WO2008005506A2 - An ebv inhibitor and methods of making and using the same - Google Patents

An ebv inhibitor and methods of making and using the same Download PDF

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Publication number
WO2008005506A2
WO2008005506A2 PCT/US2007/015502 US2007015502W WO2008005506A2 WO 2008005506 A2 WO2008005506 A2 WO 2008005506A2 US 2007015502 W US2007015502 W US 2007015502W WO 2008005506 A2 WO2008005506 A2 WO 2008005506A2
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ebna3c
peptide
another embodiment
cells
sequence
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PCT/US2007/015502
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French (fr)
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WO2008005506A3 (en
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Erle S. Robertson
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The Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention provides an Epstein Barr virus inhibitor and methods of using the same.
  • Epstein-Barr virus is a member of the herpesvirus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age have been infected. Infants become susceptible to EBV as soon as maternal antibody protection (present at birth) disappears. Many children become infected with EBV, and these infections usually cause no symptoms or are indistinguishable from the other mild, brief illnesses of childhood. In the United States and in other developed countries, many persons are not infected with EBV in their childhood years. When infection with EBV occurs during adolescence or young adulthood, it causes infectious mononucleosis 35% to 50% of the time.
  • Symptoms of infectious mononucleosis are fever, sore throat, and swollen lymph glands. Sometimes, a swollen spleen or liver involvement may develop. Heart problems or involvement of the central nervous system occurs only rarely, and infectious mononucleosis is almost never fatal. There are no known associations between active EBV infection and problems during pregnancy, such as miscarriages or birth defects. Although the symptoms of infectious mononucleosis usually resolve in 1 or 2 months, EBV remains dormant or latent in a few cells in the throat and blood for the rest of the person's life. Periodically, the virus can reactivate and is commonly found in the saliva of infected persons. This reactivation usually occurs without symptoms of illness.
  • EBV also establishes a lifelong dormant infection in some cells of the body's immune system.
  • a late event in a very few carriers of this virus is the emergence of Burkitt's lymphoma and nasopharyngeal carcinoma, two rare cancers that are not normally found in the United States. EBV appears to play an important role in these malignancies, but is probably not the sole cause of disease.
  • Laboratory tests are not always foolproof. For various reasons, false-positive and false- negative results can occur for any test. However, the laboratory tests for EBV are for the most part accurate and specific. Because the antibody response in primary EBV infection appears to be quite rapid, in most cases testing paired acute- and convalescent-phase serum samples will not demonstrate a significant change in antibody level. Effective laboratory diagnosis can be made on a single acute- phase serum sample by testing for antibodies to several EBV-associated antigens simultaneously. In most cases, a distinction can be made as to whether a person is susceptible to EBV, has had a recent infection, has had infection in the past, or has a reactivated EBV infection. Antibodies to several antigen complexes may be measured.
  • antigens are the viral capsid antigen, the early antigen, and the EBV nuclear antigen (EBNA).
  • EBNA EBV nuclear antigen
  • differentiation of immunoglobulin G and M subclasses to the viral capsid antigen can often be helpful for confirmation.
  • the optimal combination of EBV serologic testing consists of the antibody titration of four markers: IgM and IgG to the viral capsid antigen, IgM to the early antigen, and antibody to EBNA.
  • IgM to the viral capsid antigen appears early in infection and disappears within 4 to 6 weeks.
  • IgG to the viral capsid antigen appears in the acute phase, peaks at 2 to 4 weeks after onset, declines slightly, and then persists for life.
  • IgG to the early antigen appears in the acute phase and generally falls to undetectable levels after 3 to 6 months. In many people, detection of antibody to the early antigen is a sign of active infection, but 20% of healthy people may have this antibody for years.
  • Antibody to EBNA determined by the standard immunofluorescent test is not seen in the acute phase, but slowly appears 2 to 4 months after onset, and persists for life. This is not true for some EBNA enzyme immunoassays, which detect antibody within a few weeks of onset.
  • EBV antibody tests such as the early antigen test
  • this result does not necessarily indicate that a patient's current medical condition is caused by EBV infection.
  • a number of healthy people with no symptoms have antibodies to the EBV early antigen for years after their initial EBV infection. Therefore, interpretation of laboratory results is somewhat complex and should be left to physicians who are familiar with EBV testing and who have access to the entire clinical picture of a person. To determine if EBV infection is associated with a current illness, consult with an experienced physician.
  • the current invention provides a peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention further provides a composition comprising a peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention further provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 , thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
  • the present invention further provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus in a subject selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post- transplant lymphoproliferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID
  • the present invention further provides a method of inhibiting hyperproliferation of a B-cell comprising the step of inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein, thereby inhibiting hyperproliferation of a B cell.
  • FIG. 1 E3C-TAT efficiently enters LCLs.
  • Figure IA is Schematic of EBNA3C depicting the 20-amino-acid region chosen for peptide construction. Complete sequences of E3C-TAT and SCR (scrambled) peptides are indicated. Rb, retinoblastoma protein. A, CycHn A. SCF, Skpl/Cullinl/F-box protein. JK, RBP- JK. LZ, leuzine zipper.
  • Figure IB shows a micrograph of LCLs treated with 1 ⁇ M E3C-TAT peptide for 24 hours. Live cells were imaged at 10Ox magnification.
  • Figure 1 C shows a micrograph of LCLs treated with 1 ⁇ M either E3C-TAT peptide or SCR peptide for 24 hours and imaged at 600x magnification by confocal microscopy.
  • Figure 2. depicts a bar graph showing how E3C-TAT disrupts the interaction of EBNA3C with Skp2.
  • Skp2 was in vitro-translated and incubated with either GST or GST-EBNA3C amino acids 90- 190 (E3C) in binding buffer.
  • Peptide either 10 or 25 ⁇ M as indicated, was additionally added to some samples.
  • GST fusion proteins were washed thoroughly with binding buffer and bound proteins were resolved by SDS- 12% PAGE.
  • Figure 3. depicts graphs showing E3C-TAT inhibition of LCL proliferation.
  • Figure 3A are graphs showing the results of 50,000 LCL cells that were seeded into 96-well plates with either 10% or 0.5% bovine growth serum. Cultures were treated with increasing concentrations of E3C-TAT as indicated. Total cells were counted at 0, 48, and 96 hours.
  • Figure 3B are graphs showing the results of 50,000 LCL cells (from two independently established LCLs) that were seeded into 96-well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 ⁇ M. Total cells were counted at 0, 48, and 96 hours.
  • Figure 3C are bar graphs showing the results of, 500,000 LCL cells (from two independently established LCLs) that were stained with PKH26 and seeded into 6- well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 ⁇ M. Cells were assayed for extent of proliferation at 72 hours. Quantification of the cells with reduced fluorescence compared to the control is represented.
  • Figure 4. depicts graphs that E3C-TAT specifically inhibits EBV-immortalized cells.
  • Figure 4A are graphs showing the results of A, 50,000 cells that were seeded into 96-well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 ⁇ M. Total cells were counted at 0, 48, and 96 hours.
  • Figure 4B is a bar graph showing the, Primary B-lymphocytes were infected with B95.8 EBV as described in Methods and seeded into a 96- well plate. 24 wells were treated with no peptide, 24 with 20 ⁇ M E3C-TAT peptide, and 24 with 20 ⁇ M SCR peptide. Transformed wells were scored at 6 weeks.
  • Figure 5. shows that E3C-TAT inhibits B-lymphocyte outgrowth in an EBV-positive cancer patient.
  • Figure 5A is a bar graph showing PBMCs that were isolated from an EBV-positive, immunosuppressed patient and seeded into a 96-well plate as described in Methods. 24 wells were treated with no peptide, 24 with 20 ⁇ M E3C-TAT peptide, and 24 with 20 ⁇ M SCR peptide. Transformed wells were scored at 4 weeks.
  • Figure 5B shows a micrograph of representative LCL-like outgrowths from SCR peptide-treated wells.
  • Figure 5C is a graph showing the, LCL-like outgrowths from SCR peptide-treated wells, stained with either CDl 9 monoclonal or isotype -control antibody and assayed for fluorescence intensity by FACS.
  • Figure 5D depicts a Western-blot membrane showing a three independent LCL-like outgrowths from SCR peptide-treated wells were lysed in SDS-loading buffer (approximately 1 million cells) and resolved by SDS-8% PAGE. Western blotting was with either EBNA3C monoclonal or actin control antibody. BJAB cells were used as an EBV-negative control.
  • Int-C and Int-N refer to the N- and C-terminal halves of the intein; Luciferase is Firefly Luciferase and DEVD is the Caspase-3 recognition sequence.
  • Figure 6. shows that EBNA3C destabilizes the Rb protein and enhances Rb ubiquitination.
  • Figure 6A depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pA3M-Rb, 1 ⁇ g pCDNA3- p27, and 0, 5, or 15 ⁇ g pA3M-EBNA3C as indicated. Total protein was normalized by Bradford assay and resolved by SDS-PAGE.
  • Figure 6B depicts a gel and a graph showing HEK 293T cells, transfected with pA3M-Rb and either pA3M-EBNA3C (bottom gel) or vector control (top gel).
  • FIG. 6C depicts a table and gels showing HEK 293T cells, transfected with pA3M-Rb, pCDNA3-HA-Ub, and pSG5-EBNA3C as indicated. Samples were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody.
  • Figure 6D depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pCM V-HA-Rb, pCDNA3-HA-pl07, or pCDNA3-HA-pl30 as indicated. Samples were additionally transfected with 10 ⁇ g either pA3M- EBN A3C or vector control.
  • Figure 7. shows that EBNA3C regulates Rb in BJAB and SAOS-2 cells.
  • Figure 7A depicts a table and a gel showing BJAB cells, transfected with 20 ⁇ g pCMV-HA-Rb and 20 ⁇ g either pA3M- EBN A3C or vector control as indicated.
  • Figure 7B depicts micrographs and a bar graph showing SAOS-2 flat cell analysis as described in Methods. After 2 weeks of puromycin selection, 40 200x fields were monitored for flat cell formation.
  • Figure 8. shows that EBNA3C forms complexes with Rb under conditions of proteasome inhibition.
  • Figure 8A depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pCMV-HA-Rb and 10 ⁇ g either pA3M vector (left) or pA3M-EBNA3C (right). At 36 hours, samples were treated with either 20 ⁇ g/mL MG-132 (upper) or DMSO vehicle control (lower).
  • Figure 8B depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pA3M-EBNA3C and 10 ⁇ g pCMV-HA-Rb.
  • Figure 8C depicts a gel showing samples, transfected with 10 ⁇ g of the indicated pA3M-EBNA3C truncation mutants and 10 ⁇ g pCM V-H A-Rb and treated as for Figure 8B.
  • EBNA3C truncation proteins are marked by asterisks on the myc western blot.
  • Figure 9. shows that EBNA3C amino acids 140-149 are critical regulators of Rb stability.
  • Figure 9A depicts a gel showing EBNA3C constructs in-vitro translated and incubated with either GST or GST-Rb.
  • Figure 9B depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pCMV- HA-Rb and 10 ⁇ g of the indicated pA3M-EBNA3C expression plasmids.
  • EBNA3C proteins are marked by asterisks on the myc western blot
  • Figure 9C depicts a gel showing HEK 293T cells, transfected as for Figure 9B.
  • Figure 9D depicts a gel showing Rb that was in vitro-translated and incubated with 10-20 ⁇ g either GST, GST-EBNA3C 130-159, or GST-EB NA3C 130-159 F144A.
  • Figure 9E depicts a gel showing, HEK 293T cells, transfected as for Figure 9B.
  • Figure 10 shows that EBN A3C amino acids 141 - 145 are critical for both the regulation of Rb stability and SCF ⁇ 2 recruitment.
  • Figure 1OA shows an alignment of human papillomavirus type 16 E7 protein with the region of EBNA3C that regulates Rb stability. Boxed amino acids (EBNA3C 141 - 145) were mutated to alanines in the subsequent experiments.
  • Figure 1OB depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pCMV-HA-Rb and 10 ⁇ g either pA3M-EBNA3C 1 -200, pA3M- EBNA3C 1-200ILCV14S to AAAAARS (A5)7 or pA3M-EBNA3C 1 -129.
  • Figure 1OC depicts a gel showing EBNA3C 1-200 and EBNA3C 1 -200 A 5 that were in-vitro translated and incubated with either GST or GST-Rb.
  • Figure 1OD depicts a gel showing EBNA3C 1-200 and EBNA3C 1-200 A 5 were in Wire-translated and incubated with either GST or GST-Skp2.
  • Figure 1OE depicts a gel showing HEK 293T cells, transfected with 10 ⁇ g pCDNA3-HA-Rocl and 10 ⁇ g either pA3M, pA3M- EBNA3C 1-200, or pA3M EBNA3C 1-200 A 5 . After 36 hours, samples were immunoprecipitated for the myc tag on EBNA3C. IP bands were quantified and presented as the ratio of Roc] to EBNA3C.
  • Figure 11. shows that disruption of Skp2 abrogates the destabilization of Rb by EBNA3C.
  • Figure 1 I A shows a table, a Western blot membrane and a bar graph of 10 million HEK 293T cells, transfected with 10 ⁇ g pA3M-EBNA3C, 10 ⁇ g pA3M-Skp2 ⁇ F, and 10 ⁇ g pCMV-HA-Rb as indicated.
  • Figure H B shows a table, a Western blot membrane and a bar graph of 10 million BJAB cells, transfected as for Figure H A.
  • Figure HC shows a table, a Western blot membrane and a bar graph of 10 million U2OS cells, transfected as for Figure 1 IA.
  • Figure 1 I D shows a table, Western blot membranes and bar graphs of HEK 293T cells, seeded into plates and transfected with 200 nM siRNA at 24 hours. 48 hours after seeding, cells were again transfected with siRNA as well as pCMV-HA-Rb and pA3M-EBNA3C as indicated.
  • Figure 12. shows that deletion of the p27carboxy terminus abrogates EBNA3C rescue of kinase activity.
  • Figure 12A is a schematic representation showing a deletion of the carboxy-termina! 13 residues of p27. Phosphorylation on threonine-187 is a critical regulator of p27 stability via the SCF Skp2 complex.
  • Figure 12B depicts a table and gels showing U2OS cells, transfected with expression constructs for cyclin A, Cdk2, full-length p27, p27 deleted for the carboxy-terminal 13 residues, and EBNA3C as indicated.
  • Figure 13 shows that EBNA3C associates with ubiquitination activity and is itself ubiquitinated.
  • Figure 13A depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C or EBNA3C truncation expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
  • Figure 13B depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc- tagged EBNA3C truncation expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
  • Figure 13C is a schematic of EBNA3C demonstrating a region of the molecule that is essential for ubiqutination.
  • Figure 14 shows that EBNA3C amino adds 90-190 recruit SCF 5 ⁇ 2 core components.
  • Figure 14A depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C expression plasmids and point mutants as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
  • Figure 14B is a schematic of the sCF 511152 complex.
  • Figure 14C depicts a gel showing the expression plasmids for Skp2, Skpl, Cull and Rocl that were in vitro-translated and individually incubated with either GST or GST- EBNA3C 90-190 fusion proteins pre-bound to glutathione sepharose beads.
  • Figure 14D depicts gels showing HEK 293T cells, transfected with myc-tagged Skp2 (left column), myc-tagged Skp2, myc- tagged Skpl, and HA-tagged Cull (middle column), or myc-tagged Skp2, myc-tagged Skpl, HA-tagged Cull, and HA-tagged Rocl (right column) expression plarnids.
  • FIG. 14E depicts gels showing HEK 293T cells, transfected with HA- Ub, un-tagged EBNA3C, and either myc-tagged Skp2 or myc-tagged Skpl expression plasmids as indicated.
  • Cells were harvested at 36 hours and total protein was immunoprecipitated with myc- specific antibody. Samples were resolved by 12% SDS-PAGE. Western blotting was by stripping and re-probing the same membrane.
  • Figure 15. shows that EBNA3C amino acids 130-159 and 160-190 both contribute to EBNA3C ubiquitination and SCF ⁇ 2 recruitment.
  • Figure 15A depicts Westem-blot membranes showing HEK 293 T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc- specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
  • Figure 15B depicts gels showing HEK 293T cells, transfected with either myc-tagged Skp2 or both HA-taggcd Cull and HA-tagged Rod expression plasmids.
  • Cells were harvested at 36 hours and total protein was incubated with either GST, GST-EBNA3C 90-129, GST- EBNA3C 130-159, or GST-EBNA3C 160-190 fusion proteins pre-bound to Glutathione Sepharose beads.
  • Figure 15C depicts Western-blot membranes showing HEK 293T cells, transfected with HA- tagged ubiquitin, HA-tagged Rocl, and myc-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
  • Figure 16. shows that BBNA3C associates with Skp2 in cells.
  • Figure 16A depicts a Western- blot membrane showing 100 million EBV-positive LCL cells lysed in RIPA buffer and protein complexes were immunoprecipitated with Skp2-reactive serum. Samples were resolved by SDS- PAGE. Western blotting for the indicated proteins was by stripping and reprobing the same blot, L, 5% total protein lysate. CS, control serum. IP, anti-Skp2 immunoprecipitation.
  • Figure 16B depicts Western-blot membranes showing HEK 293T cells, transfected with pCDNA3-Skp2 encoding un- tagged Skp2 and pA3M-EBNA3C expression plasmids encoding either full-length EBNA3C or EBNA3C truncations as indicated. All EBNA3C proteins were tagged with the myc epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific serum. Samples were resolved by 10% SDS-PAGE and probed by western blot.
  • Figure 16C depicts micrographs showing HeLa cells transfected with plasmid expressing full-length myc-tagged EBNA3C and un-tagged Skp2.
  • Figure 17. shows that EBNA3C associates with Rocl in cells.
  • Figure 17A depicts western blot membranes of HEK 293T cells, transfected with pCDNA3-HA-Rocl encoding Rocl tagged with the HA-epitope and pA3M-EBNA3C encoding either full-length EBNA3C or EBNA3C truncations as indicated. All EBNA3C proteins were tagged with the myc epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific serum. Samples were resolved by 10% SDS-PAGE and probed by western blot.
  • Figure 17B depicts western blot membranes of HEK 293T cells, transfected with expression plasmids either for myc-tagged EBNA3C truncations or for HA-tagged Cull .
  • Cells were harvested at 36 hours and total protein was incubated with either GST or GST-Roc 1 pre-bound to Glutathione Sepharose beads. Samples were resolved by 10% SDS-PAGE and probed by western blot.
  • Figure 17C depicts western blot membranes of HEK 293T cells, transfected with pA3M-EBNA3C 1- 365 encoding EBNA3C amino acids 1 -365 tagged with the myc epitope.
  • Cells were also transfected with pCDNA3-HA-Rocl or pCDNA3-HA-Rocl 36-108 encoding full-length Rocl or a truncated form of Rocl, respectively, both tagged with the HA epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE and probed by western blot.
  • Figure 18. shows that Rocl stimulates EBNA3C ubiquitination.
  • Figure 18A depicts Western blot membranes of HEK 293T cells transfected, as indicated, with expression plasmids for HA-tagged ubiquitin, myc-tagged EBNA3C amino acids 1-365, and either HA-tagged Roc l or a Roc l mutant, C53A/C56A, which is deficient for ubiquitin recruitment.
  • Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS- PAGE. Western blotting was by stripping and re-probing the same membrane.
  • Figure 18A depicts gels of HEK 293T cells transfected, as indicated, with expression plasmids for HA-tagged ubiquitin and HA-tagged Rocl. Cells were harvested at 36 hours and cell lysates were resolved by 10% SDS-PAGE.
  • Figure 19 shows that EBNA3C stimulates p27 ubiquitination in HEK 293T cells and decreasesp27 stability in an SCF-2-dependent degradation assay.
  • Figure 18A depicts a gel showing HEK 293T cells, transfected with myc-tagged p27, HA-Ub, and un-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specif ⁇ c antibody. Samples were resolved by 10% SDS-PAGE.
  • Figure 18B depicts a gel showing HEK 293T cells, transfected with Skp2-myc, Skpl, Cull, Rocl, and Cksl expression plasmids or alternatively with an equal amount of vector DNA.
  • Cells were Iysed at 36 hours and the SCF Skp2 complex was immunoprecipitated via the myc-tag on Skp2. Immunoprecpitates were then incubated with 35S-labelled in vitro-translated p27. Degradation reactions were incubated at 30 0 C with samples taken for SDS-PAGE at the times indicated. Quantification of p27 autoradiography was by ImageQuant software. All values were normalized to the zero time point.
  • HEK 293T cells were transfected as described above, but with a Skp2 mutant deleted for the F-box (Skp2AF) replacing full-length Skp2.
  • Cells were Iysed at 36 hours and the sCF Skp2 ⁇ F complex was immunoprecipitated via the myc-tag on Skp2AF.
  • Degradation assays were as described above with samples taken at the indicated times.
  • cells were additionally transfected with an un-tagged EBNA3C expression construct.
  • HEK 293 T cells were transfected as described above with immunoprecipitation for the myc-tag on full-length Sk ⁇ 2.
  • cells were additionally transfected with an un-tagged EBNA3C expression construct.
  • FIG. 20 shows that p27, but neither Skp2 nor EBNA3C, is stabilized in LCLs by treatment with the proteasome inhibitor MG-132.
  • the Western blot membrane and the accompanying graph represent asynchronously growth of LCLs, treated for eight hours with either 10 ⁇ g/mL MG- 132 or DMSO control. Cells were harvested at the times indicated. Lysates were normalized by Bradford assay and resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same membrane. Bands were quantified, and the data for each time point was plotted in the line graph as MG-132-band intensity divided by DMSO-band intensity.
  • Figure 21 shows that EBNA3C recruits ubiquitination activity to cyclin A complexes.
  • Figure 19A depicts a graph and a gel showing HEK 293T cells, transfected with Skp2, Skpl, Cull, Rocl, and Cksl expression plasmids. Cells were additionally transfected with expression plasmids for EBNA3C amino acids 1 -365 or EBN A3C amino acids 621-992, both tagged with the myc epitope. Cells were Iysed at 36 hours, immunoprecipitated with myc-specific serum and incubated with 35S-labelled in- vitro-translated p27.
  • FIG. 19B depicts a graph showing HEK 293T cells, transfected with Skp2,, Skpl, Cull, Rocl, and Cksl expression plasmids. Cells were additionally transfected with either vector (squares) or EBNA3C (circles). Cells were lysed at 36 hours and incubated with GST-cyclin A fusion protein purified from bacteria and pre-bound to glutathione sepharose beads.
  • FIG. 21 C depicts a schematic representation of a model suggesting that the amino terminus of EBNA3C may serve as scaffolding for the assembly of cyclin A/cdk2/p27 with SCFskp2. This may facilitate p27 ubiquitination and, ultimately, degradation by the 26S proteasome.
  • the present invention provides a peptide comprising a protein transduction domain and an Epstein-Barr nuclear antigen 3C (EBNA3C) peptide.
  • EBNA3C Epstein-Barr nuclear antigen 3C
  • the present invention provides that the EBNA3C peptide is encoded by an amino acid sequence comprising HILCFVMAAR (SEQ ID NO:1).
  • the EBNA3C peptide is a variant of SEQ ID No: 1.
  • the EBNA3C peptide is an isomer of SEQ ID No: 1.
  • the EBNA3C peptide is a fragment of SEQ ID No: 1.
  • the EBNA3C peptide of the present invention consists of amino acid 140-149 of the EBNA3C protein.
  • the EBNA3C protein is a full length protein.
  • the EBNA3C peptide is a fragment of the EBNA3C protein.
  • the present invention provides that the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation.
  • the present invention provides that the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C.
  • the present invention provides that EBNA3C is a latent protein which is necessary for the efficient immortalization of primary human B cells by EBV.
  • the present invention provides that in co-operation with activated ras, EBNA3C has oncogenic activity in fibroblasts.
  • the present invention provides that EBNA3C disrupts the cyclin/CDK-pRb-E2F pathway, which regulates cell cycle progression at the restriction point (R-point) in Gl of the proliferation cycle.
  • the present invention provides that EBNA3C abrogates the mitotic spindle checkpoint.
  • the present invention provides that EBV encoded oncoprotein EBNA3C has the remarkable capacity to permit complete nuclear division even when growth inhibitory signals have been activated.
  • the present invention provides that EBNA3C overcomes the restriction checkpoint that causes cells to arrest in Gl of the cell cycle.
  • the present invention provides that EBNA3C expression is associated with progression through both S and G2 phases and the completion of mitosis.
  • the present invention provides that expression of EBNA3C disrupts the cell cycle regulatory machinery.
  • the present invention provides that EBNA3C provides internal signals that allow the nucleus to complete the cell cycle.
  • the present invention provides that EBNA3C is coupled to a protein transduction domain. In another embodiment, the present invention provides that EBNA3C is chemically linked to a protein transduction domain. In another embodiment, the present invention further provides that EBNA3C is fused to a protein transduction domain. In another embodiment, the present invention further provides a protein transduction domain is fused to the carboxy terminal of said EBNA3C peptide.
  • the present invention provides that Protein transduction domain (PTD) peptides are used to enhance cellular uptake of EBNA3C peptide of the present invention.
  • PTD Protein transduction domain
  • the PTD of the present invention is Functionalised Antennapedia (Antp amino acid 43- 58).
  • the PTD of the present invention is HlV transcriptional transactivator (TAT) (Tat amino acid 47-57).
  • the present invention provides that the HIV Tat peptide is encoded by an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO: 3).
  • the HIV Tat peptide is a variant of SEQ ID No: 3.
  • the HIV Tat peptide is an isomer of SEQ ID No: 3.
  • the HIV Tat peptide is a fragment of SEQ ID No: 3.
  • the present invention provides that one PTD is coupled to an EBN A3C peptide of the present invention. In another embodiment, the present invention provides that more than one PTD is coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1 -100 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1- 10 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1-3 PTDs are coupled to an EBN A3C peptide of the present invention. In another embodiment, the present invention provides that 2-4 PTDs are coupled to an EBNA3C peptide of the present invention.
  • the present invention provides that 3-8 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 8-15 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1-30 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 15-45 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 20-60 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 40-80 PTDs are coupled to an EBNA3C peptide of the present invention.
  • the present invention provides that 30-500 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 70-100 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 60-90 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that all PTDs utilized are encoded by the same amino acid sequence. In another embodiment, the present invention provides that the PTDs utilized are derived from different genes and thus encoded by different amino acid sequences. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides that the transporter peptide mediates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that the transporter peptide facilitates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that the transporter peptide enhances the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell.
  • the present invention provides that a PTD mediates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that a PTD facilitates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that a PTD enhances the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell.
  • a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 1 -100 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 1-5 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 2-7 folds.
  • a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 5-10 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 7-15 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 10-18 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 15-25 folds.
  • a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 20-30 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 25-50 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 40-70 folds.
  • a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 50-75 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 70-100 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 80-100 folds.
  • the present invention provides that the EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of the EBNA3C peptide and a second peptide in the carboxy terminus of the EBNA3C peptide, wherein the sequence of the second peptide comprises the sequence QRLQDIRR (SEQ ID NO: 2)
  • the second peptide is a variant of SEQ ID No: 2.
  • the second peptide is an isomer of SEQ ID No: 2.
  • the second peptide is a fragment of SEQ ID No: 2.
  • the second peptide is comprises amino acids 150-157 of the EBNA3C protein.
  • the EBNA3C peptide is encoded by the amino acids sequence TQHILCFVMAARQRLQDIRR (SEQ ID NO: 4)
  • the EBNA3C peptide is a variant of SEQ ID No: 4.
  • the EBNA3C peptide is an isomer of SEQ ID No: 4.
  • the EBNA3C peptide is a fragment of SEQ ID No: 4.
  • the EBNA3C peptide is comprises amino acids 138- 157 of the EBNA3C protein.
  • the EBNA3C-TAT peptide (E3C-TAT) is encoded by the amino acids sequence TQHILCFVMAARQRLQDIRRYGRKKRRQRRR (SEQ ID NO: 5).
  • the EBNA3C-TAT peptide is a variant of SEQ ID No: 5.
  • the EBNA3C-TAT peptide is an isomer of SEQ ID No: 5.
  • the EBNA3C-TAT peptide is a fragment of SEQ ID No: 5.
  • the EBNA3C-TAT peptide is encoded by amino acids 138-157 of the EBNA3C (SEQ ID NO: 4) and the amino acids encoded the TAT fragment (SEQ ID NO: 3).
  • the EBNA3C-TAT peptide comprises EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 4 fused to the TAT fragment encoded by the amino acids sequence of SEQ ID NO: 3.
  • the EBNA3C-TAT peptide comprises EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1 fused to the TAT fragment encoded by the amino acids sequence of SEQ ID NO: 3.
  • the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1.
  • the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 4.
  • the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1.
  • the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C-TAT peptide encoded by the amino acids sequence of SEQ ID NO: 5.
  • a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C-TAT peptide encoded by the amino acids sequence of SEQ ID NO: 5.
  • the present invention provides a composition comprising an peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention provides a composition comprising an peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4.
  • the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4.
  • the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: I .
  • the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4.
  • the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 and the sequence of the TAT fragment comprises the sequence set forth in SEQ ID NO: 3.
  • the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4 and the sequence of the TAT fragment comprises the sequence set forth in SEQ ID NO: 3.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 , thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4, thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a transporter peptide.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a PTD.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a TAT peptide.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide fused to a TAT peptide.
  • the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide fused to a TAT peptide, wherein the sequence of the EBNA3C-TAT peptide comprises the sequence set forth in SEQ ID NO: 5.
  • the methods and peptides of the present invention provide an EBNA3C peptide fused to a TAT peptide.
  • an EBNA3C peptide fused to a TAT peptide results in protein called a hybrid or fusion protein, which has characteristics that combine those of EBNA3C peptide and a TAT peptide.
  • the present invention provides that a fusion protein further comprises a "reporter” such as fluorescein isothiocyanate (FITC) or green fluorescent protein (GFP).
  • FITC fluorescein isothiocyanate
  • GFP green fluorescent protein
  • the present invention provides that a fusion protein of the invention comprises FITC- EBNA3C- TAT peptide.
  • an EBV infected cell is an EBV transformed cell.
  • the methods of the present invention provide that an EBV transformed cell is a cell that undergone cancerous transformation as a result of an EBV infection.
  • the methods of the present invention provide that an EBV infected cell contains EBV proteins.
  • the methods of the present invention provide that an EBV transformed cell contains EBV proteins.
  • the methods of the present invention provide that an EBV transformed cell expresses EBV proteins, by an animal cell upon infection by a cancer-causing virus.
  • an EBV infected B- cell is an EBV transformed B-cell.
  • the methods of the present invention provide that an EBV transformed B-cell is a B-cell that undergone cancerous transformation as a result of an EBV infection.
  • the methods of the present invention provide that an EBV infected B-cell contains EBV proteins.
  • the methods of the present invention provide that an EBV transformed B-cell contains EBV proteins.
  • the methods of the present invention provide that an EBV transformed B-cell expresses EBV proteins.
  • the present invention provides that EBV utilizes its latency proteins to drive B-lymphocyte proliferation, inducing a phenotype that closely mimics antigen-stimulated B- lymphocyte activation and expansion.
  • the present invention provides that a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell comprises inhibiting hyperproliferation of an Epstein-Barr virus-infected cell.
  • the present invention provides that hyperproliferation is an abnormally high rate of cell division.
  • the present invention provides that hyperproliferation occurs, for example: in tumours and cancers.
  • the present invention provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post-transplant lymphoproHferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C peptide of the invention.
  • an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatit
  • the present invention provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post-transplant lymphoproHferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C-TAT fusion peptide of the invention.
  • an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson
  • the present invention provides a method for treating, reducing the incidence, delaying the onset or progression, or reducing and/or abrogating the symptoms associated with a disease caused by EBV infection.
  • the present invention provides a method for treating, reducing the incidence, delaying the onset or progression, or reducing and/or abrogating the symptoms associated with a disease caused by EBV B-cell transformation.
  • the present invention provides that compositions comprising EBNA3C peptides inhibit the progression of a disease caused by EBV cell infection or cell transformation.
  • the present invention provides that compositions comprising EBNA3C peptides abrogate a disease caused by EBV cell infection or cell transformation.
  • compositions comprising EBNA3C peptides ameliorate the condition of a subject infected by EBV.
  • the present invention provides that compositions comprising EBNA3C peptides can protect a subject from an EBV infection.
  • compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV infection.
  • the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV cell transformation.
  • compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV B-cell transformation.
  • the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a malignant transformation caused by an EBV.
  • the present invention provides that compositions comprising EBN A3C peptides are administered to subjects at risk of being infected with an EBV.
  • the present invention provides that the subject is a transplant recipient.
  • the present invention provides that the subject is immunosuppressed.
  • the present invention provides that the subject resides in a developing nation.
  • the present invention provides that the disease is infectious mononucleosis.
  • the present invention provides that EBV causes infectious mononucleosis, also known as 'glandular fever 1 , 'Mono' and 'Pfeiffer's disease 1 .
  • the present invention provides that infectious mononucleosis is caused when a subject is first exposed to the virus during or after adolescence.
  • the present invention provides that it is therefore predominantly found in the developed world, as most children in the developing world are found to be already infected by around 18 months of age.
  • the present invention provides that the disease is an EBV-associated malignancy. In another embodiment, the present invention provides that EBV induces cancer formation. In another embodiment, the present invention provides that EBV causes Burkitt's lymphoma. In another embodiment, the present invention provides that EBV causes nasopharyngeal carcinoma. In another embodiment, the present invention provides that Burkitt's lymphoma is a type of Non-Hodgkin's lymphoma and is most common in equatorial Africa and is co-existent with the presence of malaria. In another embodiment, the present invention provides that Malaria infection causes reduced immune surveillance of EBV immortalised B cells, so allowing their proliferation.
  • the present invention provides that this proliferation increases the chance of a mutation to occur.
  • the present invention provides that mutations can lead to the B cells escaping the body's cell-cycle control, so allowing the cells to proliferate unchecked, resulting in the formation of Burkitt's lymphoma.
  • the present invention provides that Burkitt's lymphoma commonly affects the jaw bone, forming a huge tumour mass.
  • the present invention provides other B cell lymphomas that arise in immunocompromised subjects such as those with AIDS that are more susceptible to EBV infection.
  • the present invention provides subjects undergone organ transplantation with associated immunosuppression (Post-Transplant Lymphoproliferative Disorder (PTLPD)) that are more susceptible to EBV infection.
  • the present invention provides smooth muscle tumors that are associated with EBV in malignent patients.
  • nasopharyngeal carcinoma is a cancer found in the upper respiratory tract, most commonly in the nasopharynx, and is linked to the EBV virus.
  • the present invention provides that it is found predominantly in Southern China and Africa, due to both genetic and environmental factors.
  • the present invention provides that the disease is Chronic Fatigue Syndrome. In another embodiment, the present invention that EBV causes chronic fatigue syndrome. In another embodiment, the present invention provides that the disease is Multiple Sclerosis. In another embodiment, the present invention provides that the disease is an autoimmune disease.
  • the present invention provides a method of inhibiting hyperproliferation of a B-cell comprising the step of inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein, thereby inhibiting hyperproliferation of a B cell.
  • the present invention provides that the B-cell is infected with an Epstein Barr virus.
  • the present invention provides that the B-cell is transformed by an Epstein Ban- virus.
  • Rb degradation is an ubiquitin- proteasome complex mediated degradation.
  • the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the translation of EBNA3C mRNA with an antisense agent.
  • the antisense comprises RNAi.
  • the present invention provides that antisense comprises a Ribozyme.
  • the present invention provides that antisense comprises antisense DNA compounds.
  • the present invention provides that one skilled in the art will choose and use the optimal antisense technology according to the methods of the invention.
  • the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide. In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
  • the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4.
  • the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C- TAT peptide, wherein the sequence of an EBNA3C-TAT peptide comprises the sequence set forth in SEQ ID NO: 5.
  • the present invention provides that EBNA3C is transcriptional regulator targeting RBP-JK and Spi-1/Spi-B transcription factors to regulate viral promoters such as Cp and LMPl.
  • the present invention provides that EBNA3C regulates histone acetylation as it apparently targets and regulates both histone acetyl transferase activity and histone deacetylase complexes including HDACl , HDAC2, and the corepressors mSin3a and NcoR and thus controls cell cycle and differentiation.
  • the present invention provides that EBNA3C regulates transcription of the EBV oncogene LMPl in a cell cycle dependent manner and targets Rb-E2F regulatory pathways leading to the accumulation of cells in the S/G2 phase of the cell cycle.
  • the present invention provides that EBNA3C targets cyclin A complexes in cells and enhances cyclin A- dependent kinase activity by disrupting the cdk inhibitor p27 from kinase complexes.
  • the present invention provides that EBNA3C targets the SCF sl ⁇ 2 E3 ubiquitin ligase complex.
  • the present invention provides that components of SCF Skp2 , Skpl and Skp2, are also known as S-phase Kinase-associated Proteins that complex with cyclin A in tumor cells, but not primary cells.
  • SCF 811112 complex consists of additional core components Cull and Rocl and that this complex plays a critical role in regulating the stability of diverse cell cycle proteins which include p27, E2F, and c-myc.
  • SCF ⁇ 2 is an E3 ubiqutin ligase functionally linking specific substrates to the ubiqutin-activating and ubiquitin-conjugati ⁇ g machinery.
  • this link targets substrates for poly-ubiquitination and ultimately degradation by the 26S proteasome.
  • the present invention provides that EBNA3C regulates p27 stability by manipulating the oncoprotein Skp2 as well as other SCF components. Regulation of p27 stability by EBNA3C is most likely at the level of its interaction with cyclin A complexes, providing a potential mechanism by which EBNA3C disrupts p27 from cyclin A complexes and ultimately stimulates cyclin A-dependent kinase activity.
  • the present invention provides that EBNA3C targets cell cycle regulators resulting in deregulation of the mammalian cell cycle.
  • the present invention provides that EBNA3C manipulates the Rb-E2F axis.
  • the present invention provides that EBNA3C causes the accumulation of cells in S/G2 and prevents the induction of certain mitotic checkpoints resulting in aberrant nuclear division.
  • the present invention provides that EBNA3C regulates the activity and stability of the retinoblastoma protein (Rb).
  • the present invention provides that EBNA3C regulates Rb stability by recruiting the SCF ⁇ 2 ubiquitin ligase complex which mediates the ubiquitination and degradation of Rb.
  • the present invention provides that regulation of Rb stability is mapped to a small region within the conserved domain of EBNA3C, amino acids 140-149.
  • the present invention provides that this region of EBNA3C regulates the SCF Skp2 complex.
  • the present invention provides that EBNA3C serves as a direct link between Rb and SCF complexes.
  • the present invention provides that disruption of EBNA3C-targeted cell cycle pathways is a viable method for treating EBV-driven diseases.
  • the present invention provides that disruption of EBNA3C-targeted cell cycle pathways is a viable method for treating EBV-driven cancers.
  • the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects the proliferation of established EBV-infected cells.
  • the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects the de novo immortalization of B-lymphocytes by EBV.
  • the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects both the proliferation of established EBV-infected cell lines and the de novo immortalization of primary B-lymphocytes by EBV.
  • the present invention provides that the EBNA3C-TAT peptide inhibits the proliferation of previously established EBV-infected cell.
  • the present invention provides that the EBNA3C peptide blocks the spontaneous emergence and outgrowth of EBV-transformed cells.
  • the invention provides that a composition is a pharmaceutical composition.
  • the pharmaceutical compositions containing the EBNA3C-TAT peptide can be administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially, intra vaginal Iy or intratumorally.
  • the dosage of the EBNA3C-TAT peptide may be in the range of 0.1 -80 mg/day. In another embodiment, the dosage is in the range of 0.1 -50 mg/day. In another embodiment, the dosage is in the range of 0.1 -20 mg/day. In another embodiment, the dosage is in the range of 0.1-10 mg/day. In another embodiment, the dosage is in the range of 0.1-5 mg/day. In another embodiment, the dosage is in the range of 0.5-5 mg/day. In another embodiment, the dosage is in the range of 0.5-50 mg/day. In another embodiment, the dosage is be in the range of 5-80 mg/day.
  • the dosage is in the range of 35-65 mg/day. In another embodiment, the dosage is in the range of 35-65 mg/day. In another embodiment, the dosage is in the range of 20-60 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 45-60 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 60-120 mg/day. In another embodiment, the dosage is in the range of 120-240 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 240-400 mg/day.
  • the dosage is in a range of 45-60 mg/day. In another embodiment, the dosage is in the range of 15-25 mg/day. In another embodiment, the dosage is in the range of 5-10 mg/day. In another embodiment, the dosage is in the range of 55-65 mg/day. In one embodiment, the dosage is 20 mg/day. In another embodiment, the dosage is 40 mg/day. In another embodiment, the dosage is 60 mg/day.
  • a unit dosage form used may comprise tablets, capsules, lozenges, chewable tablets, suspensions, emulsions and the like.
  • Such unit dosage forms comprise a safe and effective amount of the desired compound, or compounds, each of which is in one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or in another embodiment, about 0.5 or 10 mg to about 210 mg/70 kg.
  • the pharmaceutically-acceptable carrier suitable for the preparation of unit dosage forms for peroral administration are well-known in the art.
  • Tablets typically comprise conventional pharmaceutically- compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder-mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets.
  • inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose
  • binders such as starch, gelatin and sucrose
  • disintegrants such as starch, alginic acid and cro
  • Capsules typically comprise one or more solid diluents disclosed above.
  • the selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention, and can be readily made by a person skilled in the art.
  • the oral dosage form comprises predefined release profile.
  • the oral dosage form of the present invention comprises an extended release tablets, capsules, lozenges or chewable tablets.
  • the oral dosage form of the present invention comprises a slow release tablets, capsules, lozenges or chewable tablets.
  • the oral dosage form of the present invention comprises an immediate release tablets, capsules, lozenges or chewable tablets.
  • the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient as known to one skilled in the art.
  • Peroral compositions may comprise liquid solutions, emulsions, suspensions, and the like.
  • the pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art.
  • Such liquid oral compositions comprise, in some embodiments, from about 0.012% to about 0.933% of the EBNA3C-TAT perptide, or in another embodiment, from about 0.033% to about 0.7%.
  • compositions for use in the methods of this invention may comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of a EBNA3C-TAT peptide and optionally, other compounds, intended for topical intranasal administration.
  • Such compositions may comprise from about 0.01% to about 10.0% w/v of a subject compound, more preferably from about 0.1 % to about 2.0, which may be used for systemic delivery of the compounds by the intranasal route.
  • compositions comprise dry powders.
  • Compositions may be formulated for atomization and inhalation administration. Such compositions may be contained in a container with attached atomizing means.
  • the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation.
  • suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra- arterial administration.
  • the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.
  • the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
  • a liposome see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
  • the pharmaceutical composition delivered in a controlled release system may be formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989).
  • polymeric materials can be used.
  • a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).
  • compositions which contain active components are well understood in the art, for example by mixing, granulating, or tablet-forming processes.
  • the active therapeutic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • additives customary for this purpose comprise vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.
  • An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compositions may also comprise preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions as needed.
  • the compositions may also comprise local anesthetics or other actives.
  • the compositions can be used as sprays, mists, drops, and the like.
  • substances which can serve as pharmaceutically-acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TweenTM brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants;
  • a pharmaceutically-acceptable carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.
  • compositions may further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone
  • disintegrating agents e.g.
  • cornstarch potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.
  • sodium lauryl sulfate permeation enhancers
  • solubilizing agents e.g., glycerol, polyethylene glycerol
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole
  • stabilizers e.g. hydroxypropyl cellulose, hyroxy propyl methyl cellulose
  • viscosity increasing agents e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum
  • sweeteners e.g. aspartame, citric acid
  • preservatives e.g., Thimerosal, benzyl alcohol, parabens
  • lubricants e.g.
  • stearic acid magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • plasticizers e.g. diethyl phthalate, triethyl citrate
  • emulsifiers e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate
  • polymer coatings e.g., poloxamers or poloxamines
  • coating and film forming agents e.g. ethyl cellulose
  • Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
  • typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, cellulose (e.g. AvicelTM, RC-591 ), tragacanth and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitan (e.g. polysorbate 80).
  • Typical preservatives include methyl paraben and sodium benzoate.
  • Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
  • Dry powder compositions may comprise propellants such as chlorofluorocarbons 12/1 1 and 12/1 14, or, in another embodiment, other fluorocarbons, nontoxic volatiles; solvents such as water, glycerol and ethanol, these include co-solvents as needed to solvate or suspend the active; stabilizers such as ascorbic acid, sodium metabisulfite; preservatives such as cetylpyridinium chloride and benzalkonium chloride; tonicity adjustors such as sodium chloride; buffers; and flavoring agents such as sodium saccharin.
  • propellants such as chlorofluorocarbons 12/1 1 and 12/1 14, or, in another embodiment, other fluorocarbons, nontoxic volatiles
  • solvents such as water, glycerol and ethanol, these include co-solvents as needed to solvate or suspend the active
  • stabilizers such as ascorbic acid, sodium metabisulfite
  • preservatives such as cetylpyridinium chloride and benzal
  • compositions may also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.)
  • polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • paniculate compositions coated with polymers e.g. poloxamers or poloxamines
  • the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors are also comprehended by the invention.
  • compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline.
  • the modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981 ; Newmark et al., 1982; and Katre et al., 1987).
  • Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound.
  • the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
  • the EBNA3C-TAT peptide of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound, and/or in combination with other agents used in the treatment and/or prevention of diseases, disorders and/or conditions, as described herein, as will be understood by one skilled in the art.
  • the compounds of the present invention can be administered sequentially with one or more such agents to provide sustained therapeutic and prophylactic effects.
  • the compounds may be administered via different routes, at different times, or a combination thereof. It is to be understood that any means of administering combined therapies which include the EBNA3C-TAT peptide of this invention are to be considered as part of this invention.
  • the additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS 1 DESK REFERENCE (PDR) (20079), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.
  • the compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient.
  • the combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • E3C-TAT had a sequence of Fitc-TQHILCFVMAARQRLQDIRRYGRKKRRQRRR with an average mass of 4356 daltons.
  • SCR had a sequence of Fitc-IQRTCQRQAIDLRHVRAMFLYGRKKRRQRRR. Purity of the peptide preparations was confirmed by HPLC and peptide identity was confirmed by MS. The 1 1 -amino-acid HIV TAT tag has been previously described (Farrell et al., 2004).
  • GST fusion proteins were purified from bulk E. coli cultures following induction with IPTG.
  • GST fusion proteins were incubated with 35 S-labeled, in vitro- translated protein in binding buffer (I x PBS, 0.1 % NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors).
  • binding buffer I x PBS, 0.1 % NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors.
  • TNT ® T7 Quick Coupled Transcription/Translation System Promega Corporation, Madison, WI.
  • RPMI 1640 (Invitrogen Corporation, Carlsbad, California). RPMI was supplemented with the indicated concentrations of bovine growth serum and either E3C-TAT or SCR peptide. Cultures were fed every 24 hours with 100 ⁇ L fresh medium supplemented with peptide. Total cells were counted with a hemocytometer at 48 and 96 hours. The optimal concentration of PKH26 (Sigma, St Louis, Missouri) for staining and viability of LCL's was determined to be 4 ⁇ M. Staining of cells was per the manufacturer's recommendation.
  • the remaining cells were divided into samples of 500,000 cells and seeded into 1 ml RPMI 1640. Media was supplemented with 10% bovine growth serum and either no peptide (control) or one of E3C-TAT or SCR peptides. Cultures were fed every 24 hours with 1 ml fresh medium supplemented with peptide. The samples were assayed 72 hours post-seeding using a FACScan flow cytometer.
  • B95.8 virus was prepared from fresh LCLs by seeding 10 million cells into 10 mL RPMI plus 10% bovine growth serum. Lytic replication was induced by treating cultures with TPA and butyrate for 6 days. Culture supernatant was harvested and mixed with clarified cell extracts prepared by freeze/thaw of the cell pellet. The pooled viral supernatant was passed through a 0.45 micron filter. Primary B-lymphocytes were prepared from donor blood. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient, and T-cells were removed by rosetting with sheep red blood cells.
  • PBMCs Peripheral blood mononuclear cells
  • B-lymphocytes were mixed with viral supernatant and seeded into 96-well plates with 100,000 cells, 50 ⁇ L viral supernatant, and 20 ⁇ M peptide per well. Wells were fed every three days with 50 ⁇ L fresh RPMI plus 10% bovine growth serum, supplemented with 20 ⁇ M peptide. At 6 weeks, wells were scored for obvious LCL outgrowth by blinded observers.
  • PBMCs were isolated from patient blood by Ficoll gradient and seeded into 96-well plates with 50,000 PBMCs, 50 ⁇ L RPMI plus 10% bovine growth serum, and 20 ⁇ M peptide per well. Wells were fed every three days with 50 ⁇ L fresh RPMI plus 10% bovine growth serum, supplemented with 20 ⁇ M peptide. At 4 weeks, wells were scored for obvious LCL outgrowth.
  • pA3M-EBNAC constructs express either full-length EBNA3C or EBNA3C truncations with a carboxy-terminal myc-tag.
  • pA3M-EBNA3C 1 -200 with amino acids 141-145 (ILCFV) mutated to alanines was prepared by standard overlap extension PCR mutagenesis.
  • the dominant negative pA3M-Skp2 ⁇ F construct was prepared by cloning PCR-amplified cDNA encoding Skp2 amino acids J 54-435 into the previously described pA3M vector.
  • ⁇ Gex-Skp2 was prepared by cloning PCR- amplified cDNA encoding full-length Skp2 into the pGex2TK vector.
  • pA3M-Rb was prepared by cloning PCR-amplified cDNA into the previously described pA3M vector.
  • GST-EBNA3C constructs express truncation mutants and pSG5-EBNA3C.
  • Constructs expressing myc-tagged cyclin A, Skpl, p27, and p27 amino acids 1-185 were prepared by cloning PCR-amplified cDNAs into pA3M vector.
  • Constructs expressing un-tagged Skpl, Skp2, and HA-tagged Roc l 36-108 were prepared by cloning PCR-amplified cDNAs into the pCDNA3.1 vector (Invitrogen Corporation, Carlsbad, California). C53A/C56A point mutations in the Rocl gene were prepared by a standard PCR primer mutagenesis method. Rabbit polyclonal antibodies reactive to cyclin A, cdk2, Skp2, Cull, and p27 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, California).
  • Antibodies reactive to p27 and Rb were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody reactive to the HA tag was purchased from Covance Research Products, Inc. (Berkeley, CA). AlO monoclonal antibody reactive to EBNA3C were used.
  • HEK 293 cells are human embryonic kidney cells transformed by adenovirus type 5 DNA; HEK 293T cells stably express the SV40 large-T antigen.
  • HEK 293T cells were transfected by electroporation with a Bio-Rad Gene Pulser in 0.4 cm-gap cuvettes at 210 Volts and 975 microfarads.
  • BJAB and U2OS cells were transfected by electroporation at 220 Volts and 975 microfarads.
  • U2OS is a human osteosarcoma cell line.
  • HEK 293, 293T, U2OS, and HeLa cells were grown in DMEM (Invitrogen Corporation, Carlsbad, California) supplemented with 10% Fetal Bovine Serum unless otherwise indicated.
  • HEK 293T cells were transfected by electroporation at 210 volts and 975 microFarads in a 0.4 ⁇ m gap cuvette.
  • LCLs were maintained in RPMI (Invitrogen Corporation, Carlsbad, California) supplemented as for DMEM.
  • Pulse-chase experiments 10 million HEK 293T cells were transfected by electroporation with 10 ⁇ g pA3M-Rb and 10 ⁇ g pA3M-EBNA3C. Samples were pulsed 2 hours in pulse-labeling medium ((DMEM deficient for Met and Cys supplemented with 10% FBS) + 150 ⁇ Ci 35 S Trans-label (PerkinElmer, Inc., Wellesley, MA) and then chased with DMEM complete for all amino acids and supplemented with 10% FBS.
  • pulse-labeling medium (DMEM deficient for Met and Cys supplemented with 10% FBS) + 150 ⁇ Ci 35 S Trans-label (PerkinElmer, Inc., Wellesley, MA)
  • SAOS-2 cells were transfected with 4 ⁇ g pBABE-puro and with pA3M-Rb and pA3M- EBNA3C as indicated by Lipofectamine 2000 (Invitrogen Corporation). 48 hours after transfection, cells were selected with puromycin.
  • GST fusion proteins were purified from bulk E. coli cultures following induction with IPTG.
  • lysates were prepared in RlPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). Lysates were pre- cleared and then rotated with either GST control or the appropriate GST fusion protein bound to Glutathione Sepharose beads.
  • GST fusion proteins were incubated with 35 S-labeled, in vitro-translated protein in binding buffer (Ix PBS, 0.1% NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors).
  • binding buffer Ix PBS, 0.1% NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors.
  • transfected HEK 293T samples cells were lysed on ice in 500 ⁇ L RIPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, supplemented with protease inhibitors).
  • RIPA buffer 0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, supplemented with protease inhibitors.
  • LCLs 100 million viable cells were lysed in 1 mL RIPA buffer.
  • Lysates were pre-cleared with either normal rabbit or normal mouse serum and then rotated with 1 ⁇ g specific antibody for 4 hours at 4°C. Immune complexes were precipitated with a 1 : 1 mixture of Protein A- and Protein G-Sepharose beads. Samples were washed, fractionated by SDS-PAGE, and then transferred to a 0.45- ⁇ m nitrocellulose membrane for western blotting. Detection was with a standard chemiluminescence protocol unless otherwise indicated. HeLa cells were transfected by LipofectamineTM 2000 reagent (Invitrogen Corporation, Carlsbad, California) with pA3M-EBNA3C and pCDNA3-Skp2.
  • RNA primers were purchased from Invitrogen Corporation (Carlsbad, CA). The sequence for Skp2 siRNA was as described (29). A GFP siRNA duplex was also prepared as a control.
  • HEK 293T cells were transfected with 200 nM siRNA at 24 hours with Lipofectamine 2000 (Invitrogen Corporation). 48 hours after seeding, cells were transfected with siRNA as well as pCMV- HA-Rb and pA3M-EBNA3C as indicated.
  • U2OS cells were seeded into 6-well plates and grown to confluence in 0.5% FBS for 48 hours prior to transfection.
  • Cells were transfected with LipofectamineTM 2000 reagent (Invitrogen Corporation, Carlsbad, California), harvested after 24 hours with a cell scraper, washed with PBS, and lysed on ice in 500 ⁇ L RIPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, 1 mM EGTA, with protease and phosphatase inhibitors). Lysates were pre-cleared and then rotated with 1 ⁇ g cyclin A antibody overnight at 4°C.
  • Cyclin A complexes were captured by rotating with Protein A-Sepharose beads and washed with RIPA buffer. Cyclin A complexes were then washed with histone buffer (25 mM Tris pH 7.5, 70 mM NaCl, 10 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, with protease and phosphatase inhibitors). Complexes were incubated in 30 ⁇ L histone wash buffer supplemented with 4 ⁇ g Histone H 1 (Upstate USA, Inc., Chicago, Illinois), 10 mM cold ATP, and 0.2 ⁇ Ci/ ⁇ L 32 P ⁇ - ATP for 30 minutes at 37°C.
  • Histone H 1 Upstate USA, Inc., Chicago, Illinois
  • the reaction was stopped by adding SDS-lysis buffer and heating to 95°C for 10 minutes. Labeled Histone Hl was resolved by 12% SDS-PAGE. Quantitation was with ImageQuant software (Amersham Biosciences Corporation, Piscataway, New Jersey).
  • the degradation reaction consisted of 20 ⁇ L concentrated BJAB extract, 10 ⁇ L in vitro- translated p27, 10 mM ATP, and SCF ⁇ 2 complex immunoprecipitated from HEK 293T cells in a total volume of 40 ⁇ L. Samples were incubated at 30°C and mixed every 30 minutes. 5 ⁇ L samples were harvested, mixed with 25 ⁇ L SDS-loading buffer, and heated at 95°C for 7 minutes.
  • Concentrated BJAB extracts were prepared by washing cells in PBS and then resuspending in 0.5 volumes (relative to pellet) low-salt buffer (50 mM HEPES pH 7.4, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, ImM PMSF, and 2 ⁇ g/mL aprotinin, pepstatin, and leupeptin).
  • Cells were sonicated with four 10 second pulses and cell debris was removed by centrifugation.
  • Glycerol was added to 10% and samples were frozen in liquid nitrogen and stored at -80 0 C until use.
  • In vitro-translation was with a rabbit reticulocyte-based kit from Promega.
  • HEK 293T cells For immunoprecipitation of SCF ⁇ 2 complexes, 20 million HEK 293T cells were transfected by electroporation (as described above) with 10 ⁇ g each of pCDNA3- myc-Skp2, pCDNA3-HA-Cul 1 , pCR3.1-Skpl , pCDNA3-HA-Rocl , pCDNA3-Cksl-FL, and 15 ⁇ g of pSG5-E3C as indicated. Samples were harvested at 36 hours, lysed in RIPA buffer (see above), and immunoprecipitated with 9E10 myc-specific ascites fluid overnight followed by a 1 :1 mixture of Protein A- and Protein G-Sepharose beads for 2 hours.
  • E3C-TAT HIV transcriptional transactivator
  • Figure IA A 20-amino-acid region of EBNA3C was chosen, amino acids 138-157, which included the aforementioned region 140-149, and which was predicted by several standard secondary structure prediction protocols to form an alpha helix.
  • a FITC tag was added to the amino terminus to enable the monitoring of peptide delivery, and a scrambled peptide was also designed (SCR) ( Figure IA).
  • EXAMPLE 2 THE E3C-TAT PEPTIDE DISRUPTS EBNA3C/SKP2 COMPLEXES IN
  • EXAMPLE 3 THE E3C-TAT PEPTIDE INHIBITS LCL GROWTH
  • EXAMPLE 4 THE E3C-TAT PEPTIDE SPECIFICALLY INHIBITS EBV-INFECTED CELLS AND EBV-MEDIATED TRANSFORMATION
  • E3C-TAT peptide could inhibit the transformation of primary B-cells by EBV in vitro.
  • Primary B-lymphocytes were prepared from donor blood as described in Methods (Robertson and Kieff, 1995). Lymphocytes were mixed with viral supernatant and seeded into 96-well plates. 24 wells were treated with no peptide, 24 with 20 ⁇ M E3C-TAT peptide, and 24 with 20 ⁇ M SCR peptide. Wells were fed every three days with fresh medium supplemented with 20 ⁇ M peptide. At 6 weeks, wells were scored for obvious LCL outgrowth by blinded observers.
  • EXAMPLE 5 THE E3C-TAT PEPTIDE INHIBITS THE OUTGROWTH OF EBV- POSITIVE LYMPHOMA CELLS ISOLATED FROM A PTLD PATIENT
  • the patient described in Figure 5 has a history of kidney transplant for polycystic kidney disease. She developed a B-cell, EBV-positive PTLD 2.5 years ago. At that time, she was treated with reduction in immunosuppression and rituximab (trade name Rituxan, an anti-CD20 monoclonal antibody) and had a complete response. One year later, she developed a new adenopathy and her EBV PCR (which had been negative for a year) was again positive. Two preliminary needle biopsies showed T cells only (no evidence of her old PTLD). However, she finally had an open biopsy which showed EBV-positive Hodgkin's disease.
  • rituximab trade name Rituxan, an anti-CD20 monoclonal antibody
  • E3C-TAT significantly inhibited LCL-like outgrowth (Figure 5A). While 18 and 19 out of 24 wells were scored positive for no-peptide control and SCR, respectively, only 6 wells were scored positive for the E3C-TAT sample after four weeks (Figure 5A). Representative LCL-like outgrowths from SCR-treated cells are shown in Figure 5B. FACS analysis confirmed that these cells are CDl 9 positive ( Figure 5C), and, importantly, three independent patient-derived LCLs tested by western blotting were EBNA3C positive, confirming the presence of EBV in the proliferating lymphoblastoid cell lines ( Figure 5D).
  • Post-transplant lymphoproliferative disease is seen in 1% to 2% of renal and liver transplant patients (Rickinson and Kieff, 2002). The incidence is even higher in heart and lung transplant recipients (3% to 8%) where T-cell suppression is more dramatic.
  • the EBV-dependence of these tumors is underscored by immunostraining which invariably demonstrates lesions with EBNAl , EBN A2, and LMPl -positive cells, consistent with the formation of LCL-like outgrowths in the absence of cytotoxic T-lymphocyte surveillance.
  • Patients who develop PTLD are generally managed by reducing the doses of immunosuppressive drugs which will result in regression of PTLD in approximately half of patients.
  • EBN A3C is essential for EBV-mediated B-lymphocyte transformation, and proliferation.
  • E3C- TAT blocks the interaction between full-length EBNA3C and cell cycle regulators, can limit LCL proliferation and abrogate the transformation of B-lymphocytes by EBV.
  • amino acids 138-157 of EBNA3C were chosen; these include the critical region 140-149 and are predicted by several standard secondary structure prediction protocols to form an alpha helix.
  • EBNA3C- targeting therapeutics can treat EBNA3C-positive B-cell cancers.
  • EXAMPLE 8 EBNA3C REGULATES RB IN HUMAN B-CELLS ASSOCIATED WITH
  • the assay was performed in a human B-cell line BJAB.
  • the hypophosphorylated form of Rb was eliminated with EBNA3C expression, while the hyperphosphorylated form was significantly reduced (Fig. 7a). This confirms that EBNA3C is capable of regulating Rb in a B-cell background and hints that the hypophosphorylated form may be most potently targeted.
  • a classic assay for abrogation of Rb function is the SAOS-2 flat cell assay.
  • SAOS-2 cells are null for Rb; consequently, the introduction of Rb results in dramatic arrest of cells and induction of the flat cell phenotype as cells exit the cell division cycle.
  • Rb expression induced flat cell formation in a dose-responsive fashion (Fig. 7b).
  • EBNA3C abrogated this phenotype, maximally reducing flat cell formation by approximately 60% in this assay (Fig. 7b, lower panel). This result corroborates the data above and provides a functional context for the destabilization of Rb by EXAMPLE 9: PROTEASOME INHIBITION STABILIZES RB IN THE CONTEXT OF
  • HEK 293T cells were treated with the proteasome inhibitor MG-132.
  • MG-132 treatment had no effect on Rb levels in the absence of EBNA3C (Fig. 8a, top left).
  • Rb levels clearly increased over an 8 hour time course suggesting protection from proteasome-dependent degradation (Fig. 8a, top right).
  • western blotting for p27 levels showed that the p27 protein was stabilized in both the presence and absence of EBNA3C (Fig. 8a, top).
  • EXAMPLE 10 THE AMINO TERMINUS OF EBNA3C BINDS RB JW VITRO AND
  • EXAMPLE 11 EBNA3C AMINO ACIDS 140-149 WITHIN THE CONSERVED HOMOLOGY DOMAIN REGULATE RB STABILITY
  • EXAMPLE 13 THE 141-145 A ⁇ MUTANT OF EBNA3C BINDS RB. BUT LACKS THE ABILITY TO RECRUIT COMPONENTS OF THE SCF SKP2 COMPLEX
  • GST-Skp2 bound with significantly higher affinity to wild-type EBNA3C as compared to the 141 - 145 A 5 mutant, suggesting that this mutation, which blocks the degradation of Rb, may function by abrogating the recruitment of SCF ⁇ 2 (Fig. 1Od)
  • this mutation significantly reduced Rocl co-immunoprecipitation, implicating these EBNA3C residues in recruitment of SCF ⁇ 2 (Fig. 5e).
  • EXAMPLE 14 EXPRESSION OF A DOMINANT-NEGATIVE SKP2 ABROGATES THE
  • Skp2 lacking the so-called F-box domain is a dominant negative for full-length Skp2. Because the region of EBNA3C that regulates SCF Skp2 is linked to Rb degradation, we tested whether dominant negative Skp2 blocks the degradation of Rb by EBNA3C. Indeed, the expression of dominant negative Skp2 blocked EBNA3C regulation of Rb (Fig. 1 Ia). Importantly, this dominant negative molecule had no discernable effect on Rb levels in the absence of EBNA3C suggesting that Skp2 may not normally play a prominent role in regulating Rb levels in the absence of EBNA3C. A similar effect was seen in BJAB cells (Fig. 1 Ib).
  • EXAMPLE 15 SKP2 SIRNA ABROGATES EBNA3C REGULATION OF RB
  • SCF Skp2 is not likely to be a potent regulator of Rb in most cells under most conditions; however, the fact that EBNA3C has exploited this association suggests that other viral-associated and non-viral human cancers might employ a similar strategy to deregulate Rb, leading to cell cycle progression and cell proliferation.
  • EXAMPLE 16 INHIBITION OF KINASE ACTIVITY BY CARBOXY-TERMINAL DELETED P27 IS NOT RESCUED BY EBNA3C
  • EBNA3C disrupts p27 from cyclin A complexes and that EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity.
  • U2OS cells were transfected with cyclin A, cdk2, p27, and EBNA3C expression constructs as indicated ( Figure 12B).
  • cyclin A complexes were immunoprecipitated and incubated with 32 P-Iabelled ATP and histone Hl substrate.
  • EBNA3C was able to rescue inhibition of kinase activity by p27 five- to ten-fold, while no reproducible rescue was seen for carboxy-terminal truncated p27 in spite of its efficient inhibition of kinase activity (Figure 12B, compare lanes 2 and 3 with 4 and 5).
  • This carboxy-terminal region of p27 is notable for the critical residue threonine- 187 which regulates p27 stability ( Figure 12A). Phosphorylation of this residue by cyclin A/cdk2 and other kinase complexes such as cyclin E/cdk2 promotes recognition of p27 by the SCF ⁇ 2 complex, resulting in poly-ubiquitination and ultimately degradation of p27. Thus, threonine-187 is likely critical for EBNA3C-mediated rescue of kinase activity.
  • EXAMPLE 17 EBNA3C IS ASSOCIATED WITH UBIOUITINATION ACTIVITY AND IS
  • HEK 293T cells known to prominently express the SCF 51* " 2 complex which regulates p27 stability, were transfected with expression constructs for HA-tagged ubiquitin and myc-tagged EBN A3C or EBNA3C truncation mutants as indicated ( Figure 13A).
  • Myc-specific immunoprecipitation resulted in the co-immunoprecipitation of high-molecular-weight ubiquitin- tagged proteins from cells expressing both full-length EBNA3C and a carboxy-terminal deleted EBNA3C, amino acids 1-365 ( Figure 13A, bottom left panel, lanes 2 and 3).
  • EXAMPLE 18 EBNA3C UBIOUITINATION IS DEPENDENT ON AMINO ACIDS 101-
  • EXAMPLE 19 LYSINE-120 IS DISPENSABLE FOR THE UBIOUITINATION OF
  • EBNA3C 101-200 may contain a critical lysine residue that accepts the poly-ubiquitin chain.
  • EBNA3C 101-200 may recruit factors and ubiquitination machinery essential for formation of the poly-ubiquitin chain.
  • the first possibility was easily addressed as there is a single lysine residue within EBNA3C amino acids 101 -200. Mutation of lysine-120 to an alanine had no effect on ubiquitination of EBNA3C amino acids 1 -200 using two independent clones of this truncation ( Figure 14A, lanes 5-7). This result suggests that, instead of providing a critical ubiquitin acceptor, EBNA3C amino acids 101-200 likely recruit the enzymatic factors essential for ubiquitination of the EBNA3C molecule.
  • EXAMPLE 20 EBNA3C AMINO ACIDS 90-190 RECRUIT COMPONENTS OF THE
  • EBNA3C amino acids 101-200 may contain an essential domain for the recruitment of ubiquitination machinery; to test whether this region of EBNA3C binds SCF Skp2 , we prepared a GST fusion protein of EBNA3C corresponding to amino acids 90-190, shown previously to interact with cyclin A, and tested whether individual in vitro-translated SCF components bind to this region of EBN A3C ( Figure 14C).
  • EBN A3C amino acids 90-190 strongly precipitated Skp2, Cull , and Rocl , but not Skpl when the proteins were expressed individually ( Figure 14C).
  • EXAMPLE 21 EBNA3C UBIOUITIN ATION AND SCF 51 " *2 RECRUITMENT ARE DEPENDENT UPON EBN A3C AMINO ACIDS 130-190
  • EXAMPLE 22 EBNA3C UBIOUITINATION AND ROCl RECRUITMENT ARE DEPENDENT UPON EBNA3C AMINO ACIDS 140-149
  • RING finger protein Rocl physically links the SCF core to the more basal El ubiquitin- activiating and E2 ubiquitin-co ⁇ jugating machinery and, consequently, is the minimal factor necessary for recruiting ubiquitination activity.
  • Rocl recruitment is tightly linked to EBNA3C ubiquitination, HEK 293T cells were transfected with expression constructs encoding EBNA3C amino acids 1-159, 1-149, 1 -139, and 1-129, each with a myc tag at the carboxy terminus ( Figure 15C). Cells were additionally transfected with expression constructs for HA-tagged ubiquitin and Rocl.
  • EXAMPLE 24 EBNA3C FUNCTIONALLY ASSOCIATES WITH THE RING FINGER
  • Rocl contains a RING finger motif which is responsible for the recruitment of E2 ubiquitin- conjugating activity to the SCF Skp2 ubiquitin ligase complex.
  • the Rocl RING finger motif conjugates three Zn 2+ ions and occupies the carboxy-terminal two-thirds of this 108-amino acid protein.
  • EXAMPLE 25 EBN A3C STIMULATES THE UBIOUITIN ATION OF EXOGENOUSLY
  • EXAMPLE 26 EBNA3C DECREASES P27 STABILITY IN AN IN VITRO
  • HEK 293T cells Poly-ubiquitination of p27 in HEK 293T cells suggested that EBNA3C may be regulating p27 stability.
  • SCF 5 * 112 - dependent in vitro degradation assay HEK 293T cells were transfected with expression constructs for Skp 1 , CuI 1 , Roc 1 , Cks 1 , and myc-tagged Skp2. After 48 hours cells were lysed and the SCF complex was immunoprecipitated with monoclonal antibody against the myc tag. The complex was mixed with ATP, concentrated BJAB extract, and in vitro-translated p27.
  • p27 stability was measured by collecting time points and resolving for autoradiography by SDS-PAGE. As shown in Figure I 9B, the SCF complex specifically resulted in time-dependent destabilization of in vitro-translated p27 ( Figure 19B, lanes 3, 5, and 7).
  • EXAMPLE 27 P27 IS STABILIZED IN LCLS BY TREATMENT WITH THE PROTEASOME INHIBITOR MG-132
  • EBNA3C As EBNA3C apparently directly binds SCF Skp2 components and as EBNA3C regulates p27 ubiquitination and stability, an attractive hypothesis is that EBNA3C might provide a physical link between p27 and SCF ⁇ 2 thereby facilitating and enhancing Skp2 recognition of the substrate.
  • the hypothesis was that cyclin A, which also strongly binds the amino terminus of EBNA3C, may serve as an intermediary molecule, in addition to EBNA3C, functionally linking p27 to the SCF ⁇ 2 complex.
  • HEK 293T cells were transfected with expression constructs encoding either the amino terminus of EBNA3C, amino acids 1 -365, or the carboxy terminus, amino acids 621-992 ( Figure 21 A, right panel). Both proteins are myc-tagged. Cells were additionally transfected with expression constructs for components of the SCF ⁇ complex ( Figure 2 IA, right panel). Samples were immunoprecipitated with myc-specific antibody and incubated with in vitro-translated p27 as for the degradation assays above.

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Abstract

The present invention provides a fusion peptide made of a protein transduction domain and an EBNA3C peptide. The present invention further provides compositions and methods based on a fusion peptide made of a protein transduction domain and an EBNA3C peptide for avoiding, treating, and inhibiting diseases caused by an Epstein Barr virus.

Description

AN EBV INHIBITOR AND METHODS OF MAKING AND USING THE SAME
FIELD OF THE INVENTION
[001] The present invention provides an Epstein Barr virus inhibitor and methods of using the same.
BACKGROUND OF THE INVENTION
[002] Epstein-Barr virus, frequently referred to as EBV, is a member of the herpesvirus family and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age have been infected. Infants become susceptible to EBV as soon as maternal antibody protection (present at birth) disappears. Many children become infected with EBV, and these infections usually cause no symptoms or are indistinguishable from the other mild, brief illnesses of childhood. In the United States and in other developed countries, many persons are not infected with EBV in their childhood years. When infection with EBV occurs during adolescence or young adulthood, it causes infectious mononucleosis 35% to 50% of the time.
[003J Symptoms of infectious mononucleosis are fever, sore throat, and swollen lymph glands. Sometimes, a swollen spleen or liver involvement may develop. Heart problems or involvement of the central nervous system occurs only rarely, and infectious mononucleosis is almost never fatal. There are no known associations between active EBV infection and problems during pregnancy, such as miscarriages or birth defects. Although the symptoms of infectious mononucleosis usually resolve in 1 or 2 months, EBV remains dormant or latent in a few cells in the throat and blood for the rest of the person's life. Periodically, the virus can reactivate and is commonly found in the saliva of infected persons. This reactivation usually occurs without symptoms of illness.
[004] EBV also establishes a lifelong dormant infection in some cells of the body's immune system. A late event in a very few carriers of this virus is the emergence of Burkitt's lymphoma and nasopharyngeal carcinoma, two rare cancers that are not normally found in the United States. EBV appears to play an important role in these malignancies, but is probably not the sole cause of disease.
[005] Laboratory tests are not always foolproof. For various reasons, false-positive and false- negative results can occur for any test. However, the laboratory tests for EBV are for the most part accurate and specific. Because the antibody response in primary EBV infection appears to be quite rapid, in most cases testing paired acute- and convalescent-phase serum samples will not demonstrate a significant change in antibody level. Effective laboratory diagnosis can be made on a single acute- phase serum sample by testing for antibodies to several EBV-associated antigens simultaneously. In most cases, a distinction can be made as to whether a person is susceptible to EBV, has had a recent infection, has had infection in the past, or has a reactivated EBV infection. Antibodies to several antigen complexes may be measured. These antigens are the viral capsid antigen, the early antigen, and the EBV nuclear antigen (EBNA). In addition, differentiation of immunoglobulin G and M subclasses to the viral capsid antigen can often be helpful for confirmation. When the "mono spot" test is negative, the optimal combination of EBV serologic testing consists of the antibody titration of four markers: IgM and IgG to the viral capsid antigen, IgM to the early antigen, and antibody to EBNA. IgM to the viral capsid antigen appears early in infection and disappears within 4 to 6 weeks. IgG to the viral capsid antigen appears in the acute phase, peaks at 2 to 4 weeks after onset, declines slightly, and then persists for life. IgG to the early antigen appears in the acute phase and generally falls to undetectable levels after 3 to 6 months. In many people, detection of antibody to the early antigen is a sign of active infection, but 20% of healthy people may have this antibody for years. Antibody to EBNA determined by the standard immunofluorescent test, is not seen in the acute phase, but slowly appears 2 to 4 months after onset, and persists for life. This is not true for some EBNA enzyme immunoassays, which detect antibody within a few weeks of onset.
[006] Finally, even when EBV antibody tests, such as the early antigen test, suggest that reactivated infection is present, this result does not necessarily indicate that a patient's current medical condition is caused by EBV infection. A number of healthy people with no symptoms have antibodies to the EBV early antigen for years after their initial EBV infection. Therefore, interpretation of laboratory results is somewhat complex and should be left to physicians who are familiar with EBV testing and who have access to the entire clinical picture of a person. To determine if EBV infection is associated with a current illness, consult with an experienced physician.
SUMMARY OF THE INVENTION
[007] In one embodiment, the current invention provides a peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
[008] In another embodiment, the present invention further provides a composition comprising a peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1.
[009J In another embodiment, the present invention further provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 , thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
[0010J In another embodiment, the present invention further provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus in a subject selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post- transplant lymphoproliferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 , thereby treating or reducing the incidence of a disease caused by an Epstein-Barr virus in a subject.
[001 1] In another embodiment, the present invention further provides a method of inhibiting hyperproliferation of a B-cell comprising the step of inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein, thereby inhibiting hyperproliferation of a B cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1. E3C-TAT efficiently enters LCLs. Figure IA is Schematic of EBNA3C depicting the 20-amino-acid region chosen for peptide construction. Complete sequences of E3C-TAT and SCR (scrambled) peptides are indicated. Rb, retinoblastoma protein. A, CycHn A. SCF, Skpl/Cullinl/F-box protein. JK, RBP- JK. LZ, leuzine zipper. Figure IB, shows a micrograph of LCLs treated with 1 μM E3C-TAT peptide for 24 hours. Live cells were imaged at 10Ox magnification. Figure 1 C, shows a micrograph of LCLs treated with 1 μM either E3C-TAT peptide or SCR peptide for 24 hours and imaged at 600x magnification by confocal microscopy.
[0013] Figure 2. depicts a bar graph showing how E3C-TAT disrupts the interaction of EBNA3C with Skp2. Skp2 was in vitro-translated and incubated with either GST or GST-EBNA3C amino acids 90- 190 (E3C) in binding buffer. Peptide, either 10 or 25 μM as indicated, was additionally added to some samples. GST fusion proteins were washed thoroughly with binding buffer and bound proteins were resolved by SDS- 12% PAGE.
[0014] Figure 3. depicts graphs showing E3C-TAT inhibition of LCL proliferation. Figure 3A are graphs showing the results of 50,000 LCL cells that were seeded into 96-well plates with either 10% or 0.5% bovine growth serum. Cultures were treated with increasing concentrations of E3C-TAT as indicated. Total cells were counted at 0, 48, and 96 hours. Figure 3B are graphs showing the results of 50,000 LCL cells (from two independently established LCLs) that were seeded into 96-well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 μM. Total cells were counted at 0, 48, and 96 hours. Figure 3C are bar graphs showing the results of, 500,000 LCL cells (from two independently established LCLs) that were stained with PKH26 and seeded into 6- well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 μM. Cells were assayed for extent of proliferation at 72 hours. Quantification of the cells with reduced fluorescence compared to the control is represented.
[0015] Figure 4. depicts graphs that E3C-TAT specifically inhibits EBV-immortalized cells. Figure 4A are graphs showing the results of A, 50,000 cells that were seeded into 96-well plates with 10% bovine growth serum. Cultures were treated with either E3C-TAT or SCR peptide at a concentration of 20 μM. Total cells were counted at 0, 48, and 96 hours. Figure 4B is a bar graph showing the, Primary B-lymphocytes were infected with B95.8 EBV as described in Methods and seeded into a 96- well plate. 24 wells were treated with no peptide, 24 with 20 μM E3C-TAT peptide, and 24 with 20 μM SCR peptide. Transformed wells were scored at 6 weeks.
[0016] Figure 5. shows that E3C-TAT inhibits B-lymphocyte outgrowth in an EBV-positive cancer patient. Figure 5A is a bar graph showing PBMCs that were isolated from an EBV-positive, immunosuppressed patient and seeded into a 96-well plate as described in Methods. 24 wells were treated with no peptide, 24 with 20 μM E3C-TAT peptide, and 24 with 20 μM SCR peptide. Transformed wells were scored at 4 weeks. Figure 5B shows a micrograph of representative LCL-like outgrowths from SCR peptide-treated wells. Figure 5C is a graph showing the, LCL-like outgrowths from SCR peptide-treated wells, stained with either CDl 9 monoclonal or isotype -control antibody and assayed for fluorescence intensity by FACS. Figure 5D depicts a Western-blot membrane showing a three independent LCL-like outgrowths from SCR peptide-treated wells were lysed in SDS-loading buffer (approximately 1 million cells) and resolved by SDS-8% PAGE. Western blotting was with either EBNA3C monoclonal or actin control antibody. BJAB cells were used as an EBV-negative control. P-LCL, patient-LCL.depicts the overall mechanism of the protease-caspase 3 probe. Int-C and Int-N refer to the N- and C-terminal halves of the intein; Luciferase is Firefly Luciferase and DEVD is the Caspase-3 recognition sequence.
[0017] Figure 6. shows that EBNA3C destabilizes the Rb protein and enhances Rb ubiquitination. Figure 6A, depicts a gel showing HEK 293T cells, transfected with 10 μg pA3M-Rb, 1 μg pCDNA3- p27, and 0, 5, or 15 μg pA3M-EBNA3C as indicated. Total protein was normalized by Bradford assay and resolved by SDS-PAGE. Figure 6B, depicts a gel and a graph showing HEK 293T cells, transfected with pA3M-Rb and either pA3M-EBNA3C (bottom gel) or vector control (top gel). Pulse- chase analysis was as described in Methods, Figure 6C depicts a table and gels showing HEK 293T cells, transfected with pA3M-Rb, pCDNA3-HA-Ub, and pSG5-EBNA3C as indicated. Samples were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Figure 6D depicts a gel showing HEK 293T cells, transfected with 10 μg pCM V-HA-Rb, pCDNA3-HA-pl07, or pCDNA3-HA-pl30 as indicated. Samples were additionally transfected with 10 μg either pA3M- EBN A3C or vector control.
[0018] Figure 7. shows that EBNA3C regulates Rb in BJAB and SAOS-2 cells. Figure 7A, depicts a table and a gel showing BJAB cells, transfected with 20 μg pCMV-HA-Rb and 20 μg either pA3M- EBN A3C or vector control as indicated. Figure 7B depicts micrographs and a bar graph showing SAOS-2 flat cell analysis as described in Methods. After 2 weeks of puromycin selection, 40 200x fields were monitored for flat cell formation.
[0019] Figure 8. shows that EBNA3C forms complexes with Rb under conditions of proteasome inhibition. Figure 8A depicts a gel showing HEK 293T cells, transfected with 10 μg pCMV-HA-Rb and 10 μg either pA3M vector (left) or pA3M-EBNA3C (right). At 36 hours, samples were treated with either 20 μg/mL MG-132 (upper) or DMSO vehicle control (lower). Figure 8B depicts a gel showing HEK 293T cells, transfected with 10 μg pA3M-EBNA3C and 10 μg pCMV-HA-Rb. At 24 hours, cells were treated with increasing concentrations of the proteasome inhibitor MG-132 for an additional 1 hour and then immunoprecipitated for the myc tag on EBNA3C. Figure 8C depicts a gel showing samples, transfected with 10 μg of the indicated pA3M-EBNA3C truncation mutants and 10 μg pCM V-H A-Rb and treated as for Figure 8B. EBNA3C truncation proteins are marked by asterisks on the myc western blot.
[0020] Figure 9. shows that EBNA3C amino acids 140-149 are critical regulators of Rb stability. Figure 9A depicts a gel showing EBNA3C constructs in-vitro translated and incubated with either GST or GST-Rb. Figure 9B depicts a gel showing HEK 293T cells, transfected with 10 μg pCMV- HA-Rb and 10 μg of the indicated pA3M-EBNA3C expression plasmids. EBNA3C proteins are marked by asterisks on the myc western blot, Figure 9C depicts a gel showing HEK 293T cells, transfected as for Figure 9B. Figure 9D depicts a gel showing Rb that was in vitro-translated and incubated with 10-20 μg either GST, GST-EBNA3C 130-159, or GST-EB NA3C 130-159 F144A. Figure 9E depicts a gel showing, HEK 293T cells, transfected as for Figure 9B.
[0021 ] Figure 10. shows that EBN A3C amino acids 141 - 145 are critical for both the regulation of Rb stability and SCF^2 recruitment. Figure 1OA shows an alignment of human papillomavirus type 16 E7 protein with the region of EBNA3C that regulates Rb stability. Boxed amino acids (EBNA3C 141 - 145) were mutated to alanines in the subsequent experiments. Figure 1OB depicts a gel showing HEK 293T cells, transfected with 10 μg pCMV-HA-Rb and 10 μg either pA3M-EBNA3C 1 -200, pA3M- EBNA3C 1-200ILCV14S to AAAAARS (A5)7 or pA3M-EBNA3C 1 -129. Figure 1OC depicts a gel showing EBNA3C 1-200 and EBNA3C 1 -200 A5 that were in-vitro translated and incubated with either GST or GST-Rb. Figure 1OD depicts a gel showing EBNA3C 1-200 and EBNA3C 1-200 A5 were in Wire-translated and incubated with either GST or GST-Skp2. Figure 1OE depicts a gel showing HEK 293T cells, transfected with 10 μg pCDNA3-HA-Rocl and 10 μg either pA3M, pA3M- EBNA3C 1-200, or pA3M EBNA3C 1-200 A5. After 36 hours, samples were immunoprecipitated for the myc tag on EBNA3C. IP bands were quantified and presented as the ratio of Roc] to EBNA3C.
[0022] Figure 11. shows that disruption of Skp2 abrogates the destabilization of Rb by EBNA3C. Figure 1 I A shows a table, a Western blot membrane and a bar graph of 10 million HEK 293T cells, transfected with 10 μg pA3M-EBNA3C, 10 μg pA3M-Skp2ΔF, and 10 μg pCMV-HA-Rb as indicated. Figure H B shows a table, a Western blot membrane and a bar graph of 10 million BJAB cells, transfected as for Figure H A. Figure HC shows a table, a Western blot membrane and a bar graph of 10 million U2OS cells, transfected as for Figure 1 IA. Figure 1 I D shows a table, Western blot membranes and bar graphs of HEK 293T cells, seeded into plates and transfected with 200 nM siRNA at 24 hours. 48 hours after seeding, cells were again transfected with siRNA as well as pCMV-HA-Rb and pA3M-EBNA3C as indicated.
[0023] Figure 12. shows that deletion of the p27carboxy terminus abrogates EBNA3C rescue of kinase activity. Figure 12A is a schematic representation showing a deletion of the carboxy-termina! 13 residues of p27. Phosphorylation on threonine-187 is a critical regulator of p27 stability via the SCFSkp2 complex. Figure 12B depicts a table and gels showing U2OS cells, transfected with expression constructs for cyclin A, Cdk2, full-length p27, p27 deleted for the carboxy-terminal 13 residues, and EBNA3C as indicated. After 24 hours, cyclin A immunoprecipitateds were captured and assayed for in vitro kinase activity toward histone Hl . Western blotting demonstrates protein expression in cell lysates prior to immunoprecipitation. Quantification of kinase activity was with ImageQuaπt software (Molecular Dynamics).
[0024] Figure 13. shows that EBNA3C associates with ubiquitination activity and is itself ubiquitinated. Figure 13A depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C or EBNA3C truncation expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot. Figure 13B depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc- tagged EBNA3C truncation expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot. Figure 13C is a schematic of EBNA3C demonstrating a region of the molecule that is essential for ubiqutination.
[0025] Figure 14. shows that EBNA3C amino adds 90-190 recruit SCF5^2 core components. Figure 14A depicts gels showing HEK 293T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C expression plasmids and point mutants as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot. Figure 14B is a schematic of the sCF511152 complex. Figure 14C depicts a gel showing the expression plasmids for Skp2, Skpl, Cull and Rocl that were in vitro-translated and individually incubated with either GST or GST- EBNA3C 90-190 fusion proteins pre-bound to glutathione sepharose beads. Figure 14D depicts gels showing HEK 293T cells, transfected with myc-tagged Skp2 (left column), myc-tagged Skp2, myc- tagged Skpl, and HA-tagged Cull (middle column), or myc-tagged Skp2, myc-tagged Skpl, HA-tagged Cull, and HA-tagged Rocl (right column) expression plarnids. Cells were harvested at 36 hours and total protein was incubated with either GST or GST-EB NA3C 90-190 fusion proteins pre-bound to glutathione sepharose beads. Figure 14E depicts gels showing HEK 293T cells, transfected with HA- Ub, un-tagged EBNA3C, and either myc-tagged Skp2 or myc-tagged Skpl expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc- specific antibody. Samples were resolved by 12% SDS-PAGE. Western blotting was by stripping and re-probing the same membrane.
[0026] Figure 15. shows that EBNA3C amino acids 130-159 and 160-190 both contribute to EBNA3C ubiquitination and SCF^2 recruitment. Figure 15A depicts Westem-blot membranes showing HEK 293 T cells, transfected with HA-tagged ubiquitin and myc-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc- specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot. Figure 15B depicts gels showing HEK 293T cells, transfected with either myc-tagged Skp2 or both HA-taggcd Cull and HA-tagged Rod expression plasmids. Cells were harvested at 36 hours and total protein was incubated with either GST, GST-EBNA3C 90-129, GST- EBNA3C 130-159, or GST-EBNA3C 160-190 fusion proteins pre-bound to Glutathione Sepharose beads. Figure 15C depicts Western-blot membranes showing HEK 293T cells, transfected with HA- tagged ubiquitin, HA-tagged Rocl, and myc-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same blot.
[0027] Figure 16. shows that BBNA3C associates with Skp2 in cells. Figure 16A depicts a Western- blot membrane showing 100 million EBV-positive LCL cells lysed in RIPA buffer and protein complexes were immunoprecipitated with Skp2-reactive serum. Samples were resolved by SDS- PAGE. Western blotting for the indicated proteins was by stripping and reprobing the same blot, L, 5% total protein lysate. CS, control serum. IP, anti-Skp2 immunoprecipitation. Figure 16B depicts Western-blot membranes showing HEK 293T cells, transfected with pCDNA3-Skp2 encoding un- tagged Skp2 and pA3M-EBNA3C expression plasmids encoding either full-length EBNA3C or EBNA3C truncations as indicated. All EBNA3C proteins were tagged with the myc epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific serum. Samples were resolved by 10% SDS-PAGE and probed by western blot. Figure 16C depicts micrographs showing HeLa cells transfected with plasmid expressing full-length myc-tagged EBNA3C and un-tagged Skp2. Cells were fixed and probed with anti-Skp2 rabbit polyclonal and anti-myc mouse monoclonal antibodies. Staining was visualized by confocal microscopy with goat anti-rabbit (green) and goat anti-mouse (red) antibodies.
[0028] Figure 17. shows that EBNA3C associates with Rocl in cells. Figure 17A depicts western blot membranes of HEK 293T cells, transfected with pCDNA3-HA-Rocl encoding Rocl tagged with the HA-epitope and pA3M-EBNA3C encoding either full-length EBNA3C or EBNA3C truncations as indicated. All EBNA3C proteins were tagged with the myc epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific serum. Samples were resolved by 10% SDS-PAGE and probed by western blot. Figure 17B depicts western blot membranes of HEK 293T cells, transfected with expression plasmids either for myc-tagged EBNA3C truncations or for HA-tagged Cull . Cells were harvested at 36 hours and total protein was incubated with either GST or GST-Roc 1 pre-bound to Glutathione Sepharose beads. Samples were resolved by 10% SDS-PAGE and probed by western blot. Figure 17C depicts western blot membranes of HEK 293T cells, transfected with pA3M-EBNA3C 1- 365 encoding EBNA3C amino acids 1 -365 tagged with the myc epitope. Cells were also transfected with pCDNA3-HA-Rocl or pCDNA3-HA-Rocl 36-108 encoding full-length Rocl or a truncated form of Rocl, respectively, both tagged with the HA epitope. Cells were harvested at 36 hours and immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS-PAGE and probed by western blot.
[0029] Figure 18. shows that Rocl stimulates EBNA3C ubiquitination. Figure 18A depicts Western blot membranes of HEK 293T cells transfected, as indicated, with expression plasmids for HA-tagged ubiquitin, myc-tagged EBNA3C amino acids 1-365, and either HA-tagged Roc l or a Roc l mutant, C53A/C56A, which is deficient for ubiquitin recruitment. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specific antibody. Samples were resolved by 10% SDS- PAGE. Western blotting was by stripping and re-probing the same membrane. Figure 18A depicts gels of HEK 293T cells transfected, as indicated, with expression plasmids for HA-tagged ubiquitin and HA-tagged Rocl. Cells were harvested at 36 hours and cell lysates were resolved by 10% SDS-PAGE.
[0030] Figure 19. shows that EBNA3C stimulates p27 ubiquitination in HEK 293T cells and decreasesp27 stability in an SCF-2-dependent degradation assay. Figure 18A depicts a gel showing HEK 293T cells, transfected with myc-tagged p27, HA-Ub, and un-tagged EBNA3C expression plasmids as indicated. Cells were harvested at 36 hours and total protein was immunoprecipitated with myc-specifϊc antibody. Samples were resolved by 10% SDS-PAGE. Figure 18B depicts a gel showing HEK 293T cells, transfected with Skp2-myc, Skpl, Cull, Rocl, and Cksl expression plasmids or alternatively with an equal amount of vector DNA. Cells were Iysed at 36 hours and the SCFSkp2 complex was immunoprecipitated via the myc-tag on Skp2. Immunoprecpitates were then incubated with 35S-labelled in vitro-translated p27. Degradation reactions were incubated at 300C with samples taken for SDS-PAGE at the times indicated. Quantification of p27 autoradiography was by ImageQuant software. All values were normalized to the zero time point. C, In the upper panel, HEK 293T cells were transfected as described above, but with a Skp2 mutant deleted for the F-box (Skp2AF) replacing full-length Skp2. Cells were Iysed at 36 hours and the sCFSkp2ΔF complex was immunoprecipitated via the myc-tag on Skp2AF. Degradation assays were as described above with samples taken at the indicated times. In the sample represented by open circles, cells were additionally transfected with an un-tagged EBNA3C expression construct. In the lower panel, HEK 293 T cells were transfected as described above with immunoprecipitation for the myc-tag on full-length Skρ2. In the sample represented by filled circles, cells were additionally transfected with an un-tagged EBNA3C expression construct.
[0031] Figure 20. shows that p27, but neither Skp2 nor EBNA3C, is stabilized in LCLs by treatment with the proteasome inhibitor MG-132. The Western blot membrane and the accompanying graph represent asynchronously growth of LCLs, treated for eight hours with either 10 μg/mL MG- 132 or DMSO control. Cells were harvested at the times indicated. Lysates were normalized by Bradford assay and resolved by 10% SDS-PAGE. Western blotting was by stripping and re-probing the same membrane. Bands were quantified, and the data for each time point was plotted in the line graph as MG-132-band intensity divided by DMSO-band intensity.
[0032] Figure 21. shows that EBNA3C recruits ubiquitination activity to cyclin A complexes. Figure 19A depicts a graph and a gel showing HEK 293T cells, transfected with Skp2, Skpl, Cull, Rocl, and Cksl expression plasmids. Cells were additionally transfected with expression plasmids for EBNA3C amino acids 1 -365 or EBN A3C amino acids 621-992, both tagged with the myc epitope. Cells were Iysed at 36 hours, immunoprecipitated with myc-specific serum and incubated with 35S-labelled in- vitro-translated p27. Degradation reactions were incubated at 300C with samples taken for SDS-PAGE at the times indicated. Quantification of p27 autoradiography was by ImageQuant software. AH values were normalized to the zero time point. The right panel confirms protein expression in cell lysates. Figure 19B depicts a graph showing HEK 293T cells, transfected with Skp2,, Skpl, Cull, Rocl, and Cksl expression plasmids. Cells were additionally transfected with either vector (squares) or EBNA3C (circles). Cells were lysed at 36 hours and incubated with GST-cyclin A fusion protein purified from bacteria and pre-bound to glutathione sepharose beads. GST-cyclin A precipitates were then incubated with 35S-labelled in vitro-translated p27. Degradation reactions were incubated at 300C with samples taken for SDS-PAGE at the times indicated. Quantification of p27 autoradiography was by ImageQuant software. All values were normalized to the zero time point. Figure 21 C depicts a schematic representation of a model suggesting that the amino terminus of EBNA3C may serve as scaffolding for the assembly of cyclin A/cdk2/p27 with SCFskp2. This may facilitate p27 ubiquitination and, ultimately, degradation by the 26S proteasome.
DETAILED DESCRIPTION OF THE INVENTION
[0033J In one embodiment, the present invention provides a peptide comprising a protein transduction domain and an Epstein-Barr nuclear antigen 3C (EBNA3C) peptide. In another embodiment, the present invention provides that the EBNA3C peptide is encoded by an amino acid sequence comprising HILCFVMAAR (SEQ ID NO:1). In another embodiment, the EBNA3C peptide is a variant of SEQ ID No: 1. In another embodiment, the EBNA3C peptide is an isomer of SEQ ID No: 1. In another embodiment, the EBNA3C peptide is a fragment of SEQ ID No: 1. Each possibility represents a separate embodiment of the present invention.
[0034] In another embodiment, the EBNA3C peptide of the present invention consists of amino acid 140-149 of the EBNA3C protein. In another embodiment, the EBNA3C protein is a full length protein. In another embodiment, the EBNA3C peptide is a fragment of the EBNA3C protein.
[0035] In another embodiment, the present invention provides that the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In another embodiment, the present invention provides that the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C.
[0036] In another embodiment, the present invention provides that EBNA3C is a latent protein which is necessary for the efficient immortalization of primary human B cells by EBV. In another embodiment, the present invention provides that in co-operation with activated ras, EBNA3C has oncogenic activity in fibroblasts. In another embodiment, the present invention provides that EBNA3C disrupts the cyclin/CDK-pRb-E2F pathway, which regulates cell cycle progression at the restriction point (R-point) in Gl of the proliferation cycle. In another embodiment, the present invention provides that EBNA3C abrogates the mitotic spindle checkpoint.
[0037] In another embodiment, the present invention provides that EBV encoded oncoprotein EBNA3C has the remarkable capacity to permit complete nuclear division even when growth inhibitory signals have been activated. In another embodiment, the present invention provides that EBNA3C overcomes the restriction checkpoint that causes cells to arrest in Gl of the cell cycle. In another embodiment, the present invention provides that EBNA3C expression is associated with progression through both S and G2 phases and the completion of mitosis. In another embodiment, the present invention provides that expression of EBNA3C disrupts the cell cycle regulatory machinery. In another embodiment, the present invention provides that EBNA3C provides internal signals that allow the nucleus to complete the cell cycle.
[0038] In another embodiment, the present invention provides that EBNA3C is coupled to a protein transduction domain. In another embodiment, the present invention provides that EBNA3C is chemically linked to a protein transduction domain. In another embodiment, the present invention further provides that EBNA3C is fused to a protein transduction domain. In another embodiment, the present invention further provides a protein transduction domain is fused to the carboxy terminal of said EBNA3C peptide.
[0039] In another embodiment, the present invention provides that Protein transduction domain (PTD) peptides are used to enhance cellular uptake of EBNA3C peptide of the present invention. In another embodiment, the PTD of the present invention is Functionalised Antennapedia (Antp amino acid 43- 58). In another embodiment, the PTD of the present invention is HlV transcriptional transactivator (TAT) (Tat amino acid 47-57).
[0040] In another embodiment, the present invention provides that the HIV Tat peptide is encoded by an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO: 3). In another embodiment, the HIV Tat peptide is a variant of SEQ ID No: 3. In another embodiment, the HIV Tat peptide is an isomer of SEQ ID No: 3. In another embodiment, the HIV Tat peptide is a fragment of SEQ ID No: 3. Each possibility represents a separate embodiment of the present invention.
[0041 ] In another embodiment, the present invention provides that one PTD is coupled to an EBN A3C peptide of the present invention. In another embodiment, the present invention provides that more than one PTD is coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1 -100 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1- 10 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1-3 PTDs are coupled to an EBN A3C peptide of the present invention. In another embodiment, the present invention provides that 2-4 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 3-8 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 8-15 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 1-30 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 15-45 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 20-60 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 40-80 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 30-500 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 70-100 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that 60-90 PTDs are coupled to an EBNA3C peptide of the present invention. In another embodiment, the present invention provides that all PTDs utilized are encoded by the same amino acid sequence. In another embodiment, the present invention provides that the PTDs utilized are derived from different genes and thus encoded by different amino acid sequences. Each possibility represents a separate embodiment of the present invention.
[0042] In another embodiment, the present invention provides that the transporter peptide mediates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that the transporter peptide facilitates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that the transporter peptide enhances the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell.
[0043] In another embodiment, the present invention provides that a PTD mediates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that a PTD facilitates the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that a PTD enhances the transport of the EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell.
[0044] In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 1 -100 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 1-5 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 2-7 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 5-10 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 7-15 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 10-18 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 15-25 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 20-30 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 25-50 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 40-70 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein- Barr virus-infected cell by 50-75 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 70-100 folds. In another embodiment, a PTD of the invention enhances the transport of an EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell by 80-100 folds.
[0045] In another embodiment, the present invention provides that the EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of the EBNA3C peptide and a second peptide in the carboxy terminus of the EBNA3C peptide, wherein the sequence of the second peptide comprises the sequence QRLQDIRR (SEQ ID NO: 2) In another embodiment, the second peptide is a variant of SEQ ID No: 2. In another embodiment, the second peptide is an isomer of SEQ ID No: 2. In another embodiment, the second peptide is a fragment of SEQ ID No: 2. In another embodiment, the second peptide is comprises amino acids 150-157 of the EBNA3C protein.
[0046] In another embodiment the EBNA3C peptide is encoded by the amino acids sequence TQHILCFVMAARQRLQDIRR (SEQ ID NO: 4) In another embodiment, the EBNA3C peptide is a variant of SEQ ID No: 4. In another embodiment, the EBNA3C peptide is an isomer of SEQ ID No: 4. In another embodiment, the EBNA3C peptide is a fragment of SEQ ID No: 4. In another embodiment, the EBNA3C peptide is comprises amino acids 138- 157 of the EBNA3C protein. Each possibility represents a separate embodiment of the present invention.
[0047] In another embodiment, the EBNA3C-TAT peptide (E3C-TAT) is encoded by the amino acids sequence TQHILCFVMAARQRLQDIRRYGRKKRRQRRR (SEQ ID NO: 5). In another embodiment, the EBNA3C-TAT peptide is a variant of SEQ ID No: 5. In another embodiment, the EBNA3C-TAT peptide is an isomer of SEQ ID No: 5. In another embodiment, the EBNA3C-TAT peptide is a fragment of SEQ ID No: 5. In another embodiment, the EBNA3C-TAT peptide is encoded by amino acids 138-157 of the EBNA3C (SEQ ID NO: 4) and the amino acids encoded the TAT fragment (SEQ ID NO: 3). In another embodiment, the EBNA3C-TAT peptide comprises EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 4 fused to the TAT fragment encoded by the amino acids sequence of SEQ ID NO: 3. In another embodiment, the EBNA3C-TAT peptide comprises EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1 fused to the TAT fragment encoded by the amino acids sequence of SEQ ID NO: 3.
[0048] In another embodiment, the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1. In another embodiment, the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 4. In another embodiment, the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C peptide encoded by the amino acids sequence of SEQ ID NO: 1. In another embodiment, the present invention further provides a recombinant fusion protein comprising a heterologous amino acid sequence fused to EBNA3C-TAT peptide encoded by the amino acids sequence of SEQ ID NO: 5. Each possibility represents a separate embodiment of the present invention.
[0049] In another embodiment, the present invention provides a composition comprising an peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the present invention provides a composition comprising an peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4. In another embodiment, the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4. In another embodiment, the present invention provides a composition comprising an peptide comprising a protein transduction domain fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: I .
[0050] In another embodiment, the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4. In another embodiment, the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 and the sequence of the TAT fragment comprises the sequence set forth in SEQ ID NO: 3. In another embodiment, the present invention provides a composition comprising a peptide comprising a TAT fragment fused to an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4 and the sequence of the TAT fragment comprises the sequence set forth in SEQ ID NO: 3.
[0051] In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, thereby inhibiting Epstein-Barr virus infected B-cell proliferation. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 , thereby inhibiting Epstein-Barr virus infected B-cell proliferation. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of the EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4, thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
[0052] In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a transporter peptide. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a PTD. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide and a TAT peptide. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide fused to a TAT peptide. In another embodiment, the present invention provides a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting an Epstein-Barr virus-infected cell with an EBNA3C peptide fused to a TAT peptide, wherein the sequence of the EBNA3C-TAT peptide comprises the sequence set forth in SEQ ID NO: 5.
[0053] In another embodiment, the methods and peptides of the present invention provide an EBNA3C peptide fused to a TAT peptide. In another embodiment, an EBNA3C peptide fused to a TAT peptide results in protein called a hybrid or fusion protein, which has characteristics that combine those of EBNA3C peptide and a TAT peptide. In another embodiment, the present invention provides that a fusion protein further comprises a "reporter" such as fluorescein isothiocyanate (FITC) or green fluorescent protein (GFP). In another embodiment, the present invention provides that a fusion protein of the invention comprises FITC- EBNA3C- TAT peptide.
[0054] In another embodiment, the methods of the present invention provide that an EBV infected cell is an EBV transformed cell. In another embodiment, the methods of the present invention provide that an EBV transformed cell is a cell that undergone cancerous transformation as a result of an EBV infection. In another embodiment, the methods of the present invention provide that an EBV infected cell contains EBV proteins. In another embodiment, the methods of the present invention provide that an EBV transformed cell contains EBV proteins. In another embodiment, the methods of the present invention provide that an EBV transformed cell expresses EBV proteins, by an animal cell upon infection by a cancer-causing virus.
[0055] In another embodiment, the methods of the present invention provide that an EBV infected B- cell is an EBV transformed B-cell. In another embodiment, the methods of the present invention provide that an EBV transformed B-cell is a B-cell that undergone cancerous transformation as a result of an EBV infection. In another embodiment, the methods of the present invention provide that an EBV infected B-cell contains EBV proteins. In another embodiment, the methods of the present invention provide that an EBV transformed B-cell contains EBV proteins. In another embodiment, the methods of the present invention provide that an EBV transformed B-cell expresses EBV proteins.
[0056] In another embodiment, the present invention provides that EBV utilizes its latency proteins to drive B-lymphocyte proliferation, inducing a phenotype that closely mimics antigen-stimulated B- lymphocyte activation and expansion.
[0057] In another embodiment, the present invention provides that a method of inhibiting the proliferation of an Epstein-Barr virus-infected cell comprises inhibiting hyperproliferation of an Epstein-Barr virus-infected cell. In another embodiment, the present invention provides that hyperproliferation is an abnormally high rate of cell division. In another embodiment, the present invention provides that hyperproliferation occurs, for example: in tumours and cancers.
[0058] In another embodiment, the present invention provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post-transplant lymphoproHferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C peptide of the invention.
[0059] In another embodiment, the present invention provides a method of treating, or reducing the incidence of a disease caused by an Epstein-Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post-transplant lymphoproHferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to a subject a composition comprising an EBNA3C-TAT fusion peptide of the invention.
[0060] In another embodiment, the present invention provides a method for treating, reducing the incidence, delaying the onset or progression, or reducing and/or abrogating the symptoms associated with a disease caused by EBV infection. In another embodiment, the present invention provides a method for treating, reducing the incidence, delaying the onset or progression, or reducing and/or abrogating the symptoms associated with a disease caused by EBV B-cell transformation. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides inhibit the progression of a disease caused by EBV cell infection or cell transformation. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides abrogate a disease caused by EBV cell infection or cell transformation. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides ameliorate the condition of a subject infected by EBV. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides can protect a subject from an EBV infection. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV infection. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV cell transformation. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a disease caused by an EBV B-cell transformation. In another embodiment, the present invention provides that compositions comprising EBNA3C peptides can protect a subject from a malignant transformation caused by an EBV.
[0061] In another embodiment, the present invention provides that compositions comprising EBN A3C peptides are administered to subjects at risk of being infected with an EBV. In another embodiment, the present invention provides that the subject is a transplant recipient. In another embodiment, the present invention provides that the subject is immunosuppressed. In another embodiment, the present invention provides that the subject resides in a developing nation.
[0062] In another embodiment, the present invention provides that the disease is infectious mononucleosis. In another embodiment, the present invention provides that EBV causes infectious mononucleosis, also known as 'glandular fever1, 'Mono' and 'Pfeiffer's disease1. In another embodiment, the present invention provides that infectious mononucleosis is caused when a subject is first exposed to the virus during or after adolescence. In another embodiment, the present invention provides that it is therefore predominantly found in the developed world, as most children in the developing world are found to be already infected by around 18 months of age.
[0063] In another embodiment, the present invention provides that the disease is an EBV-associated malignancy. In another embodiment, the present invention provides that EBV induces cancer formation. In another embodiment, the present invention provides that EBV causes Burkitt's lymphoma. In another embodiment, the present invention provides that EBV causes nasopharyngeal carcinoma. In another embodiment, the present invention provides that Burkitt's lymphoma is a type of Non-Hodgkin's lymphoma and is most common in equatorial Africa and is co-existent with the presence of malaria. In another embodiment, the present invention provides that Malaria infection causes reduced immune surveillance of EBV immortalised B cells, so allowing their proliferation. In another embodiment, the present invention provides that this proliferation increases the chance of a mutation to occur. In another embodiment, the present invention provides that mutations can lead to the B cells escaping the body's cell-cycle control, so allowing the cells to proliferate unchecked, resulting in the formation of Burkitt's lymphoma. In another embodiment, the present invention provides that Burkitt's lymphoma commonly affects the jaw bone, forming a huge tumour mass.
[0064] In another embodiment, the present invention provides other B cell lymphomas that arise in immunocompromised subjects such as those with AIDS that are more susceptible to EBV infection. In another embodiment, the present invention provides subjects undergone organ transplantation with associated immunosuppression (Post-Transplant Lymphoproliferative Disorder (PTLPD)) that are more susceptible to EBV infection. In another embodiment, the present invention provides smooth muscle tumors that are associated with EBV in malignent patients. [0065] In another embodiment, the present invention provides that nasopharyngeal carcinoma is a cancer found in the upper respiratory tract, most commonly in the nasopharynx, and is linked to the EBV virus. In another embodiment, the present invention provides that it is found predominantly in Southern China and Africa, due to both genetic and environmental factors.
[0066] In another embodiment, the present invention provides that the disease is Chronic Fatigue Syndrome. In another embodiment, the present invention that EBV causes chronic fatigue syndrome. In another embodiment, the present invention provides that the disease is Multiple Sclerosis. In another embodiment, the present invention provides that the disease is an autoimmune disease.
[0067] In another embodiment, the present invention provides a method of inhibiting hyperproliferation of a B-cell comprising the step of inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein, thereby inhibiting hyperproliferation of a B cell. In another embodiment, the present invention provides that the B-cell is infected with an Epstein Barr virus. In another embodiment, the present invention provides that the B-cell is transformed by an Epstein Ban- virus. In another embodiment, the present invention provides that Rb degradation is an ubiquitin- proteasome complex mediated degradation.
[0068] In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the translation of EBNA3C mRNA with an antisense agent. In another embodiment, the present invention provides that the antisense comprises RNAi. In another embodiment, the present invention provides that antisense comprises a Ribozyme. In another embodiment, the present invention provides that antisense comprises antisense DNA compounds. In another embodiment, the present invention provides that one skilled in the art will choose and use the optimal antisense technology according to the methods of the invention.
[0069] In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide. In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide, wherein the sequence of an EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 4. In another embodiment, the present invention provides that inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C- TAT peptide, wherein the sequence of an EBNA3C-TAT peptide comprises the sequence set forth in SEQ ID NO: 5.
[0070] In another embodiment, the present invention provides that EBNA3C is transcriptional regulator targeting RBP-JK and Spi-1/Spi-B transcription factors to regulate viral promoters such as Cp and LMPl. In another embodiment, the present invention provides that EBNA3C regulates histone acetylation as it apparently targets and regulates both histone acetyl transferase activity and histone deacetylase complexes including HDACl , HDAC2, and the corepressors mSin3a and NcoR and thus controls cell cycle and differentiation.
[0071] In another embodiment, the present invention provides that EBNA3C regulates transcription of the EBV oncogene LMPl in a cell cycle dependent manner and targets Rb-E2F regulatory pathways leading to the accumulation of cells in the S/G2 phase of the cell cycle. In another embodiment, the present invention provides that EBNA3C targets cyclin A complexes in cells and enhances cyclin A- dependent kinase activity by disrupting the cdk inhibitor p27 from kinase complexes. In another embodiment, the present invention provides that EBNA3C targets the SCFslφ2 E3 ubiquitin ligase complex. In another embodiment, the present invention provides that components of SCFSkp2, Skpl and Skp2, are also known as S-phase Kinase-associated Proteins that complex with cyclin A in tumor cells, but not primary cells. In another embodiment, the present invention provides that SCF811112 complex consists of additional core components Cull and Rocl and that this complex plays a critical role in regulating the stability of diverse cell cycle proteins which include p27, E2F, and c-myc. In another embodiment, the present invention provides that SCF^2 is an E3 ubiqutin ligase functionally linking specific substrates to the ubiqutin-activating and ubiquitin-conjugatiπg machinery. In another embodiment, the present invention provides that this link targets substrates for poly-ubiquitination and ultimately degradation by the 26S proteasome.
[0072] In another embodiment, the present invention provides that EBNA3C regulates p27 stability by manipulating the oncoprotein Skp2 as well as other SCF components. Regulation of p27 stability by EBNA3C is most likely at the level of its interaction with cyclin A complexes, providing a potential mechanism by which EBNA3C disrupts p27 from cyclin A complexes and ultimately stimulates cyclin A-dependent kinase activity.
[0073] In another embodiment, the present invention provides that EBNA3C targets cell cycle regulators resulting in deregulation of the mammalian cell cycle. In another embodiment, the present invention provides that EBNA3C manipulates the Rb-E2F axis. In another embodiment, the present invention provides that EBNA3C causes the accumulation of cells in S/G2 and prevents the induction of certain mitotic checkpoints resulting in aberrant nuclear division. In another embodiment, the present invention provides that EBNA3C regulates the activity and stability of the retinoblastoma protein (Rb).
[0074] In another embodiment, the present invention provides that EBNA3C regulates Rb stability by recruiting the SCF^2 ubiquitin ligase complex which mediates the ubiquitination and degradation of Rb. In another embodiment, the present invention provides that regulation of Rb stability is mapped to a small region within the conserved domain of EBNA3C, amino acids 140-149. In another embodiment, the present invention provides that this region of EBNA3C regulates the SCFSkp2 complex. In another embodiment, the present invention provides that EBNA3C serves as a direct link between Rb and SCF complexes. In another embodiment, the present invention provides that disruption of EBNA3C-targeted cell cycle pathways is a viable method for treating EBV-driven diseases. In another embodiment, the present invention provides that disruption of EBNA3C-targeted cell cycle pathways is a viable method for treating EBV-driven cancers. In another embodiment, the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects the proliferation of established EBV-infected cells. In another embodiment, the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects the de novo immortalization of B-lymphocytes by EBV.
[0075] In another embodiment, the present invention provides that a TAT-tagged peptide corresponding to the EBNA3C peptide of the invention affects both the proliferation of established EBV-infected cell lines and the de novo immortalization of primary B-lymphocytes by EBV. In another embodiment, the present invention provides that the EBNA3C-TAT peptide inhibits the proliferation of previously established EBV-infected cell. In another embodiment, the present invention provides that the EBNA3C peptide blocks the spontaneous emergence and outgrowth of EBV-transformed cells.
[0076] In another embodiment, the invention provides that a composition is a pharmaceutical composition. In another embodiment, the invention provides that the pharmaceutical compositions containing the EBNA3C-TAT peptide can be administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially, intra vaginal Iy or intratumorally.
[0077] Various embodiments of dosage ranges are contemplated by this invention. The dosage of the EBNA3C-TAT peptide may be in the range of 0.1 -80 mg/day. In another embodiment, the dosage is in the range of 0.1 -50 mg/day. In another embodiment, the dosage is in the range of 0.1 -20 mg/day. In another embodiment, the dosage is in the range of 0.1-10 mg/day. In another embodiment, the dosage is in the range of 0.1-5 mg/day. In another embodiment, the dosage is in the range of 0.5-5 mg/day. In another embodiment, the dosage is in the range of 0.5-50 mg/day. In another embodiment, the dosage is be in the range of 5-80 mg/day. In another embodiment, the dosage is in the range of 35-65 mg/day. In another embodiment, the dosage is in the range of 35-65 mg/day. In another embodiment, the dosage is in the range of 20-60 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 45-60 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 60-120 mg/day. In another embodiment, the dosage is in the range of 120-240 mg/day. In another embodiment, the dosage is in the range of 40-60 mg/day. In another embodiment, the dosage is in a range of 240-400 mg/day. In another embodiment, the dosage is in a range of 45-60 mg/day. In another embodiment, the dosage is in the range of 15-25 mg/day. In another embodiment, the dosage is in the range of 5-10 mg/day. In another embodiment, the dosage is in the range of 55-65 mg/day. In one embodiment, the dosage is 20 mg/day. In another embodiment, the dosage is 40 mg/day. In another embodiment, the dosage is 60 mg/day.
[0078] If the preferred mode the EBNA3C-TAT peptide formulation is administered orally, in one embodiment, a unit dosage form used may comprise tablets, capsules, lozenges, chewable tablets, suspensions, emulsions and the like. Such unit dosage forms comprise a safe and effective amount of the desired compound, or compounds, each of which is in one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or in another embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. The pharmaceutically-acceptable carrier suitable for the preparation of unit dosage forms for peroral administration are well-known in the art. Tablets typically comprise conventional pharmaceutically- compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder-mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention, and can be readily made by a person skilled in the art.
[0079] In another embodiment, the oral dosage form comprises predefined release profile. In one embodiment, the oral dosage form of the present invention comprises an extended release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises a slow release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablets, capsules, lozenges or chewable tablets. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient as known to one skilled in the art.
[0080] Peroral compositions may comprise liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Such liquid oral compositions comprise, in some embodiments, from about 0.012% to about 0.933% of the EBNA3C-TAT perptide, or in another embodiment, from about 0.033% to about 0.7%.
[0081] Compositions for use in the methods of this invention may comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of a EBNA3C-TAT peptide and optionally, other compounds, intended for topical intranasal administration. Such compositions may comprise from about 0.01% to about 10.0% w/v of a subject compound, more preferably from about 0.1 % to about 2.0, which may be used for systemic delivery of the compounds by the intranasal route.
[0082] Other compositions comprise dry powders. Compositions may be formulated for atomization and inhalation administration. Such compositions may be contained in a container with attached atomizing means.
[0083] Further, in another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra- arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.
[0084] In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
[0085] In another embodiment, the pharmaceutical composition delivered in a controlled release system may be formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).
[0086] Numerous additional administration vehicles will be apparent to those of ordinary skill in the art, including without limitation slow release formulations, liposomal formulations and polymeric matrices.
[00871 [The preparation of pharmaceutical compositions which contain active components is well understood in the art, for example by mixing, granulating, or tablet-forming processes. The active therapeutic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. In another embodiment, additives customary for this purpose comprise vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.
[0088] An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
[0089] The compositions may also comprise preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acids and bases to adjust the pH of these aqueous compositions as needed. The compositions may also comprise local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.
[00901 Some examples of substances which can serve as pharmaceutically-acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the Tween™ brand emulsifiers; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions. The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the compound is basically determined by the way the compound is to be administered. If the subject compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological saline, with a blood-compatible suspending agent, the pH of which has been adjusted to about 7.4.
[0091] In addition, the compositions may further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxy propyl methyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
[0092] Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, cellulose (e.g. Avicel™, RC-591 ), tragacanth and sodium alginate; typical wetting agents include lecithin and polyethylene oxide sorbitan (e.g. polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above. [0093] Dry powder compositions may comprise propellants such as chlorofluorocarbons 12/1 1 and 12/1 14, or, in another embodiment, other fluorocarbons, nontoxic volatiles; solvents such as water, glycerol and ethanol, these include co-solvents as needed to solvate or suspend the active; stabilizers such as ascorbic acid, sodium metabisulfite; preservatives such as cetylpyridinium chloride and benzalkonium chloride; tonicity adjustors such as sodium chloride; buffers; and flavoring agents such as sodium saccharin.
[0094] The compositions may also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
[0095] Also comprehended by the invention are paniculate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.
[0096J In some embodiments, compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. The modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981 ; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
[0097] While the EBNA3C-TAT peptide of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound, and/or in combination with other agents used in the treatment and/or prevention of diseases, disorders and/or conditions, as described herein, as will be understood by one skilled in the art. In another embodiment, the compounds of the present invention can be administered sequentially with one or more such agents to provide sustained therapeutic and prophylactic effects. In another embodiment, the compounds may be administered via different routes, at different times, or a combination thereof. It is to be understood that any means of administering combined therapies which include the EBNA3C-TAT peptide of this invention are to be considered as part of this invention. [0098] The additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS1 DESK REFERENCE (PDR) (20079), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
EXPERIMENTAL DETAILS SECTION
EXPERIMENTAL METHODS Peptide synthesis
[0099] Peptides were purchased from Alpha Diagnostic International (San Antonio, Texas). E3C-TAT had a sequence of Fitc-TQHILCFVMAARQRLQDIRRYGRKKRRQRRR with an average mass of 4356 daltons. SCR had a sequence of Fitc-IQRTCQRQAIDLRHVRAMFLYGRKKRRQRRR. Purity of the peptide preparations was confirmed by HPLC and peptide identity was confirmed by MS. The 1 1 -amino-acid HIV TAT tag has been previously described (Farrell et al., 2004).
GST pull-down assays
[00100] GST fusion proteins were purified from bulk E. coli cultures following induction with IPTG. For in vitro binding experiments, GST fusion proteins were incubated with 35S-labeled, in vitro- translated protein in binding buffer (I x PBS, 0.1 % NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors). In Wm?-translation was with the TNT® T7 Quick Coupled Transcription/Translation System (Promega Corporation, Madison, WI).
Growth assays
[00101] For growth curves, 50,000 cells were seeded into 100 μL RPMI 1640 (Invitrogen Corporation, Carlsbad, California). RPMI was supplemented with the indicated concentrations of bovine growth serum and either E3C-TAT or SCR peptide. Cultures were fed every 24 hours with 100 μL fresh medium supplemented with peptide. Total cells were counted with a hemocytometer at 48 and 96 hours. The optimal concentration of PKH26 (Sigma, St Louis, Missouri) for staining and viability of LCL's was determined to be 4 μM. Staining of cells was per the manufacturer's recommendation. Briefly, 5xlO6 cells were pelleted, washed with medium without serum, and resuspended in 0.5 mL of diluent, ensuring a single cell suspension by pipeting gently. Equal volume of PKH26 (8μM) was added to the single cell suspension. The cells were incubated at room temperature for 5 minutes and the reaction was stopped by adding 1 mL of ice cold bovine growth serum. The cells were washed 3 times with ice cold complete medium, to get rid of any free dye. Immediately following staining with PKH26, staining was analysed using a FACScan flow cytometer (BD Biosciences, San Jose, California). The remaining cells were divided into samples of 500,000 cells and seeded into 1 ml RPMI 1640. Media was supplemented with 10% bovine growth serum and either no peptide (control) or one of E3C-TAT or SCR peptides. Cultures were fed every 24 hours with 1 ml fresh medium supplemented with peptide. The samples were assayed 72 hours post-seeding using a FACScan flow cytometer.
B-cell transformation assays
[00102] B95.8 virus was prepared from fresh LCLs by seeding 10 million cells into 10 mL RPMI plus 10% bovine growth serum. Lytic replication was induced by treating cultures with TPA and butyrate for 6 days. Culture supernatant was harvested and mixed with clarified cell extracts prepared by freeze/thaw of the cell pellet. The pooled viral supernatant was passed through a 0.45 micron filter. Primary B-lymphocytes were prepared from donor blood. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient, and T-cells were removed by rosetting with sheep red blood cells. Primary B-lymphocytes were mixed with viral supernatant and seeded into 96-well plates with 100,000 cells, 50 μL viral supernatant, and 20 μM peptide per well. Wells were fed every three days with 50 μL fresh RPMI plus 10% bovine growth serum, supplemented with 20 μM peptide. At 6 weeks, wells were scored for obvious LCL outgrowth by blinded observers.
Patient samples
[00103] Total PBMCs were isolated from patient blood by Ficoll gradient and seeded into 96-well plates with 50,000 PBMCs, 50 μL RPMI plus 10% bovine growth serum, and 20 μM peptide per well. Wells were fed every three days with 50 μL fresh RPMI plus 10% bovine growth serum, supplemented with 20 μM peptide. At 4 weeks, wells were scored for obvious LCL outgrowth.
Flow cytometry
[00104] 2x l05 cells were blocked with Fc block (BD Biosciences, San Jose, California) for 15 minutes and then stained with Allophycocyanin (APC)-anti-human CD19 (1 :200 dilution, BD Biosciences) and appropriate isotype control antibody (1 :200 dilution, BD Biosciences) for 30 minutes at 4°C. Cells were collected, washed and acquired with a FACSCalibur flow cytometer (BD Biosciences) and data was analyzed with FlowJo software (Tree Star, Inc., Ashland, Oregon).
Plasmids, antibodies, and cell lines
[00105] pA3M-EBNAC constructs express either full-length EBNA3C or EBNA3C truncations with a carboxy-terminal myc-tag. pA3M-EBNA3C 1 -200 with amino acids 141-145 (ILCFV) mutated to alanines was prepared by standard overlap extension PCR mutagenesis. The dominant negative pA3M-Skp2ΔF construct was prepared by cloning PCR-amplified cDNA encoding Skp2 amino acids J 54-435 into the previously described pA3M vector. ρGex-Skp2 was prepared by cloning PCR- amplified cDNA encoding full-length Skp2 into the pGex2TK vector. pA3M-Rb was prepared by cloning PCR-amplified cDNA into the previously described pA3M vector. GST-EBNA3C constructs express truncation mutants and pSG5-EBNA3C. Constructs expressing myc-tagged cyclin A, Skpl, p27, and p27 amino acids 1-185 were prepared by cloning PCR-amplified cDNAs into pA3M vector. Constructs expressing un-tagged Skpl, Skp2, and HA-tagged Roc l 36-108 were prepared by cloning PCR-amplified cDNAs into the pCDNA3.1 vector (Invitrogen Corporation, Carlsbad, California). C53A/C56A point mutations in the Rocl gene were prepared by a standard PCR primer mutagenesis method. Rabbit polyclonal antibodies reactive to cyclin A, cdk2, Skp2, Cull, and p27 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, California).
[00106] Antibodies reactive to p27 and Rb were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody reactive to the HA tag was purchased from Covance Research Products, Inc. (Berkeley, CA). AlO monoclonal antibody reactive to EBNA3C were used.
[00107] HEK 293 cells are human embryonic kidney cells transformed by adenovirus type 5 DNA; HEK 293T cells stably express the SV40 large-T antigen. HEK 293T cells were transfected by electroporation with a Bio-Rad Gene Pulser in 0.4 cm-gap cuvettes at 210 Volts and 975 microfarads. BJAB and U2OS cells were transfected by electroporation at 220 Volts and 975 microfarads. U2OS is a human osteosarcoma cell line. HEK 293, 293T, U2OS, and HeLa cells were grown in DMEM (Invitrogen Corporation, Carlsbad, California) supplemented with 10% Fetal Bovine Serum unless otherwise indicated. HEK 293T cells were transfected by electroporation at 210 volts and 975 microFarads in a 0.4 μm gap cuvette. LCLs were maintained in RPMI (Invitrogen Corporation, Carlsbad, California) supplemented as for DMEM.
Pulse-chase experiments [00108] 10 million HEK 293T cells were transfected by electroporation with 10 μg pA3M-Rb and 10 μg pA3M-EBNA3C. Samples were pulsed 2 hours in pulse-labeling medium ((DMEM deficient for Met and Cys supplemented with 10% FBS) + 150 μCi 35S Trans-label (PerkinElmer, Inc., Wellesley, MA) and then chased with DMEM complete for all amino acids and supplemented with 10% FBS.
Flat cell assay
[00109] SAOS-2 cells were transfected with 4 μg pBABE-puro and with pA3M-Rb and pA3M- EBNA3C as indicated by Lipofectamine 2000 (Invitrogen Corporation). 48 hours after transfection, cells were selected with puromycin.
GST pull-down assays, immunoprecipitation, immunofluorescence and western blotting
GST fusion proteins were purified from bulk E. coli cultures following induction with IPTG. For pull-down assays from cell lysates, lysates were prepared in RlPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). Lysates were pre- cleared and then rotated with either GST control or the appropriate GST fusion protein bound to Glutathione Sepharose beads. For in vitro binding experiments, GST fusion proteins were incubated with 35S-labeled, in vitro-translated protein in binding buffer (Ix PBS, 0.1% NP40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors). For transfected HEK 293T samples, cells were lysed on ice in 500 μL RIPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). For LCLs, 100 million viable cells were lysed in 1 mL RIPA buffer. Lysates were pre-cleared with either normal rabbit or normal mouse serum and then rotated with 1 μg specific antibody for 4 hours at 4°C. Immune complexes were precipitated with a 1 : 1 mixture of Protein A- and Protein G-Sepharose beads. Samples were washed, fractionated by SDS-PAGE, and then transferred to a 0.45-μm nitrocellulose membrane for western blotting. Detection was with a standard chemiluminescence protocol unless otherwise indicated. HeLa cells were transfected by Lipofectamine™ 2000 reagent (Invitrogen Corporation, Carlsbad, California) with pA3M-EBNA3C and pCDNA3-Skp2. Cells were harvested at 24 hours, trypsinized, and allowed to adhere to glass slides overnight. Cells were fixed and permeabilized in methanol at -200C for 10 minutes followed by acetone at room temperature for 30 seconds. Slides were blocked with 5% goat serum and incubated at 4°C overnight with primary antibodies (9E 10 myc ascites fluid 1 : 100, Skp2 rabbit polyclonal antibody 5 μg/mL). Slides were washed with PBS and then incubated with AlexaFluor goat anti-mouse (594 nm) and goat anti-rabbit (488 nm) antibodies (Molecular Probes, Inc., Eugene, Oregon). Slides were washed and visualized with a Zeiss LSM510 confocal microscope. In vitro-translation was with the TNT® T7 Quick Coupled Transcription/Translation System (Promcga Corporation, Madison, Wisconsin) according to manufacturer's instructions. After incubation with primary antibody, bands were visualized by incubation with infrared-conjugated secondary antibodies (Alexa Fluor 680). Direct detection of the fluorescence and quantification was with the Li-Cor Odyssey scanner (Li-Cor, Inc., Lincoln,
NE).
Skp2 siRNA
[001 10] Custom RNA primers were purchased from Invitrogen Corporation (Carlsbad, CA). The sequence for Skp2 siRNA was as described (29). A GFP siRNA duplex was also prepared as a control. HEK 293T cells were transfected with 200 nM siRNA at 24 hours with Lipofectamine 2000 (Invitrogen Corporation). 48 hours after seeding, cells were transfected with siRNA as well as pCMV- HA-Rb and pA3M-EBNA3C as indicated.
Histone Hl kinase assay
[0011 1] U2OS cells were seeded into 6-well plates and grown to confluence in 0.5% FBS for 48 hours prior to transfection. Cells were transfected with Lipofectamine™ 2000 reagent (Invitrogen Corporation, Carlsbad, California), harvested after 24 hours with a cell scraper, washed with PBS, and lysed on ice in 500 μL RIPA buffer (0.5% NP40, 10 mM Tris pH 7.5, 2mM EDTA, 150 mM NaCl, 1 mM EGTA, with protease and phosphatase inhibitors). Lysates were pre-cleared and then rotated with 1 μg cyclin A antibody overnight at 4°C. Cyclin A complexes were captured by rotating with Protein A-Sepharose beads and washed with RIPA buffer. Cyclin A complexes were then washed with histone buffer (25 mM Tris pH 7.5, 70 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, with protease and phosphatase inhibitors). Complexes were incubated in 30 μL histone wash buffer supplemented with 4 μg Histone H 1 (Upstate USA, Inc., Chicago, Illinois), 10 mM cold ATP, and 0.2 μCi/μL 32P γ- ATP for 30 minutes at 37°C. The reaction was stopped by adding SDS-lysis buffer and heating to 95°C for 10 minutes. Labeled Histone Hl was resolved by 12% SDS-PAGE. Quantitation was with ImageQuant software (Amersham Biosciences Corporation, Piscataway, New Jersey).
HEK 293T ubiquitination assay
[001 12] 10 million HEK 293T cells were transfected by electroporation (as described above) with 10 μg pCDNA3-HA-Ub and 10 μg of a myc -tagged expression plasmid for EBNA3C, p27, Skp2, or Skpl . In some cases, cells were additionally transfected with 10 μg pSG5-E3C or 10 μg pCDNA3- HA-Roc l as indicated. Cells were harvested at 36 hours, lysed in RIPA buffer (see above), immunoprecipitated with 9E10 myc-specific ascites fluid, and resolved by SDS-PAGE. Ubiquitin- tagged proteins were detected by HA-specific western blot.
p27 degradation assay
[001 13] The degradation reaction consisted of 20 μL concentrated BJAB extract, 10 μL in vitro- translated p27, 10 mM ATP, and SCF^2 complex immunoprecipitated from HEK 293T cells in a total volume of 40 μL. Samples were incubated at 30°C and mixed every 30 minutes. 5 μL samples were harvested, mixed with 25 μL SDS-loading buffer, and heated at 95°C for 7 minutes. Concentrated BJAB extracts were prepared by washing cells in PBS and then resuspending in 0.5 volumes (relative to pellet) low-salt buffer (50 mM HEPES pH 7.4, 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT, ImM PMSF, and 2 μg/mL aprotinin, pepstatin, and leupeptin). Cells were sonicated with four 10 second pulses and cell debris was removed by centrifugation. Glycerol was added to 10% and samples were frozen in liquid nitrogen and stored at -800C until use. In vitro-translation was with a rabbit reticulocyte-based kit from Promega. For immunoprecipitation of SCF^2 complexes, 20 million HEK 293T cells were transfected by electroporation (as described above) with 10 μg each of pCDNA3- myc-Skp2, pCDNA3-HA-Cul 1 , pCR3.1-Skpl , pCDNA3-HA-Rocl , pCDNA3-Cksl-FL, and 15 μg of pSG5-E3C as indicated. Samples were harvested at 36 hours, lysed in RIPA buffer (see above), and immunoprecipitated with 9E10 myc-specific ascites fluid overnight followed by a 1 :1 mixture of Protein A- and Protein G-Sepharose beads for 2 hours.
Proteasome inhibition of LCLs with MG-132
[001 14] A recently immortalized LCL was passaged every 24 hours for several days to establish an asynchronous population of cells. Cells were then treated with either DMSO control or 10 μg/mL MG- 132 (Calbiochem Biochemicals) for up to 8 hours. Lysates were prepared and normalized by Bradford assay. Samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane for western blotting. After incubation with primary antibody, bands were visualized by incubation with conjugated secondary antibodies which fluoresce in the infrared region of the spectrum (Alexa Fluor 680). Direct detection of the fluorescence and quantification was with the Li-Cor Odyssey (Li-Cor, Inc., Lincoln, Nebraska).
EXAMPLE 1; EBNA3C-TAT PEPTIDES ENTER CELLS AND LOCALIZE TO BOTH THE
CYTOPLASM AND NUCLEUS
[001 15] The PTD of the HIV transcriptional transactivator (TAT) has been used to deliver a 10-amino- acid EBNA2 peptide into EBV-infected B-lymphocytes. With the idea that delivery of a short peptide might interfere with EBNA3C-cell cycle interactions thereby inhibiting cell proliferation, an EBNA3C-TAT fusion peptide called E3C-TAT was designed (Figure IA). A 20-amino-acid region of EBNA3C was chosen, amino acids 138-157, which included the aforementioned region 140-149, and which was predicted by several standard secondary structure prediction protocols to form an alpha helix. A FITC tag was added to the amino terminus to enable the monitoring of peptide delivery, and a scrambled peptide was also designed (SCR) (Figure IA).
[001 16] The kinetics for our two peptides, E3C-TAT and SCR, was similar. A representative image is shown in Figure IB, demonstrating peptide in greater than 50% of cells at 24 hours when LCLs were treated with 1 μM E3C-TAT. Essentially identical results were obtained for the SCR peptide (data not shown). Further, confocal imaging demonstrated that both peptides were distributed throughout the cytoplasm and nucleus of treated cells (Figure 1C).
EXAMPLE 2: THE E3C-TAT PEPTIDE DISRUPTS EBNA3C/SKP2 COMPLEXES IN
VITRO
[00117] To confirm that the 20-amino-acid peptide derived from EBNA3C indeed competes with full- length EBNA3C for binding to relevant cell cycle molecules, Skp2 was in vitro translated and incubated with a GST-EB NA3C fusion in the presence of increasing concentrations of either E3C- TAT peptide or SCR peptide (Figure 2). Indeed, E3C-TAT significantly disrupted Skp2 binding to GST-EBNA3C when compared to SCR control (Figure 2). Quantitative analysis of the data indicated that 10 μM E3C-TAT peptide disrupted 80% of Skp2 binding, compared to only 40% with 10 μM SCR peptide. However, it should be noted that at higher concentrations of peptide (i.e., 25 μM), nonspecific competition with the SCR peptide was observed (Figure 2).
EXAMPLE 3: THE E3C-TAT PEPTIDE INHIBITS LCL GROWTH
[001 18] To test that the E3C-TAT peptide would disrupt necessary EBNA3C interactions and inhibit LCL growth, LCL growth was monitored over a 96 period in the presence of varying concentrations of peptide. In the presence of 10% bovine growth serum, 5 and 20 μM E3C-TAT inhibited LCL growth in a dose-responsive fashion (Figure 3A, left panel). Under conditions of 0.5% serum, the peptide effect was even more potent with 1 μM E3C-TAT significantly inhibiting growth at 96 hours (Figure 3A, right panel). To confirm the specificity of this effect, the SCR peptide was introduced into two independently prepared LCLs alongside E3C-TAT, both at 20 μM levels. Again 20 μM E3C-TAT significantly inhibited LCL growth while 20 μM SCR was indistinguishable from the no-peptide control (Figure 3B).
[001 19] In addition to monitoring LCL proliferation with the aforementioned growth curve assay, proliferation was measured using PKH26, a membrane-staining dye. As cells divide, the dye is diluted in the plasma membrane resulting in reduced fluorescence intensity which can be monitored by FACS. Two independently prepared LCLs were stained with PKH26 and then maintained in dye-free medium for 72 hours. The medium also contained 10% serum and either E3C-TAT peptide or SCR control as indicated (Figure 3C). In LCLs treated with no peptide or SCR control, approximately 80% of cells demonstrated reduced fluorescence intensity, and were consequently judged to have proliferated over the 72-hour course of the assay (Figure 3C). In contrast, only 40% of E3C-TAT treated cells showed reduced fluorescence intensity (Figure 3C). This PKH26 data is consistent with the above growth curves demonstrating decreased LCL proliferation in the presence of the E3C-TAT peptide.
EXAMPLE 4: THE E3C-TAT PEPTIDE SPECIFICALLY INHIBITS EBV-INFECTED CELLS AND EBV-MEDIATED TRANSFORMATION
[00120] We next asked whether other B-cell lines not infected with EBV would be similarly inhibited by the E3C-TAT peptide. To this end, BJAB, DG75, and Loukes were compared alongside an LCL in a 96-hour growth curve experiment. While the LCL again showed significant growth inhibition in the presence of 20 μM E3C-TAT peptide, BJAB, DG75, and Loukes treated with E3C-TAT were statistically indistinguishable from no-peptide controls (Figure 4A).
[00121] To further explore the EBV dependence of E3C-TAT inhibition, we asked whether E3C-TAT peptide could inhibit the transformation of primary B-cells by EBV in vitro. Primary B-lymphocytes were prepared from donor blood as described in Methods (Robertson and Kieff, 1995). Lymphocytes were mixed with viral supernatant and seeded into 96-well plates. 24 wells were treated with no peptide, 24 with 20 μM E3C-TAT peptide, and 24 with 20 μM SCR peptide. Wells were fed every three days with fresh medium supplemented with 20 μM peptide. At 6 weeks, wells were scored for obvious LCL outgrowth by blinded observers. For the no peptide control and the SCR-treated cells, 10 and 1 1 wells or close to 50% of the wells, respectively, showed signs of obvious LCL-like outgrowth with multiple robust foci in each of the wells (Figure 4B). In contrast, for E3C-TAT-treated cells only 4 wells showed LCL-like outgrowth, with fewer foci that did not continue outgrowth over 2-3 months (Figure 4B).
EXAMPLE 5: THE E3C-TAT PEPTIDE INHIBITS THE OUTGROWTH OF EBV- POSITIVE LYMPHOMA CELLS ISOLATED FROM A PTLD PATIENT
[00122] The patient described in Figure 5 has a history of kidney transplant for polycystic kidney disease. She developed a B-cell, EBV-positive PTLD 2.5 years ago. At that time, she was treated with reduction in immunosuppression and rituximab (trade name Rituxan, an anti-CD20 monoclonal antibody) and had a complete response. One year later, she developed a new adenopathy and her EBV PCR (which had been negative for a year) was again positive. Two preliminary needle biopsies showed T cells only (no evidence of her old PTLD). However, she finally had an open biopsy which showed EBV-positive Hodgkin's disease.
[00123] As this patient was in an immunosuppressed state and had a clear EBV-driven proliferative process, we decided to test whether the E3C-TAT peptide would prevent spontaneous EBV-mediated lymphocyte outgrowth from this patient. Total PBMCs were isolated from patient blood as described in Methods and seeded into 24-well plates in the presence of either E3C-TAT or SCR peptide. Importantly, no exogenously prepared virus was added and no effort was made to either extract or inhibit T-cells as in the aforementioned transformation experiments. At four weeks, wells were scored for obvious LCL-like outgrowth by blinded observers.
[00124] E3C-TAT significantly inhibited LCL-like outgrowth (Figure 5A). While 18 and 19 out of 24 wells were scored positive for no-peptide control and SCR, respectively, only 6 wells were scored positive for the E3C-TAT sample after four weeks (Figure 5A). Representative LCL-like outgrowths from SCR-treated cells are shown in Figure 5B. FACS analysis confirmed that these cells are CDl 9 positive (Figure 5C), and, importantly, three independent patient-derived LCLs tested by western blotting were EBNA3C positive, confirming the presence of EBV in the proliferating lymphoblastoid cell lines (Figure 5D).
[00125] Post-transplant lymphoproliferative disease (PTLD) is seen in 1% to 2% of renal and liver transplant patients (Rickinson and Kieff, 2002). The incidence is even higher in heart and lung transplant recipients (3% to 8%) where T-cell suppression is more dramatic. The EBV-dependence of these tumors is underscored by immunostraining which invariably demonstrates lesions with EBNAl , EBN A2, and LMPl -positive cells, consistent with the formation of LCL-like outgrowths in the absence of cytotoxic T-lymphocyte surveillance. Patients who develop PTLD are generally managed by reducing the doses of immunosuppressive drugs which will result in regression of PTLD in approximately half of patients.
[00126] EBN A3C is essential for EBV-mediated B-lymphocyte transformation, and proliferation. E3C- TAT blocks the interaction between full-length EBNA3C and cell cycle regulators, can limit LCL proliferation and abrogate the transformation of B-lymphocytes by EBV. For this study, amino acids 138-157 of EBNA3C were chosen; these include the critical region 140-149 and are predicted by several standard secondary structure prediction protocols to form an alpha helix. Thus, EBNA3C- targeting therapeutics can treat EBNA3C-positive B-cell cancers.
EXAMPLE 5: EBNA3C EXPRESSION REDUCES THE HALF-LIFE OF THE RB
PROTEIN [00127] To test whether EBNA3C might affect the phosphorylation status of Rb, a known in vivo target of cyclin-dependent kinase activity, human embryonic kidney (HEK) 293T cells were transfected with expression plasmids for Rb, p27, and EBNA3C as indicated in Fig. 6a. Interestingly, we did not detect changes in Rb phosphorylation status; however, we did note a significant decrease in total Rb levels with increasing EBNA3C expression (Fig. 6a). To determine whether this decrease could be attributed to destabilization of the Rb protein by EBNA3C, we performed a pulse-chase analysis. 35S-labeled Rb was stable for at least 20 hours in the absence of EBNA3C with less than 20% of the Rb degraded in this time (Fig. Ib, solid line). In contrast, EBNA3C induced the degradation of greater than 60% of Rb by 20 hours clearly suggesting decreased stability of Rb in the presence of EBNA3C (Fig. 6b, dashed line). We also tested whether the ubiquitination of Rb might be enhanced by EBNA3C. Transfection with expression plasmids for Rb-myc, HA-ubiquitin, and EBNA3C demonstrated a significant and reproducible increase in Rb ubiquitination in EBNA3C-expressing cells (Fig. 6c). This suggests that EBNA3C stimulates Rb degradation by mediating the poly-ubiquitination of Rb.
EXAMPLE 7: EBNA3C-MEDIATED DEGRADATION OF RB DOES NOT EXTEND TO
THE P107 AND P130 POCKET PROTEINS
[00128] The pocket family proteins pl07 and pl 30 share some of the functions of Rb, and viral antigens which target Rb may also target these pocket family proteins. However, in contrast to Rb, which was reduced by approximately 50% in the presence of EBNA3C, pi 07 and pi 30 were marginally increased with EBNA3C expression (Fig. 6d). This data suggests that the regulation of Rb by EBNA3C is distinct from the other pocket family proteins.
EXAMPLE 8: EBNA3C REGULATES RB IN HUMAN B-CELLS ASSOCIATED WITH
EBV LATENT INFECTION
[00129] To determine if the Rb phenotype is also present in an EBV-relevant cell background, the assay was performed in a human B-cell line BJAB. The hypophosphorylated form of Rb was eliminated with EBNA3C expression, while the hyperphosphorylated form was significantly reduced (Fig. 7a). This confirms that EBNA3C is capable of regulating Rb in a B-cell background and hints that the hypophosphorylated form may be most potently targeted.
[00130] A classic assay for abrogation of Rb function is the SAOS-2 flat cell assay. SAOS-2 cells are null for Rb; consequently, the introduction of Rb results in dramatic arrest of cells and induction of the flat cell phenotype as cells exit the cell division cycle. Indeed, Rb expression induced flat cell formation in a dose-responsive fashion (Fig. 7b). Importantly, EBNA3C abrogated this phenotype, maximally reducing flat cell formation by approximately 60% in this assay (Fig. 7b, lower panel). This result corroborates the data above and provides a functional context for the destabilization of Rb by EXAMPLE 9: PROTEASOME INHIBITION STABILIZES RB IN THE CONTEXT OF
EBNA3C EXPRESSION
[00131] To test whether EBNA3C-mediated degradation of Rb was utilizing the 26S proteasome, HEK 293T cells were treated with the proteasome inhibitor MG-132. Importantly, MG-132 treatment had no effect on Rb levels in the absence of EBNA3C (Fig. 8a, top left). However, with EBNA3C expression, Rb levels clearly increased over an 8 hour time course suggesting protection from proteasome- dependent degradation (Fig. 8a, top right). As a control, western blotting for p27 levels showed that the p27 protein was stabilized in both the presence and absence of EBNA3C (Fig. 8a, top).
[00132] To test whether Rb/EBNA3C complexes might be stabilized by proteasome inhibition, cells were transfected with EBNA3C-myc and HA-Rb (Fig. 8b). In the presence of 20 μg/mL MG-132, hypophosphorylated forms of Rb preferentially co-immunoprecipitated with EBNA3C (Fig. 3b, lower panel). EBNA3C truncation mutants were also assayed for their ability to co-immunoprecipitate Rb when cells were treated with 20 μg/mL MG-132 (Fig. 8c). Indeed, the amino terminus of EBNA3C, amino acids 1-365, co-immunoprecipitated Rb, an association not seen with either the 366-620 or 621- 992 domains (Fig. 8c, lower panels).
EXAMPLE 10: THE AMINO TERMINUS OF EBNA3C BINDS RB JW VITRO AND
REGULATES RB STABILITY
[00133] We sought to map the specific region of EBN A3C that mediates its in vitro association with Rb and to determine whether this association contributes to Rb degradation. Both full-length EBNA3C and EBNA3C amino acids 1-365 strongly bound GST-Rb comparable to the 5% input control in intensity (Fig. 9a). To further refine the amino acids mediating this interaction, additional truncation mutants corresponding to EBN A3C amino acids 1-100 and 1-200 were tested. While amino acids 1- 200 gave binding similar to full-length EBN A3C, amino acids 1-100 did not bind GST-Rb at detectable levels (Fig. 9a). This suggests amino acids 101 and 200 are the primary mediator of the in vitro association between Rb and EBNA3C.
[00134] We next determined whether the region of EBNA3C that mediates in vitro binding is critical for the destabilization of Rb by EBNA3C (Fig. 9b). Interestingly, full-length EBNA3C, as well as EBN A3C amino acids 1-365 and 1-200, reproducibly destabilized Rb compared to vector control (Fig. 9b, upper panel). This effect was not seen for either EBN A3C amino acids 1-100 or 621-992 (Fig. 9b, upper panel). As amino acids 1-200, but not 1-100, mediated this effect, the data suggests than amino acids 101-200 are sufficient for this phenotype (Fig. 9b).
EXAMPLE 11: EBNA3C AMINO ACIDS 140-149 WITHIN THE CONSERVED HOMOLOGY DOMAIN REGULATE RB STABILITY
[00135] EBNA3C amino acids 1-159, but not 1-129 destabilized Rb similar to amino acids 1-365 and 1-200, suggesting that amino acids 130-159 are critical for the degradation of Rb (Fig. 9c). Importantly, a GST fusion protein corresponding to EBNA3C amino acids 130-159 bound Rb (Fig. 9d). As a control, the mutation of phenylalanine- 144 to alanine which likely disrupts the higher order structure of this region of EBNA3C, significantly reduced the binding of EBNA3C amino acids 130- 159 to Rb by greater than 50% (Fig. 9d). We further defined the specific amino acid residues, within EBN A3C amino acids 130-159, responsible for regulation of Rb. Interestingly, EBNA3C amino acids 1-159 and 1-149 destabilized Rb, an effect not seen with either 1-139 or 1-129 domains (Fig. 9e). This strongly implicates the 10 amino acids 140-149 in regulation of Rb stability.
EXAMPLE 12; EBNA3C 140-149 ALIGNS WITH THE LXCXE MOTIF IN THE AMINO
TERMINUS OF HPV- 16 E7
[00136] The E7 protein of HPV type 16 has been clearly implicated in the degradation of Rb. We therefore aligned HPV type 16 E7 with the conserved amino terminal domain of EBNA3C. Interestingly, this region of EBNA3C aligned with the region adjacent to the LxCxE motif of E7 (Fig. 10a). To determine whether this region of EBNA3C was important for its function in the destabilization of Rb, we mutated the five amino acids which aligned with the LxCxE motif of E7, specifically residues ILCFV (amino acids 141 -145 of EBNA3C), to alanines. While EBNA3C 1-200 potently destabilized Rb, the 141 -145 As mutant did not significantly reduce Rb levels, similar to vector control and EBNA3C 1-129, a domain not involved in regulating Rb activity (Fig. 10b).
EXAMPLE 13: THE 141-145 Aς MUTANT OF EBNA3C BINDS RB. BUT LACKS THE ABILITY TO RECRUIT COMPONENTS OF THE SCFSKP2 COMPLEX
[00137] We next tested whether the aforementioned 141-145 A5 mutant might disrupt the association between Rb and EBNA3C in vitro. Surprisingly, both EBNA3C 1-200 and the 141-145 A5 mutant bound strongly to GST-Rb in an in vitro binding assay (Fig. 10c). Since we had previously shown that this specific region of EBNA3C was also important for the regulation of the SCFSkp2 complex, we tested GST-Skp2 for binding to EBNA3C 1 -200 and the 141-145 A5 mutant (Fig. 1Od). In contrast to GST-Rb, GST-Skp2 bound with significantly higher affinity to wild-type EBNA3C as compared to the 141 - 145 A5 mutant, suggesting that this mutation, which blocks the degradation of Rb, may function by abrogating the recruitment of SCF^2 (Fig. 1Od) We also found that this mutation significantly reduced Rocl co-immunoprecipitation, implicating these EBNA3C residues in recruitment of SCF^2 (Fig. 5e).
EXAMPLE 14: EXPRESSION OF A DOMINANT-NEGATIVE SKP2 ABROGATES THE
DESTABILIZATION OF RB BY EBNA3C
[00138] Skp2 lacking the so-called F-box domain is a dominant negative for full-length Skp2. Because the region of EBNA3C that regulates SCFSkp2 is linked to Rb degradation, we tested whether dominant negative Skp2 blocks the degradation of Rb by EBNA3C. Indeed, the expression of dominant negative Skp2 blocked EBNA3C regulation of Rb (Fig. 1 Ia). Importantly, this dominant negative molecule had no discernable effect on Rb levels in the absence of EBNA3C suggesting that Skp2 may not normally play a prominent role in regulating Rb levels in the absence of EBNA3C. A similar effect was seen in BJAB cells (Fig. 1 Ib). In U2OS cells, expression of EBNA3C had no effect on Rb protein levels (Fig. l ie). However, exogenous expression of Skp2 enabled EBNA3C to destabilize Rb (Fig. 1 Ic). This further implicates the SCF^2 complex in EBNA3C-dependent regulation of Rb as the exogenous expression of Skp2 may compensate for a possible deficiency in the SCFSkp2 pathway.
EXAMPLE 15: SKP2 SIRNA ABROGATES EBNA3C REGULATION OF RB
[00139] To corroborate our dominant-negative Skp2 data, we decided to test whether blocking Skp2 by siRNA delivery would similarly abrogate the ability of EBNA3C to regulate Rb. When Skp2 siRNA was delivered we saw a greater than 50% knockdown of Skp2 protein levels as compared to a GFP siRNA control (Fig. H d, upper panel). This knockdown resulted in the stabilization of p27 and a modest shift of Rb to the hypophosphorylated form (Fig. 1 Id). In the presence of the Skp2 siRNA, EBNA3C did not down-regulate Rb levels, showing strong consistency with the dominant-negative Skp2 data (Fig. 1 1 d, lower panel).
[00140] Thus, a direct link between Rb and SCFSkp2 was established. The observation that both Rb and SCFSkp2 binding map to the same residues within the conserved region of EBNA3C strengthens the conclusion that EBNA3C is serving as a direct link between Rb and SCF complexes. This is further bolstered by the results obtained using the 141 -145 mutant which does not degrade Rb, despite binding Rb in vitro. SCFSkp2 is not likely to be a potent regulator of Rb in most cells under most conditions; however, the fact that EBNA3C has exploited this association suggests that other viral-associated and non-viral human cancers might employ a similar strategy to deregulate Rb, leading to cell cycle progression and cell proliferation.
EXAMPLE 16: INHIBITION OF KINASE ACTIVITY BY CARBOXY-TERMINAL DELETED P27 IS NOT RESCUED BY EBNA3C [00141] Previously it was demonstrated that EBNA3C disrupts p27 from cyclin A complexes and that EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity. These data were consistent with a model whereby EBNA3C directly competes with p27 for cyclin A binding. The deletion of the carboxy-terminal 13 amino acids of p27, including the regulatory amino acid threonine- 187, abrogates the ability of EBNA3C to rescue kinase activity (Figure 12A). U2OS cells were transfected with cyclin A, cdk2, p27, and EBNA3C expression constructs as indicated (Figure 12B). At 24 hours, cyclin A complexes were immunoprecipitated and incubated with 32P-Iabelled ATP and histone Hl substrate. In repeated experiments EBNA3C was able to rescue inhibition of kinase activity by p27 five- to ten-fold, while no reproducible rescue was seen for carboxy-terminal truncated p27 in spite of its efficient inhibition of kinase activity (Figure 12B, compare lanes 2 and 3 with 4 and 5). This carboxy-terminal region of p27 is notable for the critical residue threonine- 187 which regulates p27 stability (Figure 12A). Phosphorylation of this residue by cyclin A/cdk2 and other kinase complexes such as cyclin E/cdk2 promotes recognition of p27 by the SCF^2 complex, resulting in poly-ubiquitination and ultimately degradation of p27. Thus, threonine-187 is likely critical for EBNA3C-mediated rescue of kinase activity.
EXAMPLE 17: EBNA3C IS ASSOCIATED WITH UBIOUITINATION ACTIVITY AND IS
ITSELF UBIOUITINATED
[00142] HEK 293T cells, known to prominently express the SCF51*"2 complex which regulates p27 stability, were transfected with expression constructs for HA-tagged ubiquitin and myc-tagged EBN A3C or EBNA3C truncation mutants as indicated (Figure 13A). Myc-specific immunoprecipitation resulted in the co-immunoprecipitation of high-molecular-weight ubiquitin- tagged proteins from cells expressing both full-length EBNA3C and a carboxy-terminal deleted EBNA3C, amino acids 1-365 (Figure 13A, bottom left panel, lanes 2 and 3). These ubiquitin-tagged species were seen predominantly at higher molecular weights than full-length EBNA3C and EBNA3C 1 -365, respectively. And, importantly, some of these species were also reactive with the myc-specific western blot (Figure 13 A, dark myc western exposure). The aforementioned observations strongly suggest that these species represent poly-ubiquitinated EBNA3C and that this ubiquitination can be recapitulated by the first 365 amino acids of EBNA3C (Figure 13 A, lower panels, lanes 2 and 3). In vector control, EBNA3C 366-620, and EBNA3C 621 -992 samples, no ubiquitinated species were precipitated by myc-specific antibody (Figure 13A, left panel, lanes 1 , 4, and 5).
EXAMPLE 18: EBNA3C UBIOUITINATION IS DEPENDENT ON AMINO ACIDS 101-
200
[00143] To further define the region or regions of EBNA3C that recruit this ubiquitination activity, additional truncation mutants EBNA3C 1-100 and EBNA3C 1 -200 were transfected into HEK 293T cells and assayed as above. Interestingly, amino acids 1-100 were not ubiquitinated, while amino acids 1 -200 were potently ubiquitinated similar to amino acids 1-365 and full-length EBNA3C (Figure 13B, lanes 5-7). This result suggests that EBNA3C amino acids 1-200 are sufficient to facilitate ubiquitiation with essential residues for this phenptype residing between amino acids 101 and 200 (Figure 13C).
EXAMPLE 19: LYSINE-120 IS DISPENSABLE FOR THE UBIOUITINATION OF
EBNA3C AMINO ACIDS 1-200
[00144] At least two scenarios could explain the above data demonstrating that EBNA3C amino acids 101 -200 are essential for ubiquitination. First, EBNA3C 101-200 may contain a critical lysine residue that accepts the poly-ubiquitin chain. Alternatively, EBNA3C 101-200 may recruit factors and ubiquitination machinery essential for formation of the poly-ubiquitin chain. The first possibility was easily addressed as there is a single lysine residue within EBNA3C amino acids 101 -200. Mutation of lysine-120 to an alanine had no effect on ubiquitination of EBNA3C amino acids 1 -200 using two independent clones of this truncation (Figure 14A, lanes 5-7). This result suggests that, instead of providing a critical ubiquitin acceptor, EBNA3C amino acids 101-200 likely recruit the enzymatic factors essential for ubiquitination of the EBNA3C molecule.
EXAMPLE 20: EBNA3C AMINO ACIDS 90-190 RECRUIT COMPONENTS OF THE
SCF SKPZ UBIQUITIN LIGASE COMPLEX
[00145] As we had demonstrated above that EBNA3C regulation of cyclin A/p27 complexes is dependent upon the SCFSkp2-binding motif of p27, we decided to test whether EBNA3C might interact with SCFsltp2 in a manner that recruits ubiquitination activity to EBNA3C (Figure 14B). Above, we demonstrated that EBNA3C amino acids 101-200 may contain an essential domain for the recruitment of ubiquitination machinery; to test whether this region of EBNA3C binds SCFSkp2, we prepared a GST fusion protein of EBNA3C corresponding to amino acids 90-190, shown previously to interact with cyclin A, and tested whether individual in vitro-translated SCF components bind to this region of EBN A3C (Figure 14C). EBN A3C amino acids 90-190 strongly precipitated Skp2, Cull , and Rocl , but not Skpl when the proteins were expressed individually (Figure 14C). To confirm that the precipitation of these species was not the result of their mutual association with ubiquitin, we also tested whether this region of EBNA3C might directly bind the 76-amino acid ubiquitin protein. Importantly, EBNA3C amino acids 90-190 did not bind to mono-ubiquitin by an in vitro binding assay, nor did they non-specifically precipitate high-molecular-weight ubiquitin chains from transfected cell lysates (data not shown). [00146] As multiple components of SCF^2 bound EBNA3C in the above in vitro binding assay, we tested whether these molecules might cooperate or compete for binding to EBNA3C in cells. EBNA3C 90-190 precipitated myc-tagged Skρ2 from singly-transfected cell lysates (Figure 14D, left panel). Interestingly, as additional components of SCF^2 were expressed, the interaction between EBNA3C 90-190 and Skp2 was reduced (Figure 14D, right two panels). Indeed, with expression of myc-tagged Skpl and HA-tagged Cull, Skp2 was only weakly precipitated by EBNA3C 90-190, and Cul l was also precipitated (Figure 14D, middle panel). With the further addition of HA-tagged Rocl , Cull and Rocl formed a strong complex with EBNA3C 90-190 and the Skp2 interaction was abolished to undetectable levels (Figure 14D, right panel).
[00147] As it is known that F-box proteins are themselves ubiquitinated and may enter only transiently into SCF complexes, the above data suggests that EBNA3C may be capable of assembling active SCF ubiquitin ligase complexes. This potentially explains why Skp2 binding to EBNA3C was potentially abolished with the recruitment of additional SCF components Cull and Rocl . To test whether EBNA3C recruits ubiquitination activity to Skp2 complexes, an expression plasmid for myc-tagged Skp2 was transfected into HEK 293T cells along with expression plasmids for HA-tagged ubiquitin and EBNA3C (Figure 14E). Cells were then lysed and Skp2 complexes were immunoprecipitated with myc-specifϊc monoclonal antibody. Western blotting with either Skp2 or HA antibodies demonstrated enhanced ubiquitin-tagged Skp2 in the presence of EBNA3C (Figure 14E, left panels). As a control, we also tested Skpl ubiquitination in a similar HEK 293T transfection background (Figure 14E, right panel). While low-molecular-weight ubiquitinated forms of Skpl were easily detected, they were not enhanced by EBNA3C expression (Figure 14E, right panel). We did detect some high-molecular- weight ubiquitinated species in the EBN A3C sample by HA western blot (Figure 14E, right panel, rightmost lane); however, these species were not reactive with the Skpl -specific western blot and likely represent an unidentified protein that co-immunoprecipitates with Skpl .
EXAMPLE 21: EBNA3C UBIOUITIN ATION AND SCF51"*2 RECRUITMENT ARE DEPENDENT UPON EBN A3C AMINO ACIDS 130-190
[00148] To more precisely define the region of EBNA3C that recruits ubiquitination activity, additional expression plasmids corresponding to EBNA3C amino acids 1 -159 and 1 -129 were constructed. These plasmids, along with EBNA3C 1 -200, were transfected into HEK 293T cells and assayed for the formation of HA-ubiquitin chains. As was shown above, EBNA3C 1-200 was strongly ubiquitinated (Figure 15A, lane 5). EBNA3C 1 -159 was reproducibly ubiquitinated to a lesser extent than 1 -200, and EBNA3C 1 -129 was not ubiquitinated (Figure 15A, lanes 6 and 7). This suggests that EBNA3C amino acids 130-159 likely comprise the predominant ubiquitination recruitment domain, while residues while lie within amino acids 160-200 may also be involved in regulation of this process. Importantly, neither of these regions contains a lysine residue.
[00149] To test whether the regions 160-200 and 130-159 play a role in SCFSkp2 recruitment, GST pulldown assays were performed from cell lysates as above. HEK 293T cells were transfected with either Skp2 alone or with both Cull and Rocl . Skp2 was precipitated with GST fusion proteins corresponding to both EBNA3C 130-159 and 160-190, although reproducibly to a greater extent with 160-190 (Figure 15B, top panel). In contrast, Cull and Rocl both precipitated most strongly with EBNA3C 130-159 and to a lesser extent with 160-190 (Figure 15B, lower panel). This cumulative data suggests that the SCF51'1'2 binding domain likely encompasses multiple residues in amino acids 130- 190 which is consistent with this being the primary domain responsible for the recruitment of ubiquitination activity.
EXAMPLE 22: EBNA3C UBIOUITINATION AND ROCl RECRUITMENT ARE DEPENDENT UPON EBNA3C AMINO ACIDS 140-149
[0015O] RING finger protein Rocl physically links the SCF core to the more basal El ubiquitin- activiating and E2 ubiquitin-coηjugating machinery and, consequently, is the minimal factor necessary for recruiting ubiquitination activity. To test whether Rocl recruitment is tightly linked to EBNA3C ubiquitination, HEK 293T cells were transfected with expression constructs encoding EBNA3C amino acids 1-159, 1-149, 1 -139, and 1-129, each with a myc tag at the carboxy terminus (Figure 15C). Cells were additionally transfected with expression constructs for HA-tagged ubiquitin and Rocl. While amino acids 1-159 and 1-149 recruited both Rocl and ubiquitination activity, amino acids 1 -139 and 1 -129 recruited neither (Figure 15C). This suggests that EBNA3C amino acids 140-149 represent a minimal domain for the recruitment of ubiquitination activity and that this activity is tightly linked to the recruitment of the RING finger protein Roc 1 (Figure 15C, lanes 6 and 7).
EXAMPLE 23; EBNA3C FORMS COMPLEXES WITH SKP2 IN EBV-TRANSFORMED B-
CELLS
[0015 I ] To test the association between SCF514152 and EBNA3C in virally-infected cells, Skp2 was immunoprecipitated from a recently transformed LCL and co-immunoprecipitating species were assessed by western blot. As expected, antibody specific to Skp2 co-immunoprecipitated Skp2 and known Skp2-binding partners Cul l and cyclin A (Figure 16A). EBNA3C was also co- immunoprecipitated at a similar level to Cul l and cyclin A suggesting that EBNA3C and Skp2 may form a functional complex in LCLs (Figure 16A). The reverse co-immunoprecipitation experiment was performed by co-transfecting HEK 293T cells with myc-tagged EBNA3C and un-tagged Skp2 expression plasmids (Figure 16B). As expected, Skp2 co-immunoprecipitated with both full-length EBNA3C and EBNA3C amino acids 1 -365, using an antibody specific to the myc tag (Figure 16B, lanes 7 and 8).
[00152] To determine if Skp2 and EBNA3C exist in similar nuclear compartments, HeLa cells were transfected with a vector expressing myc-tagged EBNA3C and un-tagged Skp2. Cells were then stained with antibodies specific for myc and Skp2, respectively, and nuclei were visualized by confocal microscopy to assess co-localization. EBNA3C staining was tightly nuclear demonstrating a finely stippled pattern with exclusion of nucleoli (Figure 16C, middle panel); Skp2 staining was similarly concentrated in the nucleus (Figure 16C, right panel). Importantly, EBNA3C and Skp2 co- localized at numerous points in the visualized nuclear plane as illustrated by yellow color in the merged image (Figure 16C, right panel). These data corroborate the in vivo association between Skp2 and EBN A3C as these molecules localize to similar compartments in the cell nucleus.
EXAMPLE 24: EBNA3C FUNCTIONALLY ASSOCIATES WITH THE RING FINGER
PROTEIN ROCl
[00153] The EBNA3C/Rocl association was assayed as for EBNA3C/Skp2 by co-immunoprecipitation in HEK 293T cells (Figure 17A). Similar to Skp2, Rocl co-immunoprecipitated with both full-length EBN A3C and EBNA3C amino acids 1-365 when both components were transiently transfected into HEK 293T cells (Figure 17A, lanes 6 and 7). To corroborate this experiment, GST-tagged Roc l pulled down full-length EBNA3C and EBNA3C amino acids 1-365, but not EBNA3C amino acids 366-620 or 621-992 (Figure 17B). The efficiency of pull-down was similar to that seen for a known Rocl- interacting protein, CuI 1.
[00154] Rocl contains a RING finger motif which is responsible for the recruitment of E2 ubiquitin- conjugating activity to the SCFSkp2 ubiquitin ligase complex. The Rocl RING finger motif conjugates three Zn2+ ions and occupies the carboxy-terminal two-thirds of this 108-amino acid protein. To test whether this critical domain contributes to EBNA3C binding, we generated a Rocl truncation mutant encompassing the entire RING finger motif, but deleted for the amino-terminus of Rocl , which has previously been shown to mediate Cull binding. Interestingly, unlike full-length Rocl, Rocl amino acids 36-108 did not co-immunoprecipitate with the amino terminus of EBNA3C, implicating Rocl amino acids 1-35 in EBNA3C binding (Figure 17C, compare lanes 3 and 4 with lanes 5 and 6). This result is consistent with EBNA3C binding Rocl in a manner that leaves the RING finger motif available for ubiquitin recruitment.
[00155] During the course of the aforementioned studies, we made the observation that the delivery of a Roc l expression plasmid into EBNA3C-expressing cells significantly and reproducibly enhanced EBNA3C ubiquitination (Figure 18A, lower left panel). As Roc l binding to EBNA3C is not dependent on the well-characterized RING finger motif (Figure 17C), we decided to test whether mutations in this motif would disrupt EBNA3C ubiquitination without affecting binding. Several specific RING finger mutations which disrupt Zn2+ coordination and consequently RING finger structure have been shown to significantly impair Roc 1 -dependent ubiquitination. We chose one such mutation, C53A/C56A, and compared its properties to the wild-type. While the mutant bound EBNA3C similar to wild-type, it did not stimulate EBNA3C ubiquitination (Figure 18A). Further, the mutant protein functioned as a dominant-negative when co-expressed with the wild-type protein, abrogating the ability of wild-type Rocl to stimulate EBNA3C ubiquitination (Figure 18A). While our previous mapping data had already strongly linked Rocl binding to EBNA3C ubiquitination (Figure 15C), this functional experiment unquestionably implicates a Roc 1 -dependent ligase in EBNA3C ubiquitiation, most likely SCF51'11'2.
EXAMPLE 25: EBN A3C STIMULATES THE UBIOUITIN ATION OF EXOGENOUSLY
EXPRESSED P27 IN HUMAN CELLS
[00156] Having established that EBNA3C recruits SCF^^-associated ubiquitination activity, we tested whether p27 ubiquitination might be regulated by EBNA3C. HEK 293T cells were transfected with expression plasmids for myc-tagged p27, HA-tagged ubiquitin, and un-tagged EBNA3C. Myc-specific immunoprecipitation resulted in the co-immunoprecipiation of ubiquitin-tagged p27 protein, an effect significantly and reproducibly enhanced with EBNA3C expression (Figure 19A). This result implicates EBNA3C in upregulating the poly-ubiquitination of p27 in the context of this plasmid- based expression system.
EXAMPLE 26: EBNA3C DECREASES P27 STABILITY IN AN IN VITRO
DEGRADATION ASSAY
[00157] Poly-ubiquitination of p27 in HEK 293T cells suggested that EBNA3C may be regulating p27 stability. To more directly assess the role of EBNA3C in this process, we employed an SCF5*112- dependent in vitro degradation assay. HEK 293T cells were transfected with expression constructs for Skp 1 , CuI 1 , Roc 1 , Cks 1 , and myc-tagged Skp2. After 48 hours cells were lysed and the SCF complex was immunoprecipitated with monoclonal antibody against the myc tag. The complex was mixed with ATP, concentrated BJAB extract, and in vitro-translated p27. p27 stability was measured by collecting time points and resolving for autoradiography by SDS-PAGE. As shown in Figure I 9B, the SCF complex specifically resulted in time-dependent destabilization of in vitro-translated p27 (Figure 19B, lanes 3, 5, and 7).
[00158] We next tested whether EBNA3C plays a role in regulating this complex. First, EBNA3C was expressed with SCF components Skpl , Cull , Roc l , Cksl, and myc-tagged Skp2 deleted for the F-box domain. A similar Skp2 mutant functions as a dominant negative for wild-type Skp2 as it dissociates substrate binding from ubiquitin recruitment. For both control and EBNA3C-expressing cells, p27 remained stable (Figure 19C, upper panel). In contrast, when EBNA3C was expressed with the wild- type SCFSkp2 complex, reproducible enhancement of SCF8151*2 activity by EBNA3C was observed (Figure 19C, lower panel). This result is consistent with EBNA3C regulating p27 stability by directly targeting specific components of the SCF^152 complex, in particular Skp2 and Rocl.
EXAMPLE 27: P27 IS STABILIZED IN LCLS BY TREATMENT WITH THE PROTEASOME INHIBITOR MG-132
[00159] The aforementioned data strongly indicates that EBNA3C can stimulate p27 ubiquitination in cells and enhance p27 degradation in a SCFSkp2-dependent in vitro assay. While we did not regularly see a reduction in p27 levels with EBNA3C expression in our plasmid-driven system. It should also be noted that, in our plasmid-driven system, we routinely detected the ubiquitination of both Skp2 and EBNA3C (Figure 14E and Figures 13-15). While our in vitro degradation assay strongly support a link between p27 ubiquitination and degradation, it is less clear whether Skp2 and EBNA3C might be similarly regulated. We therefore decided to test the ability of a proteasome inhibitor, MG-132, to stabilize these proteins in asynchronously-growing LCLs, an indication of whether these molecules are being significantly directed into the ubiquitin/proteasome pathway in the context of EBV latent infection. A representative experiment is shown in the upper panel of Figure 20. Interestingly, while p27 and another molecule, Cyclin A, were stabilized by 2 hours of MG-132 treatment, neither Skp2 nor EBNA3C was stabilized over an 8 hour treatment (Figure 20, bottom panel). This suggests that either Skp2 and EBNA3C are not significantly ubiquitinated in LCLs (as they are in our plasmid-based system) or that there is a disconnect between their ubiquitination and degradation in LCLs. So, while the ubiquitination of EBNA3C in our plasmid-based system provided a convenient and powerful mechanism for mapping the recruitment of this activity, it is not yet clear that ubiquitination activity is an important regulator of the EBNA3C protein itself in cells.
EXAMPLE 28; EBNA3C RECRUITS P27 DEGRADATION ACTIVITY TO CYCLIN A
COMPLEXES
[00160] As EBNA3C apparently directly binds SCFSkp2 components and as EBNA3C regulates p27 ubiquitination and stability, an attractive hypothesis is that EBNA3C might provide a physical link between p27 and SCF^2 thereby facilitating and enhancing Skp2 recognition of the substrate. The hypothesis was that cyclin A, which also strongly binds the amino terminus of EBNA3C, may serve as an intermediary molecule, in addition to EBNA3C, functionally linking p27 to the SCF^2 complex.
[00161] To definitely confirm that the amino terminus of EBNA3C associates not only with the SCF511152 complex, but also with p27 degradation activity in cells, HEK 293T cells were transfected with expression constructs encoding either the amino terminus of EBNA3C, amino acids 1 -365, or the carboxy terminus, amino acids 621-992 (Figure 21 A, right panel). Both proteins are myc-tagged. Cells were additionally transfected with expression constructs for components of the SCF^ complex (Figure 2 IA, right panel). Samples were immunoprecipitated with myc-specific antibody and incubated with in vitro-translated p27 as for the degradation assays above. While p27 was stable in the presence of the EBNA3C carboxy terminus (Figure 21 A, left panel, dashed line), incubation with the amino terminus resulted in significant and reproducible degradation of p27 over a four-hour incubation period (Figure 21 A, left panel, solid line). This result confirms the association between the EBNA3C amino terminus and p27 degradation activity and is consistent with a model whereby EBNA3C facilitates the assembly of cyclin AJSCT^kp2 complexes which potentially stimulates p27 degradation in cells.
[00162] To further test this model, cyclin A complexes were precipitated from HEK 293T cells either in the presence or absence of EBNA3C. Complexes were then incubated with in vitro-translated p27 and other degradation reaction components as described in above experiments. Previously we demonstrated that, without the addition of exogenous SCF^^ in vitro-translated p27 is stable over the course of a two hour degradation reaction. However, if cyclin A complexes are precipitated from HEK 293T lysates with a GST-cyclin A fusion protein bound to Glutathione Sepharose beads, the degradation of p27 increases by approximately 25 percent after two hours (Figure 1OB, solid line). Importantly, expression of EBNA3C in HEK 293T cells resulted in enhancement of p27 degradation with greater than 50% degraded by two hours (Figure 2 IB, dashed line). This result is consistent with cyclin A complexes mediating p27 specific degradation activity in HEK 293T cells, an effect that is significantly enhanced by EBNA3C expression in this system. This result is also consistent with EBNA3C stabilizing the association between cyclin A and SCFSkp2 complexes which may result in p27 being targeted for degradation specifically at the level of cyclin A complexes (see model, Figure 21C).
[00163] This demonstrates that EBNA3C binds and regulates the SCFSkp2 E3 ligase complex. As SCr p is commonly deregulated in human cancers.

Claims

What is claimed:
1. A peptide comprising a protein transduction domain and an EBN A3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 [AA 140- 149 of E3C].
2. The peptide of claim 1, wherein said protein transduction domain is fused to the carboxy terminal of said EBNA3C peptide.
3. The peptide of claim 1 , wherein said protein transduction domain sequence is set forth in SEQ ID NO: 3 [AA TAT].
4. The peptide of claim 1, wherein said EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of said EBNA3C peptide and a second peptide in the carboxy terminus of said EBNA3C peptide, wherein the sequence of said second peptide comprises the sequence set forth in SEQ ID NO: 2 [AA 150-157 of E3C] .
5. A recombinant fusion protein comprising a heterologous amino acid sequence fused to the peptide as defined in claim 1.
6. A composition comprising a peptide comprising a protein transduction domain and an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 [AA 140-149 of E3C]. .
7. The composition of claim 6, wherein said protein transduction domain is fused to the carboxy terminal of said EBNA3C peptide.
8. The composition of claim 6, wherein said protein transduction domain sequence is set forth in SEQ ID NO: 3 [AA TAT].
9. The composition of claim 6, wherein said EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of said EBNA3C peptide and a second peptide in the carboxy terminus of said EBNA3C peptide, wherein the sequence of said second peptide comprises the sequence set forth in SEQ ID NO: 2 [AA 150-157 of E3C].
10. A method of inhibiting the proliferation of an Epstein-Barr virus-infected cell, comprising the step of contacting said Epstein-Barr virus-infected cell with an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 [AA 140- 149 of E3C]. , thereby inhibiting Epstein-Barr virus infected B-cell proliferation.
11. The method of claim 10, wherein said EBNA3C peptide further comprises a transporter peptide, wherein said transporter peptide mediates the transport of said EBNA3C peptide across the plasma membrane of said Epstein-Barr virus-infected cell.
12. The method of claim 1 1, wherein said linking is fusing.
13. The method of claim 1 1 , wherein said linking is conjugating.
14. The method of claim 10, wherein said EBNA3C peptide further comprises a protein transduction domain fused to the carboxy terminus of said EBNA3C peptide.
15. The method of claim 10, wherein said protein transduction domain sequence is set forth in SEQ ID NO: 3 [AA TAT].
16. The method of claim 10, wherein said EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of said EBNA3C peptide and a second peptide in the carboxy terminus of said EBNA3C peptide, wherein the sequence of said second peptide comprises the sequence set forth in SEQ ID NO: 2 [AA 150-157 of E3C].
17. The method of claim 10, wherein said Epstein-Barr virus infected cell is an Epstein-Barr virus transformed cell.
18. The method of claim 10, wherein said cell is a B-cell.
19. The method of claim 10, wherein said inhibiting proliferation is inhibiting hyperproliferation.
20. A method of treating, or reducing the incidence of a disease in a subject caused by an Epstein- Barr virus selected from: mononucleosis, Stevens-Johnson syndrome, Hepatitis, Herpes, Alice in Wonderland syndrome, Post-transplant lymphoproliferative disorder, Herpangina, Multiple Sclerosis, Chronic fatigue syndrome, Hairy leukoplakia, Common variable immunodeficiency (CVID), Kikuchi's disease, Hodgkin's disease, Non-Hodgkin's lymphoma, cerebral lymphoma, Burkitt's lymphoma, breast cancer, esophageal cancer, nasopharyngeal carcinoma, gastric cancer, lymphoma, or leiomyosarcomas in a subject comprising the step of administering to said subject a composition comprising an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set forth in SEQ ID NO: 1 [AA 140-149 of E3C], thereby treating or reducing the incidence of a disease caused by an Epstein-Barr virus in a subject.
21. The method of claim 20, further comprising the step of linking said EBNA3C peptide with a transporter peptide, wherein said transporter peptide mediates the transport of said EBNA3C peptide across the plasma membrane of an Epstein-Barr virus-infected cell.
22. The method of claim 21, wherein said linking is fusing.
23. The method of claim 21 , wherein said linking is conjugating.
24. The method of claim 20, wherein said EBNA3C peptide further comprises a protein transduction domain fused to the carboxy terminus of said EBNA3C peptide.
25. The method of claim 20, wherein said protein transduction domain sequence is set forth in SEQ ID NO: 3 [AA TAT].
26. The method of claim 20, wherein said EBNA3C peptide further comprises amino acids threonine 138 and glutamine 139 in the amino terminus of said EBNA3C peptide and a second peptide in the carboxy terminus of said EBNA3C peptide, wherein the sequence of said second peptide comprises the sequence set forth in SEQ ID NO: 2 [AA 150-157 of E3C].
27. The method of claim 20, wherein said subject is a transplant recipient.
28. The method of claim 20, wherein said subject is immunosuppressed.
29. The method of claim 20, wherein said subject resides in a developing nation.
30. A method of inhibiting hyperproliferation of a B-cell comprising the step of inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein, thereby inhibiting hyperproliferation of a B cell.
31. The method of claim 30, wherein said B-cell is infected with an Epstein Barr virus.
32. The method of claim 30, wherein said B-cell is transformed by an Epstein Barr virus.
33. The method of claims 31 , wherein degradation is an ubiquitin-proteasome complex mediated degradation.
34. The method of claim 30, wherein said inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the translation of EBNA3C mRNA with an antisense agent.
35. The method of claim 30, wherein said inhibiting EBNA3C protein induced degradation of Retinoblastoma (Rb) protein comprises inhibiting the activity of EBNA3C protein with an EBNA3C peptide, wherein the sequence of said EBNA3C peptide comprises the sequence set
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DATABASE GENPEPT [Online] LAUX G.: 'EBNAC3C latent protein' Database accession no. (CAD53421) & EMBO JOURNAL vol. 7, 1988, pages 769 - 774 *
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