may be used as the source of potential EBNAl complex polypeptides.
  • a cellular extract or extracellular fluid may be used.
  • the choice of starting material for the extract may be based upon the cell or tissue type or type of fluid that would be expected to contain EBNAl complex polypeptides.
  • Micro-organisms or other organisms are grown in a medium that is appropriate for that organism and can be grown in specific conditions to promote the expression of proteins that may interact with the target protein.
  • Exemplary starting material that may be used to make a suitable extract are: 1) one or more types of tissue derived from an animal, especially a human, 2) cells grown in tissue culture that were derived from an animal, especially a human, 3) microorganisms grown in suspension or non-suspension cultures, 4) virus-infected cells, especially EBV infected cells, 5) purified organelles (including, but not restricted to nuclei, mitochondria, membranes, Golgi, endoplasmic reticulum, lysosomes, or peroxisomes) prepared by differential centrifugation or another procedure from animal, especially human, cells, 6) serum or other bodily fluids including, but not limited to, blood, urine, semen, synovial fluid, cerebrospinal fluid, amniotic fluid, lymphatic fluid or interstitial fluid.
  • a total cell extract may not be the optimal source of EBNAl complex polypeptides.
  • Extracts are prepared by methods known to those of skill in the art.
  • the extracts may be prepared at a low temperature (e.g., 4°C) in order to retard denaturation or degradation of proteins in the extract.
  • the pH of the extract may be adjusted to be appropriate for the body fluid or tissue, cellular, or organellar source that is used for the procedure (e.g. pH 7-8 for cytosolic extracts from mammals, but low pH for lysosomal extracts).
  • the concentration of chaotropic or non-chaotropic salts in the extracting solution may be adjusted so as to extract the appropriate sets of proteins for the procedure.
  • Glycerol may be added to the extract, as it aids in maintaining the stability of many proteins and also reduces background non-specific binding.
  • Both the lysis buffer and column buffer may contain protease inhibitors to minimize proteolytic degradation of proteins in the extract and to protect the polypeptide.
  • Appropriate co-factors that could potentially interact with the interacting proteins may be added to the extracting solution.
  • One or more nucleases or another reagent may be added to the extract, if appropriate, to prevent protein-protein interactions that are mediated by nucleic acids.
  • Appropriate detergents or other agents may be added to the solution, if desired, to extract membrane proteins from the cells or tissue.
  • a reducing agent e.g. dithiothreitol or 2-mercaptoethanol or glutathione or other agent
  • Trace metals or a chelating agent may be added, if desired, to the extracting solution.
  • the extract is centrifuged in a centrifuge or ultracentrifuge or filtered to provide a clarified supernatant solution.
  • This supernatant solution may be dialyzed using dialysis tubing, or another kind of device that is standard in the art, against a solution that is similar to, but may not be identical with, the solution that was used to make the extract.
  • the extract is clarified by centrifugation or filtration again immediately prior to its use in affinity chromatography.
  • the crude lysate will contain small molecules that can interfere with the affinity chromatography. This can be remedied by precipitating proteins with ammonium sulfate, centrifugation of the precipitate, and re-suspending the proteins in the affinity column buffer followed by dialysis. An additional centrifugation of the sample may be needed to remove any particulate matter prior to application to the affinity columns.
  • an EBNAl complex polypeptides is expressed, optionally in a heterologous cell, and purified and then mixed with a potential EBNAl complex polypeptide or mixture of polypeptides to identify EBNAl complex formation.
  • the potential EBNAl complex polypeptide may be a single purified or semi-purified protein, or a mixture of proteins, including a mixture of purified or semi-purified proteins, a cell lysate, a clarified cell lysate, a semi-purified cell lysate, etc.
  • EBNAl complex polypeptide or EBNAl complex may be immobilized onto a solid support (e.g., column matrix, microtiter plate, slide, etc.).
  • the ligand may be purified.
  • a fusion protein may be provided which adds a domain that permits the ligand to be bound to a support.
  • the set of proteins engaged in a protein-protein interaction comprises a cell extract, a clarified cell extract, or a reconstituted protein mixture of at least semi-purified proteins.
  • semi-purified it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins.
  • the proteins involved in a protein- protein interaction are present in the mixture to at least about 50% purity relative to all other proteins in the mixture, and more preferably are present in greater, even 90-95%, purity.
  • the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-protein interaction.
  • the present invention contemplates a method for identifying an EBNAl complex or
  • EBNAl complex polypeptide the method comprising: (a) exposing a sample to a solid substrate having coupled thereto an EBNAl complex or EBNAl complex polypeptide under conditions which promote protein-protein interactions; (b) washing the solid substrate so as to remove any polypeptides interacting non-specifically with the EBNAl complex or EBNAl complex polypeptide; (c) eluting the polypeptides which specifically interact with the EBNAl complex or EBNAl complex polypeptide; and (d) identifying the interacting protein.
  • the interacting protein may be identified by a number of methods, including mass spectrometry, gel electrophoresis, activity assay, or protein sequencing.
  • the present invention contemplates a method for identifying a protein capable of interacting with EBNAl , an EBNAl complex polypeptide, or EBNAl complex, or fragments thereof, the method comprising: (a) subjecting a sample to protein- affinity chromatography on multiple columns, the columns having an EBNAl complex or EBNAl complex polypeptide coupled to the column matrix in varying concentrations, and eluting bound components of the extract from the columns; (b) separating the components to isolate a polypeptide capable of interacting with the EBNAl polypeptide, complex or fragment; and (c) analyzing the interacting protein by mass spectrometry to identify the interacting protein.
  • the foregoing method will use polyacrylamide gel electrophoresis to separate and/or analyze the interacting polypeptides.
  • the present invention contemplates a method for identifying an EBNAl complex or EBNAl complex polypeptide the method comprising: (a) subjecting a cellular extract or extracellular fluid to protem-affinity chromatography on multiple columns, the columns having an EBNAl complex or EBNAl complex polypeptide coupled to the column matrix in varying concentrations, and eluting bound components of the extract from the columns; (b) gel-separating the components to isolate an interacting protein; wherein the interacting protein is observed to vary in amount in direct relation to the concentration of coupled polypeptide or fragment; (c) digesting the interacting protein to give corresponding peptides; (d) analyzing the peptides by MALDI-TOF mass spectrometry or post source decay to determine the peptide masses; and (d) performing correlative database searches with the peptide, or peptide fragment
  • the foregoing method may include the further step of including the identifies of any interacting proteins into a relational database.
  • proteins that interact with an EBNAl or EBNAl complex polypeptide may be identified using affinity chromatography. Some examples of such chromatography are described in USSN 09/727,812, filed November 30, 2000, and the PCT Application filed November 30, 2001 and entitled “Methods for Systematic Identification of Protein-Protein Interactions and other Properties", which claims priority to such U.S. application.
  • an EBNAl complex polypeptide may be attached by a variety of means known to those of skill in the art.
  • the polypeptide may be coupled directly (through a covalent linkage) to commercially available pre-activated resins as described in Formosa et al, Methods in Enzymology 1991, 208, 24-45; Sopta et al, J. Biol. Chem. 1985, 260, 10353-60;
  • polypeptide may be tethered to the solid support through high affinity binding interactions.
  • a tag such as GST
  • the fusion tag can be used to anchor the polypeptide to the matrix support, for example Sepharose beads containing immobilized glutathione. Solid supports that take advantage of these tags are commercially available.
  • the amount of cell extract applied to the column may be important for any embodiment. If too little extract is applied to the column and the interacting protein is present at low concentration, the level of interacting protein retained by the column may be difficult to detect. Conversely, if too much extract is applied to the column, protein may precipitate on the column or competition by abundant interacting proteins for the limited amount of protein ligand may result in a difficulty in detecting minor species.
  • the columns functionalized with an EBNAl or EBNAl complex polypeptide are loaded with protein extract from an appropriate source that has been dialyzed against a buffer that is consistent with the nature of the expected interaction.
  • the pH, salt concentrations and the presence or absence of reducing and chelating agents, trace metals, detergents, and co-factors may be adjusted according to the nature of the expected interaction. Most commonly, the pH and the ionic strength are chosen so as to be close to physiological for the source of the extract.
  • the extract is most commonly loaded under gravity onto the columns at a flow rate of about 4-6 column volumes per hour, but this flow rate can be adjusted for particular circumstances in an automated procedure.
  • the volume of the extract that is loaded on the columns can be varied but is most commonly equivalent to about 5 to 10 column volumes.
  • a control column may be included that contains the highest concentration of protein ligand, but buffer rather than extract is loaded onto this column. The elutions (eluates) from this column will contain polypeptide that failed to be attached to the column in a covalent manner, but no proteins that are derived from the extract.
  • the columns may be washed with a buffer appropriate to the nature of the interaction being analyzed, usually, but not necessarily, the same as the loading buffer.
  • An elution buffer with an appropriate pH, glycerol, and the presence or absence of reducing agent, chelating agent, cofactors, and detergents are all important considerations.
  • the columns may be washed with anywhere from about 5 to 20 column volumes of each wash buffer to eliminate unbound proteins from the natural extract.
  • the flow rate of the wash is usually adjusted to about 4 to 6 column volumes per hour by using gravity or an automated procedure, but other flow rates are possible in specific circumstances.
  • the interactions between the extract proteins and the column ligand should be disrupted. This is performed by eluting the column with a solution of salt or detergent. Retention of activity by the eluted proteins may require the presence of glycerol and a buffer of appropriate pH, as well as proper choices of ionic strength and the presence or absence of appropriate reducing agent, chelating agent, trace metals, cofactors, detergents, chaotropic agents, and other reagents.
  • the elution may be performed sequentially, first with buffer of high ionic strength and then with buffer containing a protein denaturant, most commonly, but not restricted to sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride.
  • a protein denaturant most commonly, but not restricted to sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride.
  • the column is eluted with a protein denaturant, particularly SDS, for example as a 1% SDS solution.
  • SDS wash and omitting the salt wash, may result in SDS-gels that have higher resolution (sharper bands with less smearing).
  • SDS wash results in half as many samples to analyze.
  • the volume of the eluting solution may be varied but is normally about 2 to 4 column volumes. For 20 ml columns, the flow rate of the eluting procedures are most commonly about 4 to 6 column volumes per hour, under gravity,
  • EBNAl complexes may be isolated using immunoprecipitation.
  • the cells expressing an EBNAl complex polypeptide are lysed under conditions which maintain protein-protein interactions, and an EBNAl complexes are isolated.
  • Suitable tags for immunoprecipitation experiments include HA, myc, FLAG, HIS, GST, protein A, protein G, etc.
  • Immunoprecipitation from a cell lysate or other protein mixture may be carried out using an antibody specific for an EBNAl complex or EBNAl complex polypeptide or using an antibody which recognizes a tag to which an EBNAl complex polypeptide is fused (e.g., anti-HA, anti-myc, anti-FLAG, etc.).
  • Antibodies specific for a variety of tags are known to the skilled artisan and are commercially available from a number of sources.
  • immunoprecipitation may be carried out using the appropriate affinity resin (e.g., beads functionalized withNi, glutathione, Fc region of IgG, etc.).
  • Test compounds which modulate a protein-protem interaction involving an EBNAl complex polypeptide may be identified by carrying out the immunoprecipitation reaction in the presence and absence of the test agent and comparing the level and/or activity of the EBNAl complex between the two reactions.
  • the proteins from the extract that were bound to and are eluted from the affinity columns or that are immunoprecipitated may be most easily resolved for identification by an electrophoresis procedure, but this procedure may be modified, replaced by another suitable method, or omitted.
  • any of the denaturing or non-denaturing electrophoresis procedures that are standard in the art may be used for this purpose, including SDS-PAGE, gradient gels, capillary electrophoresis, and two-dimensional gels with isoelectric focusing in the first dimension and SDS-PAGE in the second.
  • the individual components in the column eluent are separated by polyacrylamide gel electrophoresis.
  • protein bands or spots may be visualized using any number of methods know to those of skill in the art, including staining techniques such as Coomassie blue or silver staining, or some other agent that is standard in the art.
  • autoradiography can be used for visualizing proteins isolated from organisms cultured on media containing a radioactive label, for example 35 SO 2" or 35 [S]methionine, that is incorporated into the proteins.
  • a radioactive label for example 35 SO 2" or 35 [S]methionine
  • Protein bands that are derived from the extract (i.e. it did not elute from the control column that was not loaded with protein from the extract) and bound to an experimental column that contained polypeptide covalently attached to the solid support, and did not bind to a control column that did not contain any polypeptide, may be excised from the stained electrophoretic gel and further characterized. Eluates from the affinity chromatography columns or irnmunoprecipitates may also be analyzed directly without resolution by electrophoretic methods by proteolytic digestion with a protease in solution, followed by applying the proteolytic digestion products to a reverse phase column and eluting the peptides from the column.
  • the disulf ⁇ de bonds of the protein may be desirable to reduce the disulf ⁇ de bonds of the protein followed by alkylation of the free thiols prior to digestion of the protein with protease.
  • the reduction may be performed by treatment of the gel slice with a reducing agent, for example with dithiothreitol, whereupon, the protein is alkylated by treating the gel slice with a suitable alkylating agent, for example iodoacetamide.
  • the protein Prior to analysis by mass spectrometry, the protein may be chemically or enzymatically digested.
  • the protein sample in the gel slice may be subjected to in-gel digestion. Shevchenko A. et al, Mass Spectrometric Sequencing of Proteins from Silver Stained Polyacrylamide Gels. Analytical Chemistry 1996, 58, 850-858.
  • One method of digestion is by treatment with the enzyme trypsin.
  • the resulting peptides are extracted from the gel slice into a buffer.
  • the peptide fragments may be purified, for example by use of chromatography.
  • a solid support that differentially binds the peptides and not the other compounds derived from the gel slice, the protease reaction or the peptide extract may be used.
  • the peptides may be eluted from the solid support into a small volume of a solution that is compatible with mass spectrometry (e.g. 50% acetonitrile/0.1% trifluoroacetic acid).
  • mass spectrometry e.g. 50% acetonitrile/0.1% trifluoroacetic acid.
  • the preparation of a protein sample from a gel slice that is suitable for mass spectrometry may also be done by an automated procedure.
  • mass spectrometers may be used within the present invention. Representative examples include: triple quadrupole mass spectrometers, magnetic sector instruments (magnetic tandem mass spectrometer, JEOL, Peabody, Mass), ionspray mass spectrometers (Bruins et al, Anal Chem. 59:2642-2647, 1987), electrospray mass spectrometers (including tandem, nano- and nano-electrospray tandem) (Fenn et al, Science 246:64-71, 1989), laser desorption time-of-flight mass spectrometers (Karas and Hillenkamp, Anal. Chem.
  • MALDI ionization is a technique in which samples of interest, in this case peptides and proteins, are co-crystallized with an acidified matrix.
  • the matrix is typically a small molecule that absorbs at a specific wavelength, generally in the ultraviolet (UV) range, and dissipates the absorbed energy thermally.
  • UV ultraviolet
  • a pulsed laser beam is used to transfer energy rapidly (i.e., a few ns) to the matrix.
  • MALDI is considered a "soft- ionization" method that typically results in singly-charged species in the gas phase, most often resulting from a protonation reaction with the matrix.
  • MALDI may be coupled in-line with time of flight (TOF) mass spectrometers. TOF detectors are based on the principle that an analyte moves with a velocity proportional to its mass. Analytes of higher mass move slower than analytes of lower mass and thus reach the detector later than lighter analytes.
  • the present invention contemplates a composition comprising an EBNAl complex polypeptide and a matrix suitable for mass spectrometry.
  • the matrix is a nicotinic acid derivative or a cinnamic acid derivative.
  • MALDI-TOF MS is easily performed with modern mass spectrometers.
  • samples of interest in this case peptides or proteins, are mixed with a matrix and spotted onto a polished stainless steel plate (MALDI plate).
  • MALDI plate polished stainless steel plate
  • MALDI plates can presently hold up to 1536 samples per plate. Once spotted with sample, the MALDI sample plate is then introduced into the vacuum chamber of a MALDI mass spectrometer. The pulsed laser is then activated and the mass to charge ratios of the analytes are measured utilizing a time of flight detector. A mass spectrum representing the mass to charge ratios of the peptides/proteins is generated.
  • MALDI can be utilized to measure the mass to charge ratios of both proteins and peptides.
  • proteins a mixture of intact protein and matrix are co-crystallized on a MALDI target (Karas, M. and Hillenkamp, F. Anal. Chem. 1988, 60 (20) 2299-2301).
  • the spectrum resulting from this analysis is employed to determine the molecular weight of a whole protein. This molecular weight can then be compared to the theoretical weight of the protein and utilized in characterizing the analyte of interest, such as whether or not the protein has undergone post-translational modifications (e.g., example phosphorylation).
  • MALDI mass spectrometry is used for determination of peptide maps of digested proteins.
  • the peptide masses are measured accurately using a
  • the ensemble of the peptide masses observed in a protein digest may be used to search protein/DNA databases in a method called peptide mass fingerprinting.
  • protein entries in a database are ranked according to the number of experimental peptide masses that match the predicted trypsin digestion pattern.
  • Commercially available software utilizes a search algorithm that provides a scoring scheme based on the size of the databases, the number of matching peptides, and the different peptides. Depending on the number of peptides observed, the accuracy of the measurement, and the size of the genome of the particular species, unambiguous protein identification may be obtained.
  • Statistical analysis may be performed upon each protein match to determine the validity of the match.
  • Typical constraints include error tolerances within 0.1 Da for monoisotopic peptide masses, cysteines may be alkylated and searched as carboxyamidomethyl modifications, 0 or 1 missed enzyme cleavages, and no methionine oxidations allowed.
  • Identified proteins may be stored automatically in a relational database with software links to SDS-PAGE images and ligand sequences. Often even a partial peptide map is specific enough for identification of the protein. If no protein match is found, a more error-tolerant search can be used, for example using fewer peptides or allowing a larger margin error with respect to mass accuracy.
  • mass spectroscopy methods such as tandem mass spectrometry or post source decay may be used to obtain sequence information about proteins that cannot be identified by peptide mass mapping,or to confirm the identity of proteins that are tentatively identified by an error-tolerant peptide mass search. (Griffin et al, Rapid Commun. Mass. Spectrom. 1995, 9, 1546-51).
  • EBNAl complex polypeptide and a binding partner may be detected by a variety of methods. Modulation of the formation of EBNAl complexes may be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled polypeptides or binding partners, by immunoassay, or by chromatographic detection. Methods of isolating and identifying EBNAl complexes described in Section 4 may be incorporated into the detection methods. Typically, it will be desirable to immobilize an EBNAl complex polypeptide or its binding partner to facilitate separation of EBNAl complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Binding of an EBNAl complex polypeptide to a binding partner may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein may be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S- transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the binding partner, e.g.
  • the test compound and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated.
  • the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of EBNAl complex polypeptide or binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • EBNAl complex polypeptide or its binding partner may be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated polypeptide molecules may be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the binding partner, or which are reactive with the EBNAl complex polypeptide and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.
  • the enzyme may be chemically conjugated or provided as a fusion protein with the binding partner.
  • the binding partner may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of EBNAl complex polypeptide trapped in the EBNAl complex may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol.
  • a fusion protein comprising the EBNAl complex polypeptide and glutathione-S-transferase may be provided, and EBNAl complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
  • antibodies against the EBNAl complex polypeptide such as anti-EBNAl, USP7, PRMT5, CK2, NAPl, and/or TAF I ⁇ antibodies, may be used.
  • the EBNAl complex polypeptide to be detected in the EBNAl complex may be "epitope-tagged" in the form of a fusion protein that includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety.
  • myc-epitopes e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157
  • pFLAG system International Biotechnologies, Inc.
  • pEZZ-protein A system Pharmacia, NJ
  • the protein or the set of proteins engaged in a protein-protein, protein-substrate, or protein-nucleic acid interaction comprises a reconstituted protein mixture of at least semi-purified proteins.
  • semi- purified it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins.
  • the proteins involved in a protein-substrate, protein-protein or nucleic acid-protein interaction are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity.
  • the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure activity resulting from the given protein-substrate, protein-protein interaction, or nucleic acid-protein interaction.
  • the use of reconstituted protein mixtures allows more careful control of the protein-substrate, protein-protein, or nucleic acid-protein interaction conditions.
  • the system may be derived to favor discovery of modulators of particular intermediate states of the protein-protein interaction.
  • a reconstituted protein assay may be carried out both in the presence and absence of a candidate agent, thereby allowing detection of a modulator of a given protein-substrate, protein-protein, or nucleic acid-protein interaction.
  • Assaying biological activity resulting from a given protein-substrate, protein-protein or nucleic acid-protein interaction, in the presence and absence of a candidate modulator may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. Typically, it will be desirable to immobilize one of the EBNAl complex polypeptides to facilitate separation of EBNAl complexes from uncomplexed forms of one of the proteins, as well as to accommodate automation of the assay.
  • a fusion protein may be provided which adds a domain that permits the protein to be bound to an insmuble matrix.
  • protein-protein interaction component fusion proteins n be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with a potential interacting protein, e.g. an 35 S-labeled polypeptide, and the test compound and incubated un ⁇ conditions conducive to complex formation .
  • a potential interacting protein e.g. an 35 S-labeled polypeptide
  • the test compound and incubated un ⁇ conditions conducive to complex formation .
  • the beads are was ⁇ d to remove any unbound interacting protein, and the matrix bead-bound radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are dissociated, e.g. when microtitre plate is used.
  • the complexes may be dissociated from the matrix, separated by SDS-PAGE gel, and the level of interacting polypeptide found in the matrix-bound fraction
  • an EBNAl complex polypeptide may be used to generate an two-hybrid or interaction trap assay (see also, U.S. Patent NO: 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene
  • a first hybrid gene comprises the coding sequence for a DNA- binding domain of a transcriptional activator may be fused in frame to the coding sequence for a "bait" protein, e.g., an EBNAl complex polypeptide of sufficient length to bind to a potential interacting protein.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to a gene encoding a "fish" protein, e.g., a potential interacting protein of sufficient length to interact with the protein-protein interaction component polypeptide portion of the bait fusion protein.
  • bait and fish proteins are able to interact, e.g., form a protein-protein interaction component complex, they bring into close proximity the two domains of the transcriptional activator. This proximity causes transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene may be detected and used to score for the interaction of the bait and fish proteins.
  • the host cell also contains a first chimeric gene which is capable of being expressed in the host cell.
  • the gene encodes a chimeric protein, which comprises (a) a DNA-binding domain that recognizes the responsive element on the reporter gene in the host cell, and (b) a bait protein (e.g., an EBNAl complex polypeptide).
  • a second chimeric gene is also provided which is capable of being expressed in the host cell, and encodes the "fish" fusion protein.
  • both the first and the second chimeric genes are introduced into the host cell in the form of plasmids.
  • the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid.
  • the DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein may be derived from transcriptional activators having separable DNA-binding and transcriptional activation domains.
  • transcriptional activators having separable DNA-binding and transcriptional activation domains.
  • these separate DNA-binding and transcriptional activation domains are known to be found in the yeast GAL4 protein, and are known to be found in the yeast GCN4 and ADR1 proteins.
  • Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention, and include, for example, the LexA and VP16 proteins.
  • DNA-binding domains may be used in the subject constructs; such as domains of ACE1, ⁇ cl, lac repressor, jun or fos.
  • the DNA-binding domain and the transcriptional activation domain may be from different proteins.
  • LexA DNA binding domain provides certain advantages. For example, in yeast, the LexA moiety contains no activation function and has no known affect on transcription of yeast genes. In addition, use of LexA allows control over the sensitivity of the assay to the level of interaction (see, for example, the Brent et al. PCT publication WO94/10300).
  • any enzymatic activity associated with the bait or fish proteins is inactivated, e.g., dominant negative or other mutants of a protein-protein interaction component can be used.
  • an EBNAl complex polypeptide of the EBNAl complex if any, between the bait and fish fusion proteins in the host cell, causes the activation domain to activate transcription of the reporter gene.
  • the method is carried out by introducing the first chimeric gene and the second chimeric gene into the host cell, and subjecting that cell to conditions under which the bait and fish fusion proteins and are expressed in sufficient quantity for the reporter gene to be activated.
  • the formation of an EBNAl complex containing a EBNAl complex polypeptide results in a detectable signal produced by the expression of the reporter gene.
  • the EBNAl complex of interest is generated in whole cells, taking advantage of cell culture techniques to support the subject assay.
  • the EBNAl complex of can be constituted in a prokaryotic or eukaryotic cell culture system.
  • Advantages to generating the EBNAl complex in an intact cell includes the ability to screen for modulators of the level or activity of the EBNAl complex which are functional in an environment more closely approximating that which therapeutic use of the modulator would require, including the ability of the agent to gain entry into the cell.
  • certain of the in vivo embodiments of the assay are amenable to high throughput analysis of candidate agents.
  • the EBNAl complexes and EBNAl complex polypeptides can be endogenous to the cell selected to support the assay.
  • some or all of the components can be derived from exogenous sources.
  • fusion proteins can be introduced into the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the fusion protein itself or mRNA encoding the fusion protein.
  • the reporter gene construct can provide, upon expression, a selectable marker.
  • Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of the protein-protein interaction.
  • the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable.
  • specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain, western blots or an intrinsic activity.
  • the product of the reporter gene is detected by an intrinsic activity associated with that product.
  • the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence. 6. Characterization of Complexes
  • the present invention provides methods for characterizing the subject EBNAl complexes. Such methods may comprise determining the three- dimensional structure of an EBNAl complex, or by mapping the interactions between an EBNAl complex polypeptide and a binding partner, e.g. as by mass spectrometric analysis of mteracting regions of the polypeptides, or by mutagenic analysis, as described in Section 3, to determine which residues of the polypeptides interact.
  • NMR may be used to determine structure information of an EBNAl complex, an interaction site of EBNAl, or an EBNAl or other EBNAl complex polypeptide interacting site on an EBNAl complex polypeptide prepared in accordance with the compositions and methods described herein.
  • NMR may be used, for example, to determine the three dimensional structure, the conformational state, the aggregation level, the state of protein folding/unfolding or the dynamic properties of a polypeptide. Changes in these properties due to interaction with other molecules can also be monitored using NMR.
  • Polypeptides in aqueous solution usually populate an ensemble of 3 -dimensional (3D) structures which can be determined by NMR.
  • the 2-dimensional 1H- V5 N HSQC (Heteronuclear Single Quantum Correlation) spectrum provides a diagnostic fingerprint of conformational state, aggregation level, state of protein folding, and dynamic properties of a polypeptide (Yee et al, PNAS 99, 1825-30 (2002)).
  • the ensemble of solution structures is one of very closely related conformations. In this case one peak is expected for each non-proline residue with a dispersion of resonance frequencies with roughly equal intensity. Additional pairs of peaks from side-chain NH 2 groups are also often observed, and correspond to approximately the number of Gin and Asn residues in the protein.
  • This type of HSQC spectra usually indicates that the protein is amenable to structure determination by NMR methods.
  • x-ray crystallography may be used to determine structure information of an EBNAl complex, an interaction site of EBNAl, or an EBNAl or other EBNAl complex polypeptide interacting site on an EBNAl complex polypeptide prepared in accordance with the compositions and methods described herein.
  • x-ray diffraction of a crystallized form of a polypeptide can be used, for example, to determine the three dimensional structure of a polypeptide or to determine the space group of the crystal of the polypeptide.
  • X-ray crystallography techniques generally require that the protein molecules be available in the form of a crystal.
  • Crystals may be grown from a solution containing a purified polypeptide, or a fragment thereof (e.g., a stable domain), by a variety of conventional processes. These processes include, for example, batch, liquid, bridge, dialysis, vapour diffusion (e.g., hanging drop or sitting drop methods). See for example, McPherson, 1982 John Wiley, New York; McPherson, 1990, Eur. J. Biochem.
  • native crystals of a polypeptide may be grown by adding precipitants to a concentrated solution of the polypeptide.
  • the precipitants are added at a concentration just below that necessary to precipitate the protein.
  • Water may be removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
  • the formation of crystals is dependent on a number of different parameters, including pH, temperature, protein concentration, the nature of the solvent and precipitant, as well as the presence of added ions or ligands to the protein.
  • sequence of the polypeptide being crystallized will have a significant affect on the success of obtaining crystals.
  • Crystallization robots may automate and speed up the work of reproducibly setting up large number of crystallization experiments. Once some suitable set of conditions for growing a crystal are found, variations of the conditions may be systematically screened in order to find the set of conditions which allows the growth of sufficiently large, single, well ordered crystals. In certain instances, a polypeptide may be co-crystallized with a compound that stabilizes the polypeptide.
  • a polypeptide may be co-crystallized with another molecule in order to provide a crystal suitable for determining the structure of the complex.
  • a crystal of the EBNAl complex or EBNAl complex polypeptide may be soaked in a solution containing the other molecule in order to form co-crystals by diffusion of the other molecule into the crystal of the polypeptide.
  • the structure of the EBNAl complex or EBNAl complex polypeptide obtained in the presence and absence of another molecule may be compared to determine structural information about the complex or polypeptide and aid in identification of draggable regions.
  • x-ray beams may be produced by synchrotron rings where electrons (or positrons) are accelerated through an electromagnetic field while traveling at close to the speed of light. Because the admitted wavelength may also be controlled, synchrotrons may be used as a tunable x-ray source (Hendrickson WA., Trends Biochem Sci 2000 Dec; 25(12):637-43). For less conventional Laue diffraction studies, polychromatic x-rays covering a broad wavelength window may be used to observe many diffraction intensities simultaneously (Stoddard, B. L., Curr. Opin. Struct Biol 1998 Oct; 8(5):612-8). Neutrons may also be used for solving protein crystal structures (Gutberlet T, Heinemann U & Steiner M., Acta Crystallogr D 2001 ;57: 349-54).
  • mass spectrometry may be used to determine structure information of an EBNAl complex, an interaction site of EBNAl, or an EBNAl or other EBNAl complex polypeptide interacting site on an EBNAl complex polypeptide prepared in accordance with the compositions and methods described herein.
  • mass spectrometry can be used, for example, to determine the amino acid sequence, to obtain a peptide map, to identify post-translational modifications (e.g., phosphorylation, etc.) of a polypeptide, or to identifying regions of the polypeptide that interact with other molecules, including other polypeptides, nucleic acids and small molecules.
  • an EBNAl complex or EBNAl complex polypeptide may be subjected to limited proteolysis prior to analysis by mass spectrometry.
  • Limited proteolysis of a polypeptide may be used to identify and/or isolate stable domains of a protein that are suitable for structural characterization using NMR analysis or x-ray crystallography.
  • Limited proteolysis of a polypeptide may be performed by incubating a protein with at least one concentration of a proteolytic enzyme for an amount of time suitable to produce proteolytic cleavage of the protein of interest.
  • digestion of the polypeptide may be carried out by incubation with two or more proteolytic enzymes, at two or more concentrations of enzyme, and/or for varying amounts of time.
  • Such reactions may be carried out in solution or by exposing the polypeptide to an immobilized proteolytic enzyme to facilitate isolation of the polypeptide fragments from the digestion mixture.
  • the digestion products may be analyzed and/or isolated using electrophoretic or chromatographic techniques. Proteolytically stable fragments resulting from the enzymatic digestion may be identified based on the mass of the peptide as determined by mass spectrometry.
  • Modulators of EBNAl complexes and other structurally related molecules, and EBNAl complex polypeptides may be identified and developed as set forth below and otherwise using techniques and methods known to those of skill in the art.
  • the modulators of the invention may be employed, for instance, to inhibit and treat EBV-mediated diseases or disorders.
  • the modulators of the invention may also serve as modulators of EBV- mediated diseases or disorders via action on an EBNAl complex complex.
  • the modulators of the invention may elicit a change in any of the activities selected from the group consisting of (a) a change in the level of an EBNAl complex, (b) a change in the activity of an EBNAl complex, (c) a change in the stability of an EBNAl complex, (d) a change in the conformation of an EBNAl complex, (e) a change in the activity of at least one polypeptide comprising an EBNAl complex, (f) a change in the conformation of at least one polypeptide comprising an EBNAl complex, (g) where the reaction mixture is a whole cell, a change in the intracellular localization of an EBNAl complex or an EBNAl complex polypeptide thereof, (h) where the reaction mixture is a whole cell, a change the transcription level of a gene dependent on an EBNAl complex, and (i) where the reaction mixture is a whole cell, a change in second messenger levels in the cell.
  • an EBNAl complex or EBNAl complex polypeptide is contacted with a test compound, and the activity of the EBNAl complex or EBNAl complex polypeptide in the presence of the test compound is determined, wherein a change in the activity of the EBNAl complex or EBNAl complex polypeptide is indicative that the test compound modulates the activity of EBNAl complex or EBNAl complex polypeptide.
  • Compounds to be tested for their ability to act as modulators of EBNAl complexes or EBNAl complex polypeptides can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly.
  • Compounds for use with the above- described methods may be selected from the group of compounds consisting of lipids, carbohydrates, polypeptides, peptidomimetics, peptide-nucleic acids (PNAs), small molecules, natural products, aptamers and polynucleotides.
  • the compound is a polynucleotide.
  • said polynucleotide is an antisense nucleic acid. In other embodiments, said polynucleotide is an siRNA.
  • the compound comprises an EBNAl complex polypeptide or polynucleotide encoding an EBNAl complex polypeptide as described above in Section 3. In certain embodiments, the compound may be a member of a library of compounds.
  • Assay formats for EBNAl complex formation or enzymatic activity of an EBNAl complex complex or EBNAl complex polypeptides can be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Simple binding assays can also be used to detect agents which, by disrupting the formation of EBNAl complexes, or the binding of an EBNAl complex or EBNAl complex polypeptide to a substrate, can serve as a modulator.
  • an assay for a modulator of an EBNAl complex polypeptide is a competitive assay that combines an EBNAl complex polypeptide and a potential modulator with EBNAl complex polypeptides, recombinant molecules that comprise an EBNAl complex, EBNAl complex, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
  • EBNAl complex polypeptides can be labeled, such as by radioactivity or a colorimetric compound, such that the number of molecules of an EBNAl complex polypeptide bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential modulator.
  • Assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. Assays may also employ any of the methods for isolating, preparing and detecting EBNAl complexes as described above in Sections 4 and 5. In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time.
  • Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target.
  • potential modifiers e.g., modulators of EBNAl complexes may be detected in a cell-free assay generated by constitution of a functional EBNAl complex in a cell lysate.
  • the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.
  • methods for identifying a compound that modulates a virus-associated disease or disorder comprising: (i) contacting an EBNAl complex with a test compound; and (ii) assessing the extent of said EBV-mediated disease or disorder, wherein a modulation in the extent of said EBV-mediated disease or disorder in the presence of said test compound indicates that the test compound may be a candidate therapeutic for said EBV-mediated disease or disorder.
  • EBV-mediated cancer could be evaluated by medical diagnostic techniques known to one of skill in the art, such as, for example, biopsy, anti-EBV early antigen serum titer, serum lactate dehydrogenase levels, immunophenotyping, and the like.
  • an EBNAl complex or an EBNAl complex polypeptide may be identified and/or assayed using a variety of methods well known to the skilled artisan.
  • information about the activity of non-essential genes may be assayed by creating a null mutant strain of bacteria expressing a mutant form of, or lacking expression of, a protein of interest.
  • the resulting phenotype of the null mutant strain may provide information about the activity of the mutated gene product.
  • Essential genes may be studied by creating a bacterial strain with a conditional mutation in the gene of interest.
  • the bacterial strain may be grown under permissive and non-permissive conditions and the change in phenotype under the non-permissive conditions may be used to identify and/or assay the activity of the gene product.
  • the activity of an EBNAl complex or EBNAl complex polypeptide may be assayed using an appropriate substrate or binding partner or other reagent suitable to test for the suspected activity.
  • the assay is typically designed so that the enzymatic reaction produces a detectable signal.
  • mixture of a kinase with a substrate in the presence of 32 P will result in incorporation of the 32 P into the substrate.
  • the labeled substrate may then be separated from the free 32 P and the presence and/or amount of radiolabeled substrate may be detected using a scintillation counter or a phosphorimager.
  • Similar assays may be designed to identify and/or assay the activity of a wide variety of enzymatic activities. Based on the teachings herein, the skilled artisan would readily be able to develop an appropriate assay for an EBNAl complex or EBNAl complex polypeptide.
  • the activity of an EBNAl complex or EBNAl complex polypeptide may be determined by assaying for the level of expression of RNA and or protein molecules. Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product. Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g., fluorescence, luminescence, enzymatic activity, etc.). Depending on the particular situation, it may be desirable to detect the level of transcription and/or translation of a single gene or of multiple genes.
  • Transcription levels may be determined, for example, using Northern blots, hybridization to an oligonucleotide array or by assaying for the level of a resulting protein product.
  • Translation levels may be determined, for example, using Western blotting or by identifying a detectable signal produced by a protein product (e.g.,
  • the biological activity of an EBNAl complex or EBNAl complex polypeptide can be assessed by monitoring changes in the phenotype of the targeted cell.
  • the detection means can include a reporter gene construct which includes a transcriptional regulatory element that is dependent in some form on the level of an EBNAl complex or EBNAl complex polypeptide.
  • the EBNAl complex can be provided as a fusion protein with a domain that binds to a DNA element of the reporter gene construct.
  • the added domain of the fusion protein can be one which, through its DNA-binding ability, increases or decreases transcription of the reporter gene. Which ever the case may be, its presence in the fusion protein renders it responsive to an EBNAl complex or EBNAl complex polypeptide. Accordingly, the level of expression of the reporter gene will vary with the level of expression of an EBNAl complex or EBNAl complex polypeptide.
  • the reporter gene construct can provide, upon expression, a selectable marker.
  • a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable.
  • the reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.
  • the product of the reporter gene can be an enzyme which confers resistance to antibiotic or other drug, or an enzyme which complements a deficiency in the host cell (i.e. thymidine kinase or dihydrofolate reductase).
  • aminoglycoside phosphotransferase encoded by the bacterial transposon gene Tn5 neo can be placed under transcriptional control of a promoter element responsive to the level of an EBNAl complex or EBNAl complex polypeptide present in the cell.
  • Such embodiments of the subject assay are particularly amenable to high through-put analysis in that proliferation of the cell can provide a simple measure of inhibition of the EBNAl complex or EBNAl complex polypeptide. 7.a. Identification of Modulators of Kinase Activity
  • the methods of the present invention may comprise an assay for identifying modulators of protein kinase activity. Such methods may be useful in evaluating compounds for their ability to modulate an EBNAl complex comprising kinase activity, for example an EBNAl complex comprising CK2.
  • Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP), to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively.
  • ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate.
  • the inorganic phosphate is then attached to the targeted protein.
  • the protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
  • In vitro assays for evaluating the efficacy of a test molecule to inhibit the activity of a kinase may be carried out using a purified kinase polypeptide or polypeptide complex.
  • the purified kinase may be obtained by recombinant production of full length molecules, or biologically active variants or derivatives thereof. Methods for production of recombinant polypeptides are described above.
  • Host cells for recombinant production of kinase polypeptides include, without limitation, bacteria, such as E. coli, yeast, insect cells (using, for example, the baculovirus system), mammalian cells, or other eukaryotic cells.
  • the polypeptide members of a multi-subunit kinase complex may be co-produced in the same host cell, where the host cell is co-transfected with DNA encoding each polypeptide.
  • the kinase polypeptide or polypeptides complex can be purified from the host cell (or culture medium if it is secreted); typically, this will be accomplished by expressing each polypeptide with a "tag" sequence such as hemaglutinin ("HA"), His (polyhistidine such as hexahistidine), myc or FLAG, and purifying the tagged polypeptide via affinity chromatography using, for example, a nickel column for polyhistidine, or a mono- or polyclonal antibody for myc or FLAG.
  • Post-translational modifications which may be necessary for kinase activity may occur naturally in the host cell during production, or may be carried out in vitro using purified components.
  • Cdk2/cyclin A co-expression of Cdk2/cyclin A in insect cells using a baculovirus expression system will result in isolation of an active kinase complex (i.e., containing the activating threonine phosphorylation) from the cells.
  • active kinase complex i.e., containing the activating threonine phosphorylation
  • separately expressed Cdk2 and cyclin A polypeptides may be mixed and incubated with the Cdk activating kinase, or 'Cak', in the presence of ATP, to produce an activated Cdk2/cyclin A pair.
  • Kinase substrates useful in the assays of the invention may be proteins, protein fragments or peptides (Kemp, Design and Use of Peptide Substrates for Protein Kinases. Methods in Enzymology. 200: 121-134, (1991)).
  • Substrates for many protein kinases are commercially available, for example Histone HI is a commonly used substrate for serine/threonine protein kinases, and many oncogenes have been shown to be phosphorylated on tyrosine residues.
  • a peptide library wherein each peptide contains at least one serine, threonine and/or tyrosine residue may be used as a substrate for a protein kinase of unknown specificity in order to identify a substrate polypeptide which may be used in a kinase assay (Songyang, Z. et al., Curr. Biol. 4: 973-982 (1994) and Songyang & Cantley, Methods Mol. Biol. 87: 87-98 (1998)).
  • a mixed library of peptides may be subjected to phosphorylation by a protein kinase in the presence of ATP.
  • the phosphorylated peptides are then separated from the rest of the library and subjected to sequence analysis. Individual phosphorylated peptides may be used as the substrate for future kinase assays, or a consensus substrate sequence for that kinase may be determined based on analysis of all peptides from the library which were capable of acting as a substrate for that kinase. Typically, methods of measuring protein kinase activity are based on the radioactive detection method. In these methods, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of ⁇ - 32 P-ATP or ⁇ - 32 P-GTP. Often, a general and inexpensive substrate such as histone or casein is used.
  • the reaction is stopped and the phosphorylated substrate is separated from free phosphate using gel electrophoresis or by binding the substrate to a filter and washing to remove excess radioactively-labeled free ATP.
  • the amount of radio-labeled phosphate incorporated into the substrate may measured by scintillation counting or by phosphorimager analysis.
  • phosphorylation of a substrate may be detected by immunofluorescence using antibodies specific for a phosphoserine, phosphothreonine or phosphotyrosine residue (e.g., anti-phosphoserine, Sigma #P3430; anti-phosphothreonine, Sigma #P3555; and anti-phosphotyrosine, Sigma #P3300).
  • an assay for determining an agent which is an modulator of kinase activity is carried out in solution.
  • An active kinase is mixed with Gamma-labeled ATP (such as 32 P-ATP), a substrate (such as histone HI, casein, etc.), and the test molecule(s), which may be added to the solution either simultaneously or successively. After a period of incubation, the substrate can be isolated and assayed for the amount of label it contains.
  • Gamma-labeled ATP such as 32 P-ATP
  • a substrate such as histone HI, casein, etc.
  • biotinylated substrate histone HI or retinoblastoma peptide, for example
  • streptavidin is attached to non-porous beads coated with streptavidin and filled with scintillation fluid.
  • the beads can be incubated with active kinase, gamma-labeled ATP, and the test molecule(s) using microtiter plates (such as 96 well plates or 384 well plates).
  • the substrate can be attached to wells of a microtiter plate, and active holoenzyme complex, gamma-labeled ATP (or other suitable detection agent), and the test molecule(s) can be added sequentially or simultaneously. After a short incubation (on the order of seconds to minutes), the solution can be removed from each well and the plates can be washed and then measured for the amount of labeled gamma phosphate added to the substrate by the activity of the kinase.
  • the substrate can be attached to beads as an alternative to attaching it to the bottom of each well; the beads can then be removed from solution after incubation with the test molecule, labeled ATP, and active holoenzyme complex, and the amount of label incorporated in to the substrate can then be measured.
  • test molecule will be evaluated over a range of concentrations, and a series of suitable controls can be used for accuracy in evaluating the results. In some cases, it may be useful to evaluate two or more test molecules together to assay for the possibility of "synergistic" effects.
  • the ability of a test agent to inhibit the ability of a kinase to phosphorylate a substrate can be accomplished by measuring the activity of the substrate molecule. For example, if the substrate molecule is activated upon phosphorylation by the kinase, the activity of the substrate molecule can be assayed as a means for determining the activity of the kinase. A decrease in the level of substrate activation upon incubation of the kinase with a test molecule would be indicative of a kinase inhibitor.
  • the assay may be carried out by detecting induction of a cellular second messenger of the substrate (e.g., intracellular Ca , diacylglycerol, IP 3 , etc.), detecting a catalytic/enzymatic activity of the substrate, detecting the induction of a reporter gene (comprising a substrate- responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a target-regulated cellular response.
  • a cellular second messenger of the substrate e.g., intracellular Ca , diacylglycerol, IP 3 , etc.
  • detecting a catalytic/enzymatic activity of the substrate detecting the induction of a reporter gene (comprising a substrate- responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase
  • the methods of the present invention may comprise an assay for identifying modulators of methyltransferase activity. Such methods may be useful in evaluating compounds for their ability to modulate an EBNAl complex comprising methyltransferase activity, for example an EBNAl complex comprising PRMT5.
  • Methyltransferase activity may be determined by measuring the transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate. For example, the assay may be carried out by mixing [ 3 H]AdoMet (NEN catalog No. Netl 1 ), the donor substrate, with an acceptor substrate and the methyltransferase.
  • Methyltransferase acceptor substrates useful in the assays of the invention include nucleic acids, such as oligonucleotides of RNA or DNA, particularly those that contain at least one cytosine (C) nucleotide (Smith, SS, et al, Proc. Natl. Acad. Sci. USA 89: 4744-4748 (1992)).
  • nucleic acids such as oligonucleotides of RNA or DNA, particularly those that contain at least one cytosine (C) nucleotide (Smith, SS, et al, Proc. Natl. Acad. Sci. USA 89: 4744-4748 (1992)).
  • a test molecule may added to the methyltransferase assay before, or concurrently with, the addition of the acceptor substrate.
  • the level of methyltransferase activity is determined and compared to the level of activity in the absence of the test molecule. A decrease in the amount of [ Hjmethyl incorporated into the acceptor substrate indicates that the test molecule has methyltransferase inhibitory activity.
  • USP7 (HAUSP 1) is involved in the ubiquitin-mediated proteolysis system which is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells.
  • Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair.
  • One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins.
  • This process is catalyzed by a ubiquitin-activating enzyme (El) and a ubiquitin-conjugatmg enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s).
  • E2 ubiquitin-activating enzyme
  • E2 ubiquitin-conjugatmg enzyme
  • E3s auxiliary substrate recognition proteins
  • the application provides assays that can be used to screen for drags that modulate USP7 activity.
  • the drag screening assay will identify compounds that modulate the ability of USP7 to deubiquitinate p53.
  • ubiquitinated p53 p53-Ub
  • the assay indicates that the test compound modulates USP7 activity.
  • the p53-Ub substrate may be labeled to facilitate detection of the substrate.
  • either or both of the p53 and Ub moieties may be labeled with a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • either or both of the p53 and Ub moieties may be tagged to facilitate isolation of the polypeptides or polypeptide complex from a reaction mixture.
  • Suitable polypeptides include, for example, a poly His or glutathione S-transferase tag.
  • Indirect measurement of ubiquitination of the p53 polypeptide can also be accomplished by detecting a biological activity associated with the p53 polypeptide that is either attenuated by ubiquitin-conjugation or destroyed along with the p53 polypeptide by a ubiquitin- dependent proteolytic processes.
  • the USP7 assays may be carried out using a reconstituted protein mixture, a cell lysate and/or a whole cell. 8. Therapeutics
  • the methods and compositions described herein may be used for the treatment or prevention of diseases or disorders associated with a variety of viral infections.
  • the methods and compositions described herein may be used to treat or prevent viral infections (or diseases or disorders associated therewith) in any type of organism that is subject to infection by a virus, including, for example, humans, animals (e.g., mammals, birds, rodents, amphibians, etc.), and plants. Accordingly, the methods and compositions of the invention have utility in wide ranging fields such as, for example, agriculture, livestock, crops, medical treatments, combating bio-terrorism, etc.
  • Examples of disease causing viruses that may be treated in accord with the compositions and methods described herein include: Papovaviridae (papilloma viruses, polyoma viruses); Herpesviridae (herpes simplex virus (HSN) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses'); Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAN or HTLN- III/LAN, See Rattier, L. et al, Nature, Nol. 313, Pp. 227-284 (1985); Wain Hobson, S. et al, Cell, Nol. 40: Pp.
  • Papovaviridae papilloma viruses, polyoma viruses
  • Herpesviridae herpes simplex virus (HSN) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses'
  • HIN-2 See Guyader et al, Nature, Nol. 328, Pp. 662-669 (1987); European Patent Publication No. 0 269 520; Chakraborti et al, Nature, Vol. 328, Pp. 543-547 (1987); and European Patent Application No. 0 655 501
  • other isolates such as HIV-LP (International Publication No. WO 94/00562 entitled "A Novel Human Immunodeficiency Virus”); Picornaviridae (e.g., polio viruses, hepatitis A virus, (Gust, LD., et al, Intervirology, Vol. 20, Pp.
  • entero viruses human coxsackie viruses, rhinoviruses, echoviruses
  • Calciviridae e.g., strains that cause gastroenteritis
  • Togaviridae e.g., equine encephalitis viruses, rubella viruses
  • Flaviridae e.g., dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronaviridae e.g., coronaviruses
  • Rhabdoviridae e.g., vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g., ebola viruses
  • Paramyxoviridae e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g., influenza viruses
  • Bungaviridae e.g., Hantaan viruses, bunga viruses, phleboviruse
  • Genomic information for over 900 viral species is available from TIGR and/or NCBI, including, for example, information about deltaviruses, retroid viruses, satellites, dsDNA viruses, dsRNA viruses, ssDNA viruses, ssRNA negative-strand viruses, ssRNA positive-strand viruses, unclassified bacteriophages, and other unclassified viruses.
  • the methods and compositions described herein may be used for combating viral based biological warfare agents.
  • viral based biological warfare agents include, for example, filovirases (e.g., ebola or Marburg), arenaviruses (e.g., Lassa and Machupo), hantaviras, smallpox (variola major), hemorrhagic fever virus, Nipah virus, and alphaviruses (e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis).
  • filovirases e.g., ebola or Marburg
  • arenaviruses e.g., Lassa and Machupo
  • hantaviras e.g., smallpox (variola major)
  • hemorrhagic fever virus e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis.
  • the methods and compositions described herein may be used for promoting food freshness and or combating or preventing food contamination.
  • viral contaminants that may lead to foodborne illnesses include, for example, hepatitis A, norwalk-like viruses, rotaviras, astroviruses, calciviruses, adenovirases, and parvoviruses.
  • it may be desirable to administer or formulate the compositions of the invention in conjunction with other therapeutic agents.
  • Exemplary therapeutic agents include, for example, anti-inflammatory agents, immunosuppressive agents, and/or anti-infective agents (such as for example, antibiotic, antiviral, and/or antifungal compounds, etc.).
  • anti-inflammatory drags include, for example, steroidal (such as, for example, cortisol, aldosterone, prednisone, methylprednisone, triamcinolone, dexamethasone, deoxycorticosterone, and fluorocortisol) and non-steroidal anti-inflammatory drags (such as, for example, ibuprofen, naproxen, and piroxicam).
  • steroidal such as, for example, cortisol, aldosterone, prednisone, methylprednisone, triamcinolone, dexamethasone, deoxycorticosterone, and fluorocortisol
  • non-steroidal anti-inflammatory drags such as, for example, ibuprofen, naproxen, and piroxicam.
  • immunosuppressive drags include, for example, prednisone, azathioprine (Imuran), cyclosporine (Sandimmune, Neoral), rapamycin, antithymocyte globulin, daclizumab, OKT3 and ALG, mycophenolate mofetil (Cellcept) and tacrolimus (Prograf, FK506).
  • antibiotics include, for example, sulfa drugs (e.g., sulfanilamide), folic acid analogs (e.g., trimethoprim), beta-lactams (e.g., penacillin, cephalosporins), aminoglycosides (e.g., stretomycin, kanamycin, neomycin, gentamycin), tetracyclines (e.g., chlorotetracycline, oxytetracycline, and doxycycline), macrolides (e.g., erythromycin, azithromycin, and clarithromycin), lincosamides (e.g., clindamycin), streptogramins (e.g., quinupristin and dalfopristin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and moxifloxacin), polypeptides (e.g., polypeptid
  • antiviral agents include, for example, vidarabine, acyclovir, gancyclovir, valganciclovir, nucleoside-analog reverse transcriptase inhibitors (e.g., ZAT, ddl, ddC, D4T, 3TC), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, delavirdine), protease inhibitors (e.g., saquinavir, ritonavir, indinavir, nelfinavir), ribavirin, amantadine, rimantadine, relenza, tamiflu, pleconaril, and interferons.
  • nucleoside-analog reverse transcriptase inhibitors e.g., ZAT, ddl, ddC, D4T, 3TC
  • non-nucleoside reverse transcriptase inhibitors e.g., nevirapine,
  • antifungal drags include, for example, polyene antifungals (e.g., amphotericin and nystatin), imidazole antifungals (ketoconazole and miconazole), triazole antifungals (e.g., fluconazole and itraconazole), flucytosine, griseofulvin, and terbinafine.
  • polyene antifungals e.g., amphotericin and nystatin
  • imidazole antifungals ketoconazole and miconazole
  • triazole antifungals e.g., fluconazole and itraconazole
  • flucytosine e.g., griseofulvin
  • terbinafine e.g., fluconazole and itraconazole
  • the subject methods and compositions may be used to treat a subject who is infected with a herpesvirus. (HPV), particularly a high risk HPV such as HPV-16, HPV-18, HPV-31 and HPV-33.
  • HPV herpesvirus
  • treatment of low risk HPV conditions e.g., particular topical treatment of cutaneous or mucosal low risk HPV lesions, is also contemplated.
  • the subject method can be used to inhibit pathological progression of herepesvirus infection, such as preventing or treating diseases or disorders associated with Herpes simplex I (e.g., fever blisters), Herpes simplex II (e.g., genital herpes), Varicella-Zoster (e.g., chickenpox, shingles), Cytomegalovirus (congenital abnormalities, e.g.
  • Epstein-Barr virus e.g., infectious mononucleosis (IM), Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC)
  • EBV Epstein-Barr virus
  • IM infectious mononucleosis
  • BL Burkitt's lymphoma
  • NPC nasopharyngeal carcinoma
  • herpesviras-infected cells which have become, or are at risk of becoming, transformed and/or immortalized, e.g. cancerous, e.g. Burkitt's lymphoma (BL) or nasopharyngeal carcinoma (NPC), or as an adjunct to chemotherapy, radiation, surgical or other therapies for eliminating residual infected or pre-cancerous cells.
  • a modulator of EBNAl complex formation such as a portion of an EBNAl protein or a USP7 protein, may be added to ex vivo or in vitro cells and tissues to, e.g., protect the cells from viral contamination or from spreading of a viral contamination.
  • Cells and tissues treated in this manner may be used, e.g., for administering to a subject, such as in a graft transplant, or for analysis, such as forensic analysis.
  • a biopsy obtained from a subject may be treated as described to prevent contamination or spreading of a viral infection.
  • Inhibitors of EBNAl complexes may also be added to blood in blood banks or to other cells.
  • compositions of this invention include any modulator identified according to the methods of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Methods of making and using such pharmaceutical compositions are also included in the invention.
  • the pharmaceutical compositions of the invention can be administered orally, systemically, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.
  • Parenteral administration and “administered parenterally” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, infra-articular, subcapsular, subarachnoid, intraspinal and infrastemal injection and infusion.
  • Systemic administration refers to the administration of a subject supplement, composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 9. Diagnostics
  • compositions and methods may be incorporated into any of a variety of diagnostic reagents or methods, for example, for detecting an EBNAl complex in a cell extract, or for evaluating the stage of an EBV-associated disease or disorder to which the level of an EBNAl complex or EBNAl complex polypeptide is correlated.
  • the invention provides a method for detecting the presence of an EBNAl complex or EBNAl complex polypeptide in a biological sample. Detection of an EBNAl complex or EBNAl complex polypeptide, particularly in a mammal, and especially in a human, may provide a diagnostic method for diagnosis of a EBV-mediated disease or disorder. In general, the method involves contacting the biological sample with a compound or an agent capable of detecting an EBNAl complex polypeptide or a nucleic acid encoding an EBNAl complex polypeptide.
  • the present invention contemplates a method for detecting the presence of an EBNAl complex or EBNAl complex polypeptide in a sample, the method comprising: (a) providing a sample to be tested for the presence of the complex; (b) contacting the sample with an antibody reactive against an EBNAl complex or EBNAl complex polypeptide under conditions which permit association between the antibody and its ligand; and (c) detecting interaction of the antibody with its ligand, thereby detecting the presence of an EBNAl complex or EBNAl complex polypeptide in the sample.
  • the present invention contemplates a method for diagnosing a patient suffering from an EBV-mediated disease or disorder, comprising: (a) obtaining a biological sample from a patient; (b) detecting the presence or absence of an EBNAl complex or EBNAl complex polypeptide; and (c) diagnosing a patient suffering from an EBV-mediated disease or disorder based on the presence of an EBNAl complex or EBNAl complex polypeptide in the patient sample.
  • the diagnostic assays of the invention may also be used to monitor the effectiveness of an anti-EB V treatment in an individual suffering from an EBV-mediated disease or disorder.
  • the presence and/or amount of an EBNAl complex can be detected in an individual suffering from an EBV-mediated disease or disorder before and after treatment with anti-EBV therapeutic agent.
  • Any change in the level of a polynucleotide encoding an EBNAl complex polypeptide or an EBNAl complex or EBNAl complex polypeptide after treatment of the individual with the therapeutic agent can provide information about the effectiveness of the treatment course.
  • no change, or a decrease, in the level of a polynucleotide encoding an EBNAl complex polypeptide or an EBNAl complex or EBNAl complex polypeptid present in the biological sample will indicate that the therapeutic is successfully combating the EBV-mediated disease or disorder.
  • kits for detecting an EBNAl complex or EBNA complex polypeptide and for treating and diagnosing EBV-mediated diseases or disorders may comprise one or more compounds that modulate an EBNAl complex, e.g., for use in treating a patient having an EBV-mediated disease or disorder.
  • Compounds comprising nucleic acids may be included in a plasmid or a vector, e.g., a viral vector.
  • Other kits comprise an EBNAl complex or EBNAl complex polypeptide of the invention.
  • Still other kits may comprise reagents for detecting an EBNAl complex or EBNAl complex polypeptide.
  • the compositions comprising the kits may be formulated into pharmaceutical compositions comprising a pharmaceutically acceptable excipient.
  • Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods.
  • this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.
  • Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.
  • Example 1 Isolation of EBNAl Complex Polypeptides Using an EBNAl Affinity Column
  • EBNAl complex polypeptides were isolated using a column in which EBNAl was bound to the resin.
  • EBNAl (lacking most of the Gly Ala repeat region) was expressed as a hexahistidne fusion from a baculovirus. It was purified from insect cell nuclei on a metal chelating column, followed by a heparin agarose column. The purified EBNAl was then covalently linked to the affinity column resin using standard affinity resin preparation techniques.
  • Figure 1 depicts a flowchart of the procedure used to isolate EBNAl complex polypeptides from human cell extracts using the EBNAl affinity column, or a control affinity column.
  • TATA-box binding protein (TBP) affinity column was used as control for cellular proteins that bind nonspecifically to proteins with the same charge as EBNAl, while an affinity column containing empty resin was used as a control for cellular proteins that bind nonspecifically to the resin.
  • the eluted cellular proteins from each column were visualized by silver-stained SDS-polyacrylamide gel electrophoresis ( Figure 2).
  • Figure 2 By comparing the cellular proteins that bound to EBNAl, TBP and empty columns, it was observed that USP7, karyopherin ⁇ 3, NAPl, importin ⁇ , TAFI ⁇ and casein kinase II bound specifically to EBNAl ( Figure 2).
  • Figure 3 depicts a gel electrophoresis profile of cellular polypeptides that bound to an EBNAl affinity column after a first pass of human cell extract (lane 1) and a second pass of the EBNAl affinity column eluate visualized in lane 1 (lane 2) through the column.
  • the EBNAl complex polypeptides that were isolated from human cell extract via an EBNAl affinity column as described in Figure 1 are shown in lane 1 of silver-stained SDS- polyacrylamide gel.
  • the proteins in the eluate visualized in lane 1 were then reapplied to an EBNAl column, eluted in lM aCl and visualized by silver-stained SDS- polyacrylamide gel electrophoresis (lane 2).
  • Insect cells were co-infected with baculoviruses expressing USP7 and EBNAl, or a EBNAl deletion mutant, and metabolically labeled.
  • the EBNAl deletion mutants also lacked most of the Gly Ala repeat in addition to the indicated deletions.
  • EBNAl was then immunoprecipitated from insect cell lysates and visualized along with co- immunoprecipitated proteins by autoradiography of SDS-polyacrylamide gels.
  • Figure 4 depicts the results of immunoprecipitation of EBNAl from insect cells in which EBNAl or an EBNAl mutant and USP7 were co-expressed.
  • the bottom panel depicts autoradiograph visualization of the EBNAl immunoprecipitation results.
  • the position of USP7 is indicated with an asterisk. Each lane is labeled with the polypeptides that were co- expressed in the insect cells. USP7 was consistently observed to co-precipitate with EBNAl and several EBNAl mutants but was disrupted by deletion of EBNAl amino acids 395-450.
  • the top panel of Figure 4 depicts a diagram of the EBNAl polypeptide in which the position of the USP7 binding site, as determined from the immunoprecipitation results, is indicated.
  • EBNAl complex polypeptides were isolated using the tandem affinity purification (TAP) method (Puig, O, et al. (2001) Methods 24: 218-229). TAP-tagged EBNAl was expressed in human 293 cells, then purified from the cell lysates, along with associated human proteins, on IgG and calmodulin columns.
  • Figure 5 depicts a flowchart of the tandem affinity purification (TAP) tagging method used to isolate EBNAl complex polypeptides from human cells. TAP-tagged EBNAl and the EBNAl deletion mutant, ⁇ 395-450, were purified from human cells as described in Figure 5.
  • TAP tandem affinity purification
  • Epstein-Barr virus is a ubiquitous human ⁇ herpesvirus that persists for the life of the host. As part of its latent infectious cycle, EBV immortalizes the host cell and, in doing so, predisposes the cell to malignant transformation. As a result, EBV is associated with several types of cancer. EBV genomes are maintained in latently infected replicating cells as circular DNA episomes that replicate once per cell cycle and segregate stably during cell division (reviewed in Kieff and Rickinson (2001) Epstein-Barr Virus and its Replication, Fields Virology, DM Knipe and PM Howley (eds.) Lippincott Williams and Wilkins, Philadelphia, Pennsylvania, pp. 2511-2573).
  • Epstein-Barr nuclear antigen 1 (EBNAl) is the only viral protein required to maintain the EBV genomes in proliferating cells, which it does by binding to recognition sites in the family of repeats (FR) and dyad symmetry (DS) elements of the latent origin of DNA replication, oriP.
  • EBNAl binding to the DS is necessary to initiate DNA replication from this element.
  • EBNAl binding to the FR element is important for the partitioning of the EBV episomes during cell division and also activates the expression of other viral latency genes.
  • EBNAl In addition to its functions at oriP, EBNAl has been shown to repress its own transcription, and to promote the development of B-cell lymphomas in transgenic mice, suggesting a direct role for EBNAl in cell transformation (Wilson, et al, 1996 EMBO J., 15, 3117-3126.). While fulfilling all of its functions, EBNAl avoids detection by host cytotoxic T lymphocytes (CTL). This ability to hide from the immune system is biologically important as it enables the persistence of latently infected cells that express EBNAl in the absence of other EBV antigens.
  • CTL host cytotoxic T lymphocytes
  • EBNAl has no apparent enzymatic activities and is thought to fulfill its functions by mediating interactions with specific host cellular proteins. However, few of these cellular protein interactions have been identified. To date only yeast one- and two-hybrid approaches have been used to screen for EBNAl -interacting proteins; these screens have identified importin ⁇ (also called karyopherin ⁇ 2 or Rchl), karyopherin ⁇ l, P32/TAP and EBP2 as EBNAl binding proteins (Kim, A. et al. (1997) Virology 239: 340-351 ; Wang, Y. et al (1997) Virology 236: 18-29; Fischer, N. et al (1997) J Biol. Chem.
  • EBP2 is a component of the cellular mitotic chromosomes and EBNAl appears to attach to EBP2 in order to partition EBV plasmids (Shire, K. et al. (1999) J Virol. 73: 2587-2595; Wu, H. et al. (2000) EMBO Rep. 1: 140-144; Kapoor, P. et al (2001) EMBO J. 20: 222-230).
  • Proteins found to be specifically and reproducibly retained on the EBNAl columns were identified by MALDI-ToF mass spectrometry. Two of these proteins, importin ⁇ and P32/TAP, were previously identified as EBNAl -interacting proteins in two-hybrid screens (Kim, A. et al. (1997) Virology 239: 340-351; Wang, Y. et al. (1997) Virology 236: 18-29; Fischer, N. et al. (1997) J. Biol. Chem. 272: 3999-4005; Shire, K. et al. (1999) J Virol. 73: 2587-2595). In addition, previously unknown interactions were detected with USP7, karyopherin ⁇ 3, karyopherin ⁇ 2 (not obvious in
  • NAPl nucleosome assembly protein 1
  • TAF-I ⁇ and ⁇ form template activating factor, which can activate replication and transcription from chromatin templates through histone interactions (Okuwaki, M. et al. (1998) J Biol. Chem. 273: 34511-34518).
  • TAF-I ⁇ also known as SET, has been found to regulate p21 Clpl (Estanyol, J.M. et al. (1999) J Biol. Chem. 27 : 33161-33165).
  • CK2 ⁇ and ⁇ ' are two of the three subunits of the CK2 serine/threonine kinase (Litchfield, D.W. (2002) Biochem. J).
  • pp32 is an acidic nuclear protein that forms part of the INHAT complex (Seo, S.B. et al. (2001) Cell 104: 119-130).
  • EBP2 which was found to bind EBNAl by other methods (Shire, K. et al. (1999) J Virol. 73: 2587-2595), was not among the proteins retained on the EBNAl column.
  • EBNAl is a highly basic protein (pi of 11), we expected that some cellular proteins containing acidic regions would interact with EBNAl through nonspecific ionic interactions.
  • TBP TATA binding protein
  • USP7 also immunoprecipitated with a version of EBNAl containing a longer Gly- Ala repeat region (EBNAl GA; lane 13).
  • EBNAl GA Gly- Ala repeat region
  • the USP7 band was not detected in anti-EBNAl immunoprecipitates from cells expressing either USP7 alone, or EBNAl alone (lanes 8 and 15).
  • the USP7-EBNA1 interaction does not require other human proteins.
  • TAP-tagging which was originally developed for use in yeast, involves expressing the protein of interest fused at the C-terminus to a calmodulin binding peptide, followed by a TEV protease cleavage site and a protein A IgG binding domain (Rigaut, G. et al. (1999) Nat. Biotechnol. 17: 1030-1032).
  • the tagged protein is isolated from cell lysates on IgG resin, eluted by TEV protease cleavage, further purified on calmodulin resin and eluted with EGTA. Because the native elution conditions in this procedure enable protein complexes to remain intact through the purification, this is a powerful method for profiling in vivo protein interactions.
  • TAP-tagged EBNAl was purified from cell lysates and co-purifying proteins were separated by SDS-PAGE and identified by MALDI-ToF mass spectrometry (Figure 10A).
  • This method identified a subset of the cellular proteins that interacted with EBNAl in the affinity column approach, namely USP7, importin ⁇ , CK2 ( , ⁇ ' and P subunits) and P32/TAP.
  • PRMT5 was also found to be retained on EBNAl affinity columns but was only eluted with SDS.
  • the plasmids were recovered from cells 3 days post-transfection, linearized and incubated with the methylation-sensitive enzyme Dpnl, which digests the unreplicated plasmids leaving replicated plasmids intact. The amount of Dpnl-resistant plasmid was then quantified by Southern blotting. As shown in Figure 12B, oriP plasmids were replicated more efficiently in the presence of ⁇ 395-450 than EBNAl; replication levels calculated from 4 experiments were 4-fold higher on average in the presence of ⁇ 395-450 than with EBNAl, which could account for the increased plasmid levels seen in the plasmid maintenance assays.
  • C33A cells were cotransfected with the oriP plasmids expressing EBNAl or ⁇ 395-450 and with pFRTKCAT, and the level of CAT gene expressed was determined by measuring the acetylation rates for each cell lysate using equal amounts of protein. Results from 4 experiments indicated that ⁇ 395-450 was less active than EBNAl in these assays, yielding 65% (+/- 7%) of the transcription activation levels of EBNAl. These results suggest that the interaction with USP7 increases the transcriptional activity of EBNAl. Cellular localization and abundance of USP7 and EBNAl.
  • USP7 was very abundant in Raji cells with approximately 130,000 copies per cell (+/- 68,000 copies/cell) calculated from 4 separate experiments. USP7 was also abundant in the C33A cells where the functional assays were performed, with a copy number calculated at 50,000 copies per cell. USP7 is several fold more abundant than EBNAl in the Raji cells, which our experiments indicate is present at 18,000 copies per cell and that has previously been reported to be present at 37,000 copies per cell (Sternas et al, (1990) J Virol. 64: 2407-2410)). The relative abundance of EBNAl and USP7 is consistent with the possibility that a significant portion of EBNAl could be bound by USP7 in EBV infected cells.
  • TAF I ⁇ and ⁇ are used in multiple ways to affect cellular gene expression and cell cycle progression.
  • TAF I ⁇ and ⁇ have been shown to be components of the INHAT complex that inhibits histone acetylation (Seo, S.B. et al. (2001) Cell 104: 119-130), and TAF I ⁇ , also know as SET, has been shown to be associated with myeloid leukemia (Nagata, K. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 4279-4283) and to interact with p21 C ⁇ pl to potentially regulate cell cycle progression (Estanyol, J.M. et al. (1999) J Biol. Chem.
  • CK2 is a serine/threonine kinase that has many cellular targets and is implicated in several cellular pathways including cell cycle progression, malignant transformation and regulation of apoptosis (Litchfield, D.W. (2002) Biochem. J). Thus the interaction of EBNAl with CK2 may affect any of these pathways.
  • the EBNAl -CK2 interaction may also reflect the regulation of EBNAl function by CK2 phosphorylation, as EBNAl is known to be phosphorylated on serine residues (Frappier, L. et al. (1991) J. Biol Chem. 266 : 7819-7826) and contains three putative CK2 sites.
  • PRMT5 (also know as JBPl) is a protein arginine methyltransferase and the human homologue of the fission yeast Skbl (Pollack, B.P. et al (1999) J. Biol. Chem. 17 A: 31531- 31542). Increasing evidence points to the importance of arginine methylation in regulating a variety of protein functions (McBride, A.E. et al (2001) Cell 106: 5-8).
  • ubiquitination would stimulate EBNAl replication activity, presumably by affecting specific protein interactions. This could be due to monoubiquitination rather than poyubiquitination, since monoubiquitination is emerging as a post-translation modification that can regulate protein function without affecting turnover (Spence, J. et al. (2000) Cell 102: 67-76; Hoege, C. et al. (2002) Nature 419: 135-141).
  • the second model assumes the effects are due to deubiquitination of cellular proteins by USP7. In this model EBNAl would bring USP7 to oriP where it could deubiquitinate, one or more cellular proteins that affect the activation of replication. This could mean either that ubiquitination makes a cellular factor more active for DNA replication or that ubiquitination inactivates, or targets for proteasomal destruction, a negative regulator of DNA replication.
  • EBNAl efficiently sequesters or inactivates USP7, then we would expect EBNAl to destabilize p53, thereby promoting cell cycle progression and inhibiting apoptosis. Such effects may be important for host cell immortalization by EBV as well as in the development of EBV- associated tumours.
  • the possibility that EBNAl directly contributes to these processes is supported by the fact that some EBV tumours only express EBNAl and by the ability of EBNAl to induce B cell neoplasia in transgenic mice (Wilson, J. et al. (1996) EMBO J. 15: 3117-3126).
  • EBNAl (lacking most of the Gly Ala repeat region) was expressed as a hexahistidine fusion from a baculovirus.
  • EBNAl was purified from insect cell nuclei on a metal chelating column, followed by a heparin agarose column (Frappier, L. et al. (1991) J. Biol Chem. 266: 7819-7826).
  • the ⁇ 325-376 EBNAl mutant (lacking amino acids 325-376 in addition to most of the Gly Ala repeat) was expressed as a hexahistidine fusion from pET15b in E. coli and purified as for EBNAl.
  • HeLa S3 cells (lOg; National Cell Culture Center) were lysed in 11 mis of 10 mM HEPES, pH 7.5, 1.5 mM MgCl 2 , 10 mM KC1, 0.5 mM DTT, and complete protease inhibitors (Roche). 10 mis of 50 mM HEPES, pH 7.5, 1.5 mM MgCl 2 , 1.26 M NaCl, 0.5 mM DTT, 0.6 mM EDTA, 75% glycerol was added, and the lysate was Dounce homogenized.
  • the extract was clarified by centrifugation for 3 hours at 64,000 X g, then dialyzed overnight against 50 mM HEPES, pH 7.5, 20 % glycerol, 0.5 mM DTT, 5 MM MgCl 2 , and 75 mM KC1.
  • CaCl Prior to loading onto affinity columns, CaCl was added to 4 mM and lysates were incubated with RNase A and DNase I (1.4 ⁇ g/mg of lysate) for 30 minutes at 25 ° C to remove nucleic acid.
  • Purified EBNAl or ⁇ 325-376 was covalently linked to Affigel-10 (BioRad) by incubating 2 mg protein per mg of Affigel in buffer C (20 mM HEPES, pH 7.5, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 1 M NaCl) and blocked as previously described
  • Coupled resin was washed with buffer C and equilibrated in buffer C containing 0.1 M NaCl (buffer D) before pouring 40 ⁇ l micro-columns. 400 ⁇ l of HeLa lysate (at 14 mg/ml) was applied to the columns. Columns were washed with 400 ⁇ l of buffer D and 160 ⁇ l buffer D containing 1% Triton X-100, then sequentially eluted with buffer C and 1% SDS.
  • the buffer C eluates from 3 columns were pooled, dialyzed against buffer C containing 200 mM NaCl and reapplied to an EBNAl affinity column. This column was washed and eluted as described above. Column eluates were analyzed by SDS-PAGE and silver staining. Mass SpecttOmetry. Gel slices containing the protein bands were identified by first reducing in DTT, alkylating in iodoacetamide, then subjecting to in-gel trypsin hydrolysis.
  • the peptides were purified and analyzed by MALDI-ToF mass spectrometry using a cyano- 4-hydroxycinnamic acid matrix (Sigma) on a Voyager DE-STR instrument (Applied Biosystems) (Mann, M. et al. (1993) Biol Mass Spectrom. 22: 338-345). Identification of the proteins using this mass fingerprinting data was carried out using the ProFound software (http://129.85.19.192/profound_bin/WebProFound.exe).
  • the baculoviras expressing EBNAl with small Gly-Ala repeat and lacking amino acids 395-450 was constructed by QuickChange mutagenesis (Stratagene) of EBNAl in pFastBac (Gibco). Baculoviras was generated
  • pMZI expresses proteins with C-terminal TAP-tags (Rigaut, G. et al. (1999) Nat. Biotechnol. 17: 1030-1032) in mammalian cells under the control of an ecdysone inducible promoter.
  • 293T cells at 60% confluence in 150-mm dishes were cotransfected by calcium phosphate precipitation with 8 ⁇ g pMZI expressing EB ⁇ A1 or an EB ⁇ A1 mutant and 8 ⁇ g pVgRxR (Invitrogen), which encodes the ecdysone receptor heterodimer.
  • the precipitate was removed 15 h post transfection and expression of the EB ⁇ A1 proteins was induced by adding medium containing 3 ⁇ M Ponasterone A (Invitrogen).
  • the cells were harvested 22 to 48 h later, and a whole cell extract was prepared from 4 x 10 7 cells as described in Xiao et al. (Xiao, H. et al.
  • Protein complexes were eluted with 10 mM Tris-HCl pH 8, 100 mM ⁇ aCl, 2 mM EGTA, 10 mM ⁇ -mercaptoethanol, 1 mM imidazole, 0.1% TritonX-100, concentrated by lyophilization and analyzed by SDS-PAGE and silver staining. Protein bands were identified by MALDI-ToF mass spectrometry.
  • USP7 expression and purification A baculovirus expressing USP7 with an ⁇ - terminal hexahistidine tag, was constructed using USP7 cD ⁇ A in pET-3a (Everett, R. et al. (1997) EMBO J. 16: 1519-1530). USP7 was purified from insect cell lysates on a Talon cobalt column (Clontech), then dialyzed against 20 mM Tris-HCl, pH 7.6, 50 mM ⁇ aCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol. The resulting USP7 was greater than 90% pure.
  • EB ⁇ A1 Ubiquitination and Deubiquitination of EB ⁇ A1.
  • 5 ⁇ g of purified EB ⁇ A1 was ubiquitinated at room temperature in the following: 66% rabbit reticulocyte lysate (Promega), 1.5 ⁇ g ubiquitin, 260 ⁇ M ALLN, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, 100 mM NaCl. Aliquots were taken just before and at 1, 15, 30, and 60 minutes after addition of EBNAl.
  • EBNAl Functional Assays The construction of pc3oriPEBNAl and pc3oriP, which contain the EBV oriP sequence and express EBNAl or no protein, respectively, have been previously described (Shire et al, 1999). Plasmid pc3oriP ⁇ 395-450 was constructed by QuickChange mutagenesis (Stratagene) of pc3oriPEBNAl. Transient replication, plasmid maintenance, and transcriptional activation assays were performed as previously described (Wu, H. et al. (2002) J Virol. 76: 2480-2490).
  • C33A cells were transfected with 10 ⁇ g of pc3oriP plasmids and, 72 h post-transfection, plasmids were recovered, linearized, digested with Dpnl and analyzed by Southern blotting.
  • C33A cells were transfected with 1 ⁇ g of plasmid and, after 14 days of selection in G418, plasmids were linearized, digested with Dpnl and analyzed by Southern blotting. Plasmid bands were visualized by autoradiography and quantified by phosphorimager analysis using ImageQuant software (Molecular Dynamics).
  • C33A cells were transfected with 5 ⁇ g of pc3oriP plasmids and 2 ⁇ g of the pFRTKCAT reporter construct. 24 hours later, cell lysates were prepared and 50 ⁇ g of each was assayed for chloramphenicol acetyltransferase activity using several reaction times as previously described (Ceccarelli, D.F. et al. (2000) J Virol. 74: 4939-4948). Reaction products at each time point were quantified by phosphorimager analysis and used to determine the acetylation rate.
  • Lysates were clarified by sonication and centrifugation, and 50 ⁇ g of each was separated by SDS-PAGE and Western blotted using R4 EBNAl antisera (raised against EBNAl 452-641). The same blots were also probed with anti-IRF-1 antibody (C20, Santa Cruz Biotechnologies).
  • C33A cells expressing EBNAl were generated by transfecting C33A cells with pc3oriPEBNAl and growing the cells under selection for the plasmid. The cells were then grown on coverslips to 60% confluence, fixed with formaldehyde (5%, v/v, in PBS containing 2% sucrose) and permeabilized with acetone: methanol (70:30) at -20°C. Coverslips were washed with PBS and blocked for 1 hour in 10% BSA.
  • the cells were stained with anti-EBNAl monoclonal antibody OTlx at a 1:150 dilution and anti-USP7 rabbit antibody r201 at a 1 :200 dilution, followed by staining with goat anti-mouse-Texas Red labelled (Molecular Probes) and goat anti-rabbit-FITC labeled (Gibco) secondary antibodies (at 1/200 and 1/30 dilutions, respectively). Cells were then counterstained with 4,6-diamidino-2-phenylindole (DAPI). The slides were mounted in 5 ⁇ l of antifade solution and observed at 400-fold magnification using a Leica DMR microscope and Openlab software.
  • DAPI 4,6-diamidino-2-phenylindole
  • DUBs also generate the pool of free ubiquitin both by liberating ubiquitin from precursor ubiquitin- fusion proteins and by recycling ubiquitin from the branched polyubiquitin chains of degraded proteins (reviewed in Wing, S. S. (2003) Int.JBiochem.Cell Biol. 35, 590-605; Wilkinson, K. D. (1997) FASEBJ. 11, 1245-1256; D'Andrea A and Pellman D. (1998) Crit Rev Biochem Mol Biol 33, 337-352).
  • the DUBs are comprised of two groups of enzymes, the UCHs (Ubiquitin C-terminal Hydrolyases) and the USPs (Ubiquitin Specific Processing Proteases; referred to as UBPs in yeast).
  • the UCHs are small, closely related proteases (20-30 kDa in size) that are generally involved in cleaving ubiquitin from small processed peptides.
  • the USPs are more numerous, much larger in size (60-300 kDa) and are thought to have specific protein targets. USPs can be identified by conserved sequences within the active site, but sequences outside of the catalytic domain are highly divergent, likely reflecting their role in mediating interactions with different protein targets (D'Andrea A and Pellman D.
  • ICPO herpes simplex virus type I immediate early protein
  • ICPO also called Vmwl 10
  • ICPO is an E3 ubiquitin ligase (Boutell, C, et al. (2002) J. Virol. 76, 841-850) that is important for induction of the lytic infectious cycle.
  • ICPO promiscuously activates gene expression (Everett, R. D.
  • USP7 is also the target of another herpes virus protein, namely the EBNAl protein of Epstein-Barr virus (EBV).
  • EBNAl binds to the latent origin of DNA replication and governs the replication and segregation of the EBV episomes during latent infection (Yates, J. L. (1988) Cancer Cells 6, 197-205; Frappier, L. (2003) Viral plasmids in mammalian cells. In Funnell, B. E. and Phillips, G., editors. The Biology ofPlasmids, ASM Press, Washington, D.C.).
  • EBNAl also activates the expression of other viral latency genes and is implicated in the immortalization of host cells by EBV (Wilson, J., et al.
  • USP7 regulated the turnover of ⁇ 53.
  • USP7 was isolated on a ⁇ 53 affinity column and subsequently shown to co- immunoprecipitate withp53. Overexpression of USP7 stabilized ⁇ 53 and induced p53- dependent growth repression and apoptosis. These effects were likely due to deubiquitination of p53 by USP7, since the over expression of USP7 resulted in increased levels of deubiquitinated p53, and since dominant negative effects on p53 levels and ubiquitination were observed using a catalytically inactive USP7 point mutant.
  • TRAF domain tumor necrosis factor-receptor associated factor
  • USP7 baculovirus was constructed using full length USP7 cDNA in a pET-3a vector.
  • the cDNA was excised with Ncfel and Hind ⁇ l, and cloned into the same sites of ⁇ ET-15b ( ⁇ ovagen), downstream of the hexahistidine tag.
  • ⁇ terminally tagged USP7 was excised from pET-15b using Xbal and Hind ⁇ l, and cloned between the Xbal and Hindill sites of the pFastBac vector (GIBCO).
  • Bacmid D ⁇ A was prepared and used to transfect Spodopterafrugiperda (Sf9) insect cells to generate baculovirus according to manufacturer's specifications.
  • Culture medium containing the baculovirus was harvested 5 days post-transfection and amplified once. Hi5 insect cells were seeded into 27 150 cm 2 culture flasks at 5 X 10 6 cells per flask, and infected with USP7 baculovirus.
  • Cells were harvested 50 hours post-infection, washed in 50 ml of phosphate buffered saline, and lysed in 50 mM ⁇ aH PO , 300 mM NaCl, 5 mM ⁇ - mercaptoethanol, 10% glycerol, l% NP-40,1.5 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride (PMSF), ImM benzamidine-hydrochloride, pH 7.0, using a Dounce homogenizer.
  • the cellular extract was clarified by centrifugation at 64,000 x g for 30 minutes at 4 ° C, before loading onto a 4 ml Talon cobalt column (Clontech).
  • tagged USP7 was eluted from the column using 15 ml of buffer A containing 150 mM imidazole. Pertinent fractions were pooled and dialyzed overnight against 20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 1 mM 1,4-dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), and 5% glycerol. Expression and purification of USP7 fragments.
  • USP7 fragments coding for amino acids 1 to 205, 1 to 580, 56 to 205 and 202 to 580 were PCR amplified and cloned between the Ndel and BamHI sites of pET15b (Novagen), downstream of the hexahistidine tag. Proteins were expressed at 18° C in BL21(DE3)pLysS cells after overnight induction with 0.5 mM IPTG.
  • Cells were lysed by sonication in buffer B (50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl, 1 % NP-40,20 mM imidazole, and 1 mM PMSF), and lysates were clarified by centrifugation for 30 minutes in an SS34 Sorvall rotor at 15, 000 rpm. Clarified lysates were applied to a Ni-NTA sepharose column (Qiagen) and, after extensive washing with buffer B lacking detergent, the his-tagged proteins were eluted with buffer B containing 250 MM imidazole.
  • buffer B 50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl, 1 % NP-40,20 mM imidazole, and 1 mM PMSF
  • Clarified lysates were applied to a Ni-NTA sepharose column (Qiagen) and, after extensive washing with buffer B lacking detergent, the his-tagged proteins were eluted
  • the eluted proteins were incubated with thrombin to remove the histidine tag, then passed through a nickel column to separate cleaved from uncleaved fractions.
  • the flowthrough from the second nickel column was incubated with benzamidine sepharose to remove thrombin, and dialyzed against 20 mM Tris-HCl, pH 7.6, 0.2 M NaCl.
  • BL21(DE3) ⁇ LysS cells were transformed with pGEX2T-Ub52 (Everett, R., et al. (1997) EMBO Journal 16,1519-1530) and expression of GST-Ub52 was induced by addition of 0.1 mM IPTG at 37° C for two hours.
  • Cells were lysed by sonication in NETN buffer (20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM PMSF), and the lysate was clarified by centrifugation for 15 minutes in an SS34 Sorvall rotor at 15,000 rpm at 4°C.
  • the supernatant was incubated for 2 hours at 4°C with 0.5 ml glutathione sepharose beads (Amersham Pharmacia) equilibrated inNETN plus 0.5% milk powder.
  • the glutathione sepharose beads were washed extensively with NETN, and once with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 MM MgC12, before eluting in 100 mM Tris-HCl, pH 8.0, 120 mM NaCl, 20 mM reduced glutathione.
  • the eluate was dialyzed overnight against 20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.5 mM DTT.
  • Cleaved and uncleaved substrates were resolved by SDS-PAGE (12.5%) and visualized by staining with SYPRO-Orange (Molecular Probes) and scanning with a STORM860 laser scanner (Molecular Dynamics) set at blue fluorescence/chemifluorescence (850 N). Bands were quantified with ImageQuant 5.0 software.
  • EB ⁇ A1 Ubiquitination and Deubiquitination of EB ⁇ A1.
  • 5 ⁇ g of purified EB ⁇ A1 was ubiquitinated at room temperature in the following: 66% rabbit reticulocyte lysate (Promega), 1.5 ⁇ g ubiquitin (Sigma), 260 ⁇ M ALL ⁇ (Sigma), 50 mM Tris-HCl (pH 7.5), 5 mM MgC12, 1 mM DTT, 1 mM ATP, 10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, 100 mM ⁇ aCl Aliquots were taken just before and at 1, 15, 30, and 60 minutes after addition of EB ⁇ A1.
  • EBNAl Affinity columns EBNAl (lacking most of the Gly Ala repeat region) was expressed in insect cells with an N-terminal hexahistidine tag and purified. Purified EBNAl was coupled to Affigel-10 at a concentration of 1 mg EBNAl/ml resin. 25 ⁇ l affinity columns were equilibrated in buffer C (20 MM TrisHCI, pH 7.6, 0.2 M NaCl) before addition of 64 ⁇ g of the partially proteolyzed USP7 or an equimolar mixture of individually -expressed and purified USP7 fragments (400 pmol each fragment).
  • ICPO Affinity Columns Amino acids 594 to 775 of ICPO of HSV-1 was expressed as a GST-fusion in BL21(DE3)pLysS cells from pGEX-E52 (Meredith M, et al (1994) Virology 200, 457-469). After a 4 hour induction at room temperature with 0.1 mM IPTG, cells from 25 ml of culture were lysed by sonication in PBS plus 0.5 % Triton X-100 and complete protease inhibitors (Roche). After clarification of the lysate by centrifugation, the supernatant was incubated with 100 ⁇ l of glutathione sepharose beads (Amersham Pharmacia) overnight at 4°C.
  • the beads were washed 3 times with PBS, twice with PBS containing 300 mM NaCl, and twice more with PBS, before being resuspended in PBS. 25 ⁇ l columns were generated from the ICPO-bound beads, and these were equilibrated in buffer C and used to assess binding of partially proteolysed USP7 fragments as described for EBNAl affinity columns.
  • EBNAl peptide An EBNAl fragment coding for amino acids 395 to 450 was PCR amplified and cloned between the Ndel and BamHI sites of pET 15b (Novagen), downstream of the hexahistidine tag. The EBNAl peptide was expressed at 25°C in BL21(DE3) ⁇ LysS cells after a 4 hour induction with 0.5 mM IPTG.
  • p53 peptide Human p53 fragments encoding amino acids 311 to 393 and 355 to 393 were cloned between theN ⁇ and BamHI sites of pET15b downstream of a hexhistidine tag. p53 proteins were overexpressed in BL21-CodonPlus E.coli (Stratagene) upon addition of IroM IPTG. Three hours after induction at 25 °C, cells were lysed by sonication in buffer D and the tagged peptides were purified from the clarified lysate on Talon cobalt columns as described for the EB ⁇ A1 peptide.
  • the concentration of the p53 311-393 peptide was determined by OD280 and the concentration of the ⁇ 53 355- 393 peptide (which lacks aromatic residues) was measured by comparing with protein standards on a SDS-PAGE gel.
  • Tryptophan Fluorescence Assay Fluorescence measurements were performed in an Aviv ATF105 ratio spectrofluororneter. Peptide binding titrations were performed by stepwise mixing of the target peptide (monomeric p53 355-393, tetrameric p53 311-393, or EB ⁇ A1 395-450) with the USP7 1-205 fragment, using a Microlab 500 series automated titrator. Binding reactions contained 1 ⁇ M USP7 in 2 ml of 20 mM Tris, pH 7.6, 200 mM ⁇ aCl.
  • I (([LT] - ((-([PT] - [L ⁇ ] + K d ) + SQRT(([P T ] - [L ⁇ ] + K d )2 + 4K d [L T ]))/2))/[P T ]) (l ⁇ - lo) + lo.
  • EBNAl was quickly converted to a ladder of higher molecular weight conjugates, typical of a heterogenously ubiquitinated protein.
  • the high molecular weight EBNAl conjugates were reduced to the position of EBNAl alone (lanes 6 and 7).
  • Western blot of the lysate lacking EBNAl showed that the EBNAl antibody did not cross-react with proteins in the lysate (lane 1).
  • the results indicate that EBNAl can be efficiently ubiquitinated in vitro and can be deubiquitinated by the purified USP7.
  • USP7 remains partially active even at 520 mM NaCl, as 80 % of the substrate was cleaved in this condition upon overnight incubation with a 1:1000 molar ratio of USP7:GST-Ub52. USP7 was also found to be quite tolerant of high pH, as it exhibited complete GST-Ub52 cleavage activity at pHs between 7.0 and 9.5 ( Figure 15C). A marked decrease in activity was seen at pH 6.2, however.
  • CaCl did not irreversibly inhibit USP7 activity; wild type activity was restored when 10 mM EDTA was added after preincubation with the divalent cation.
  • USP7 was homogeneous with an apparent molecular weight of 128,815 Da, which approximates the calculated molecular weight of 130,436 Da for hexahistidine-tagged USP7. Therefore full length USP7 is monomeric under the conditions tested.
  • USP7 has been shown to bind p53, the ICPO protein of herpes simplex and the EBNAl protein of Epstein-Barr virus. While the p53 -binding region of USP7 has been mapped to the N-terminus between amino acids 53 to 208 (Hu, M., et al. (2002) Cell 111, 1041-1054), the regions responsible for binding ICPO and EBNAl have not been determined.
  • UBP6 and human USP10 were even less active; UBP6 cleaved 50% of the Ub-CEP80 or Ub-DHFR substrates after a 2 hour incubation at a 1:1 molar ratio (Park KC, et al. (1997) Arch Biochem Biophys 347, 78- 84), and 50% cleavage of a linear ubiquitin hexamer by USP10 required approximately a 30 minute incubation at a 1:1 molar ratio (Soncini, C, et al. (2001) Oncogene 20, 3869-3879). USP7 is quite tolerant to high salt conditions and pH variation, with complete activity seen at pH 7-9.5. In comparison, UCH-8 (Baek, S. H., et al (1997) Biochem J
  • this domain mediates interactions with EBNAl .
  • the region C-terminal to the catalytic domain can be divided into two protease-resistant domains, mapping approximately to amino acids 622-801 and amino acids 885-1061. The first of these domains binds ICPO, while a function has yet to be identified for the second domain.
  • the fact that EBNAl and ICPO interact with different domains of USP7 argues against the possibility that these interactions occur due to a nonspecifically sticky USP7 domain, and indicates that EBN and HSN have developed different mechanisms for targeting the same cellular protein.
  • Epstein-Barr virus efficiently immortalizes cells as part of its latent infectious cycle and this process involves a few different EBV latency proteins (reviewed in Kieff, E. and Rickinson, A. B. (2001) Epstein-Barr Virus and its Replication. Fields Virology, DM Knipe and PM Howley (eds.) Lippincott Williams and Wilkins, Philadelphia, Pennsylvania). Whether or not EB ⁇ A1 plays a direct role in this process is supported by the findings that transgenic mice expressing EB ⁇ A1 have a tendency to develop B-cell lymphomas (Wilson, J., et al (1996) EMBO 15, 3117-3126). Cellular transformation by viruses (eg.
  • adenovirus, SV40 and papillomaviruses typically involves the targeting of p53, therefore it is surprising that, to date, none of the EBV proteins known to contribute to cell immortalization have been shown to act through p53. Our data suggest that EB ⁇ A1 may contribute to host cell immortalization by EBV by sequestering USP7, thereby destabilizing p53.
  • WO 00/45168 also incorporated by reference are the following: WO 00/45168, WO 00/79238, WO 00/77712, EP 1047108, EP 1047107, WO 00/72004, WO 00/73787, WO00/67017, WO 00/48004, WO 00/45168, WO 00/45164, U.S.S.N. 09/720,272; PCT/CA99/00640; U.S.

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Abstract

La présente invention concerne des complexes composés d'EBNA1 et d'au moins un polypeptide des complexes d'EBNA1, ainsi que des méthodes d'utilisation associées. On utilise des complexes d'EBNA1 purifiés et des polypeptides des complexes d'EBNA1, ainsi que des variantes des complexes d'EBNA1 telles que des complexes de fragments polypeptidiques ou de fusions polypeptidiques, ainsi que des molécules chimères comprenant des composants polypeptidiques des complexes d'EBNA1. Cette invention concerne également des méthodes de préparation de ces complexes d'EBNA1 et des polypeptides des complexes d'EBNA1, des méthodes de détection, d'identification et de purification des complexes d'EBNA1, des méthodes d'identification de composés qui modulent les complexes d'EBNA1 et les polypeptides des complexes d'EBNA1 ainsi que des méthodes de traitement ou de diagnostic de troubles induits par le virus d'Epstein-Barr utilisant les complexes d'EBNA1 et les polypeptides des complexes d'EBNA1 ainsi que des composants qui les modulent.
PCT/CA2003/001019 2002-07-16 2003-07-16 Nouvelles interactions de la proteine du virus d'epstein-barr ebna1, compositions et methodes associees WO2004007536A2 (fr)

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CN108061798A (zh) * 2016-11-07 2018-05-22 国药中生生物技术研究院有限公司 检测HBsAg/anti-HBs复合物体系中有效成分的量的方法和应用
WO2024022466A1 (fr) * 2022-07-29 2024-02-01 Wuxi Biologics (Shanghai) Co., Ltd. Ebna1 modifié à fonction améliorée pour l'expression de protéines dans des cellules de mammifère

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006082313A2 (fr) * 2005-02-07 2006-08-10 Centre National De La Recherche Scientifique Epitopes t cd4+ des antigenes de latence de type i et ii du virus epstein-barr aptes a etre reconnus par la majorite des individus db la population caucasienne et leurs applications
FR2881746A1 (fr) * 2005-02-07 2006-08-11 Centre Nat Rech Scient Epitopes t cd4+des antigenes de latence de type i et ii du virus epstein-barr aptes a etre reconnus par la majorite des individus de la population caucasienne et leurs applications
WO2006082313A3 (fr) * 2005-02-07 2007-01-11 Centre Nat Rech Scient Epitopes t cd4+ des antigenes de latence de type i et ii du virus epstein-barr aptes a etre reconnus par la majorite des individus db la population caucasienne et leurs applications
WO2006096989A3 (fr) * 2005-03-17 2007-09-13 Ca Nat Research Council Vecteurs d'expression pour l'expression genetique transitoire et cellules mammaliennes les exprimant
EP1861498A2 (fr) * 2005-03-17 2007-12-05 National Research Council Of Canada Vecteurs d'expression pour l'expression genetique transitoire et cellules mammaliennes les exprimant
EP1861498A4 (fr) * 2005-03-17 2009-06-24 Ca Nat Research Council Vecteurs d'expression pour l'expression genetique transitoire et cellules mammaliennes les exprimant
US8551774B2 (en) * 2005-03-17 2013-10-08 National Research Council Of Canada Expression vectors containing a truncated epstein barr nuclear antigen 1 lacking the Gly-Gly-Ala domain for enhanced transient gene expression
CN108061798A (zh) * 2016-11-07 2018-05-22 国药中生生物技术研究院有限公司 检测HBsAg/anti-HBs复合物体系中有效成分的量的方法和应用
CN108061798B (zh) * 2016-11-07 2019-11-08 国药中生生物技术研究院有限公司 检测HBsAg/anti-HBs复合物体系中有效成分的量的方法和应用
WO2024022466A1 (fr) * 2022-07-29 2024-02-01 Wuxi Biologics (Shanghai) Co., Ltd. Ebna1 modifié à fonction améliorée pour l'expression de protéines dans des cellules de mammifère

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