EP1068314A1 - Highly active forms of interferon regulatory factor proteins - Google Patents

Highly active forms of interferon regulatory factor proteins

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Publication number
EP1068314A1
EP1068314A1 EP99914389A EP99914389A EP1068314A1 EP 1068314 A1 EP1068314 A1 EP 1068314A1 EP 99914389 A EP99914389 A EP 99914389A EP 99914389 A EP99914389 A EP 99914389A EP 1068314 A1 EP1068314 A1 EP 1068314A1
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Prior art keywords
irf
ser
modified
protein
regulatory factor
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French (fr)
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John Hiscott
Rongtuan Lin
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HISCOTT, JOHN
LIN, RONGTUAN
Jewish General Hospital
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Jewish General Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Interferons are a large family of multifunctional secreted proteins involved in antiviral defence, cell growth regulation and immune activation (63) .
  • Virus infection induces the transcription and synthesis of multiple IFN genes (33,52,63); newly synthesized IFN interacts with neighbouring cells through cell surface receptors and the JAK-STAT signalling pathway, resulting in the induction of over 30 new cellular proteins that mediate the diverse functions of the IFNs (17,35,39,58).
  • IFNs Interferon Regulatory Factors
  • IRF-1 and IRF-2 are the best characterized members of this family, originally identified by studies of the transcriptional regulation of the human IFN-3 gene (22,23,30,47) . Their discovery preceded the recent expansion of this group of IFN-responsive proteins which now include seven other members: IRF-3, IRF-4/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ISGF3 ⁇ /p48 and ICSBP (48) . Structurally, the Myb oncoproteins share homology with the IRF family, although its relationship to the IFN system is unclear (62) . Recent evidence also demonstrates the presence of virally encoded analogue of cellular IRFs - vIRF in the genome of human herpes virus 8 (HHV8) (55) .
  • HHV8 human herpes virus 8
  • IRF-1 was shown to have an important role in the antiviral effects of IFNs (44,54). IRF-1 binds to the ISRE element present in many IFN-inducible gene promoters and activates expression of some of these genes (54) . However, activation of ISG genes by IFNA and IFNB was shown to be mediated generally by the multiprotein ISGF3 complex (31,36,38) . The binding of this complex to DNA is mediated by another member of the IRF family, ISGF3 ⁇ /p48, which in
  • IFN-treated cells interacts with phosphorylated STAT1 and STAT2 transcription factors forming the heterotrimeric complex ISGF3 (8,39,62).
  • the homozygous deletion of p48 in mice abolished the sensitivity of these mice to the antiviral effects of IFNs, and virus-infected macrophages from p48-/- mice showed an impaired induction of IFNA and IFNB genes (31) .
  • the ICSBP gene is expressed exclusively in the cells of the immune system (18,64) and its expression can be enhanced by IFN ⁇ .
  • ICSBP was shown to form a complex with IRF-1 and inhibit the transactivating activity of IRF-1 (9,59).
  • the homozygous deletion of ICSBP in mice leads to defects in myeloid cell lineage development and chronic myelogous leukemia (34) .
  • Another ly ⁇ phoid specific Pip/LSIRF/IRF-4 was identified (19,43,66) that interacts with phosphorylated PU.l, a member of the Ets family of transcription factors (15).
  • the Pip/PU.l heterodimer can bind to the immunoglobulin light chain enhancer and function as a B cell specific transcriptional activator.
  • Expression of Pip/LSIRF was induced by antigenic stimulation but not by IFN, and Pip/LSIRF/IRF-4 -/- mice failed to develop mature T and B cells (46) .
  • a novel member of the IRF family was recently identified by its ability to bind to an ISRE-like element in the promoter region of the Qp gene of EBV (69) .
  • IRF-3 Another unique member of the human IRF family, IRF-3 was characterized recently (2) .
  • the IRF-3 gene encodes a 55-kDa protein which is expressed constitutively in all tissues.
  • IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. The relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. Recombinant IRF-3 binds to the ISRE element of the IFN-induced gene ISG-15 and stimulates this promoter in transient expression assays.
  • IRF-3 binds to the IE and PRDIII regions of the IFNA and IFNB promoters respectively, but has different effects on their transcriptional activity (56) . While the induction of the IFNA4 promoter activated by IRF-1 or virus infection was inhibited in the presence of IRF-3, the fusion protein containing the IRF-3 DNA binding domain and the RelA(p65) transactivation domain effectively activated both IFNA and IFNB promoters. In contrast, co-expression of IRF-3 and RelA plasmids transactivated the IFNB gene promoter, but not the promoter of the IFNA4 gene (56) .
  • the Qp promoter region of the EBV-encoded gene EBNA-1 contains an ISRE-like element that is responsive to the IRF-1 and IRF-2 as well as to IFN-cv.
  • a yeast one-hybrid screen technique a new factor was recently isolated that binds specifically to the Qp ISRE.
  • the amino acid sequence of this protein is identical to the IRF-7 protein present in the Genbank database ( (69) ; accession number U73036) .
  • the Pitha group has also identified a novel IRF whose sequence is identical to that of IRF-7.
  • IRF-7 shows highest homology to IRF-3.
  • ORFs open reading frames
  • IRF-7H A new IRF-7 isoform, IRF-7H, was recently identified by Pitha' s group ( (70) , accession number AF076494) .
  • vi tro translated IRF-7 encodes a protein of 68 kDa (69, 72) .
  • vi tro translated IRF-7 protein binds effectively to the Qp ISRE, it doesn't seem to affect transcription of Qp-driven reporter constructs in a transient transcription assay (72).
  • IRF- 7 expression is not generally constitutive but can be effectively induced by IFN-c. in fibroblast cells, B-cells and other cells of lymphoid origin (70, 71) .
  • IRF-3 in uninfected cells, IRF-3 is present mainly in the cytoplasm, virus infection induced phosphorylation of IRF-7, resulting in cytoplasmic to nuclear translocation of phosphorylated IRF-7 and activated gene transcription (70, 71). Recent studies indicate that virus-stimulated phosphorylation of IRF-3 results in the activation of IFNc.4 and IFN/3 gene transcription in murine cells.
  • IFNo.4 and IFN/3 subsequently feed back on cells through the IFN receptor, stimulate the Jak-STAT pathway and lead to the IFN-responsive activation of another member of the IRF family - IRF-7; up-regulation of IRF-7 production then mediates the induction of non-IFN ⁇ .4 gene expression (71) .
  • the present invention relates to IRF proteins that have been modified in the carboxy-terminus domain (transactivation domain) by modification of serine and/or threonine sites. Modification may be achieved by phosphorylation of serine and/or threonine, or by replacement of serine and/or threonine residues with residues having acidic side-chains, preferably carboxylic acid-containing side-chains, such as aspartic acid or glutamic acid residues.
  • modified proteins may be mutants of IRF-3 and IRF-7, including chimeric proteins having portions of both IRF-3 and IRF-7, and post-translationally modified (phosphorylated) IRF-3 protein, the phosphorylation being induced by Sendai virus infection.
  • the present invention provides a modified interferon regulatory factor (IRF) protein, the protein comprising at least one modified serine or threonine phosphoacceptor site in the carboxy-terminus domain, preferably wherein cytokine gene activation by the modified IRF is increased relative to cytokine gene activation by a corresponding wild type IRF protein.
  • the present invention also provides a pharmaceutical composition comprising an effective amount of the interferon regulatory factor (IRF) protein according to the invention, together with a pharmaceutically acceptable carrier, for the treatment of a viral infection, for example, an influenza infection, a herpes infection or an HIV infection.
  • the present invention further provides use of the interferon regulatory factor (IRF) protein according to the invention to activate a cytokine gene, preferably wherein the cytokine gene is an interferon gene or a chemokine gene.
  • IRF interferon regulatory factor
  • IRF-3 expression plasmid CMVBL-IRF3 (lanes 1 and 2) or CMVBL vector alone (lanes 3 and 4) , both at 5 ⁇ g were transiently transfected into 293 cells by the calcium phosphate method.
  • rtTA-IRF-3 cells selected as described in the Example, were induced to express IRF-3 by doxycycline treatment for 24h. At 24h after Dox addition, cells were infected with Sendai virus for 4, 8, 12, 16, 20, or 24h (lanes 2-7) or were left uninfected (lane 1) . IRF-3 protein was detected in whole cell extracts (10 ⁇ g) by immunoblot. Two forms of IRF-3 were detected, designated as form I and form II.
  • rtTA-IRF-3 cells were infected with Sendai virus for 16 hours (lanes 4-8) or were left uninfected (lanes 1-3) .
  • Whole cell extracts from untreated cells (20 ⁇ g) or Sendai virus infected cells (60 ⁇ g) were incubated with 0.3 units of potato acidic phosphatase (PPA, lanes 2, 3, 7 and 8) or 5 units of calf intestinal alkaline phosphatase (CIP, lanes 4 and 5) in the absence (lanes 1, 2, 4, 6 and 7) or presence of phosphatase inhibitors (lanes 3, 5 and 8) .
  • PPA potato acidic phosphatase
  • CIP calf intestinal alkaline phosphatase
  • Phosphorylated IRF-3 protein appears as a distinct band in immunoblots, migrating more slowly than IRF-3 forms I and II.
  • Thick solid lines and thin dashed lines indicate included and excluded sequences, respectively.
  • the N-terminal IRF homology domain, the nuclear export signal (NES) and C-terminal IRF association domain are indicated.
  • (B) Expression plasmids (5 ⁇ g each) encoding wild type and deletion mutants of IRF-3 (as indicated above the lanes) were transiently transfected into 293 cells; at 24h post transfection, cells were infected with Sendai virus for 16h (lanes 2, 4, 6, 8, and 10) or left uninfected (lanes 1, 3, 5, 7, and 9) .
  • Whole cell extracts (20 ⁇ g) were prepared from infected and control cells and analyzed by immunoblotting for IRF-3 forms I and II and for the presence of phosphorylated IRF-3 (P-IRF-3) with anti-IRF-3 antibody.
  • the point mutations are indicated below the sequence: (2A: S396A/S398A; 3A: S402A/T404A/S405A; 5A: S396A/S398A/S402A/T404A/S405A) ; 5D S396D/S398D/S402D/T404D/S405D; J2A: S385A/S386A; NES: S145A/S146A) .
  • GFP-IRF-3 The subcellular localization of the GFP-IRF-3 (A and B) , GFP-IRF-3 (5A) (C and D) , GFP-IRF-3 (5D) (E and F) and GFP-IRF-3 (NES) (G and H) was analyzed in uninfected (A, C, E, and G) and Sendai virus infected COS-7 cells at 16h after infection.
  • GFP fluorescence was analyzed in living cells with a Leica fluorescence microscope using 4Ox objective.
  • FIG. 6 Transactivation of PRDI/PRDIII and ISRE containing promoters by IRF-3.
  • 293 cells were transfected with IFN/3-CAT (A and B) or ISG15-CAT (C) reporter plasmids and the various expression plasmids as indicated below the bar graph.
  • CAT activity was analyzed at 48h post-transfection with 100 ⁇ g (IFNj ⁇ -CAT) or 10 ⁇ g (ISG15-CAT) of total protein extract for l-2h at 37°C.
  • Relative CAT activity was measured as fold activation (relative to the basal level of reporter gene in the presence of CMV-B1 vector alone after normalization with co-transfected -Gal activity) ; the values represent the average of three experiments with variability shown in the error bar.
  • the rtTA, IRF-3 and IRF-3 (5D) cells were cultured in the presence or absence of Dox as indicated. After 30 hours, cells were either left untreated, infected with Sendai virus (80HAU/ml) for 16 hours, or treated with IFN-a/ ⁇ (100 IU/ml) .
  • the neutralizing antibody for type I IFN (Sigma) was added at the time of Dox addition.
  • Total RNA was isolated from each sample and analyzed by RPA using the hCK5 kit (Pharmingen) .
  • IRF-3 ⁇ N ( ⁇ 9-133) (B) or IRF-3 ⁇ N2A (C) expression plasmids were transiently transfected into 293 cells; at 24h post transfection, cells were infected with Sendai virus and treated for 12h with calpain inhibitor I (100 ⁇ M, lanes 2 and 5) or MG132 proteasome inhibitor (40 ⁇ M, lanes 3 and 6) .
  • Ethanol the solvent for calpain inhibitor I and MG132, was added to the cells as control (lanes 1 and 4) .
  • Endogenous (A) and transfected (B and C) IRF-3 proteins were detected in whole cell extracts (20 ⁇ g) by immunoblot .
  • Figure 9. IRF-3 interacts with CBP in virus infected cells.
  • (B) 293 cells were transfected with wild type and deletion mutants of IRF-3 expression plasmid (5 ⁇ g, as indicated above the lanes) or left untransfected (lanes 1 and 8) .
  • cells were infected with Sendai virus for 16h (lanes 1, 3-8, and 10-13) or left uninfected (lanes 1 and 9) .
  • Whole cell extracts 300 ⁇ g, except lane 1, which was 600 ⁇ g) were immunoprecipitated with anti-CBP antibody A22 (lanes 1-6) or with preimmune serum (lane 7) .
  • the immunoprecipitated complexes were run on 5% SDS-PAGE and different forms of IRF-3 in the precipitates were analyzed by immunoblotting with anti-IRF-3 antibody.
  • Figure 10 The cDNA sequence encoding IRF-3 (5D) , together with the amino acid sequence of IRF-3 (5D) .
  • IRF-7 (1-246) /IRF-3 (5D) (132-427) chimeric protein together with the amino acid sequence of the IRF-7 (1-246) /IRF-3 (5D) (132-427) chimeric protein.
  • IRF-7 (1-246) /IRF-3 (132-427) (IRF-7N-IRF-3 (5D) C in Figure 14).
  • nucleotide sequence means a DNA or RNA molecule or sequence, and can include, for example, a cDNA, genomic DNA, or synthetic DNA sequence, a structural gene or a fragment thereof, or an mRNA sequence, that encodes an active or functional polypeptide.
  • DNA, RNA or polypeptide sequences are "substantially homologous” or “structurally equivalent” when there is at least about 85% (preferably at least about 90%, more preferably at least about 95%) identity between the nucleotides or amino acids over a defined length of the molecule.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Appropriate hybridization conditions are within the knowledge of a person skilled in the art. See, for example, Maniatis et al . , Molecular Cloning, A Laboratory Manual. Cold Spring Harbour Laboratory, New York (1982); Brown, T. A., Gene Cloning: An Introduction (2nd Ed.) Chapman & Hall, London (1990) .
  • results disclosed herein show that phosphorylation represents an important post-translational modification of IRF-3 leading to cytoplasmic-to-nuclear translocation of phosphorylated IRF-3, stimulation of DNA binding and transcriptional activity, association of IRF-3 with the transcriptional co-activator CBP/p300, and ultimately proteasome mediated degradation. More specifically, the results disclosed herein show that, following Sendai virus infection, IRF-3 may be post-translationally modified by protein phosphorylation at multiple serine and threonine residues, located in the carboxy-terminus of IRF-3.
  • modification of functionally relevant (phosphoacceptor) serine and threonine sites may be by phosphorylation
  • the modification may also be a mutation represented by replacement of at least one of these functionally relevant serine or threonine residues with an amino acid having a carboxylic acid in its side chain, preferably aspartic acid or glutamic acid, more preferably aspartic acid.
  • the preferred mutant form of IRF-3 is that having aspartic acid residues in at least one of postions 396, 398, 402, 404 and 405 of the sequence, more preferably in positions 396, 398, 402, 404 and 405 of the sequence (IRF-3 (5D) ) ( Figure 10).
  • the preferred mutant form of IRF-7 is that having aspartic acid residues in at least one of positions 477 and 479 of the sequence, more preferable in positions 477 and 479 of the sequence (IRF-7 (2D) ) ( Figure 12).
  • chimeric proteins comprising a carboxy-terminus domain of one modified IRF protein, modified as discussed above, and an amino-terminal domain of another IRF protein.
  • the amino-terminus of IRF-7 is fused to the carboxy-terminus of modified IRF-3. It is more preferred that the carboxy-terminus of modified IRF-3 is that of IRF-3 (5D) .
  • a chimeric protein comprising residues 1 to 246 of IRF-7 and residues 132 to 427 of IRF-3 (5D) ( Figure 13).
  • nucleotide sequences within the scope of the invention are those which encode a protein of the invention.
  • the nucleotide sequence is a coding DNA sequence as defined in Figure 10 or a DNA sequence which is hybridizable under stringent conditions with the complement of the coding DNA sequence of Figure 10, which DNA encodes IRF-3 (5D) .
  • the nucleotide sequence is a coding DNA sequence as defined in Figure 12 or a DNA sequence which is hybridizable under stringent conditions with the complement of the coding DNA sequence of Figure 12, which DNA encodes IRF-7 (2D) .
  • the nucleotide sequence is a coding DNA sequence as defined in Figure 13 or a DNA sequence which is hybridizable under stringent conditions with the complement of the coding DNA sequence of Figure 13, which DNA encodes IRF-7 (1-246) /IRF-3 (132-427) chimeric protein.
  • IRF-7 (1-246) /IRF-3 (132-427) chimeric protein.
  • a combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of Ser-396 and Ser-398 residues eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404 and Ser-405 were also targets.
  • Phosphorylation results in the cytoplasmic to nuclear translocation of IRF-3, DNA binding and increased transcriptional activation. Substitution of the Ser/Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI/PRDIII or ISRE regulatory elements. Use of phosphomimetic Glu for this purpose is also possible. Phosphorylation also appears to represent a signal for virus mediated degradation, since the virus induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser/Thr cluster or by proteasome inhibitors.
  • virus infection resulted in the association of IRF-3 with the CBP coactivator, as detected by co-immunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins.
  • Mutation of the residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP.
  • Sendai virus dependent phosphorylation of IRF-3 was detected, occurring in a cluster of Ser and Thr sites in the carboxyl-terminal end of the protein.
  • the residues implicated in this regulatory phosphorylation event are Ser-396/Ser-398/Ser-402/Thr-404/Ser-405, particularly the Ser-396/Ser-398 amino acids.
  • Phosphorylation of the IRF-3 in the Ser-Thr cluster resulted in the cytoplasmic to nuclear translocation of IRF-3; nuclear translocation was blocked by mutation of the phosphorylated amino acids.
  • Sendai virus infection induced the DNA binding and transactivation potential of IRF-3.
  • IRF-3 containing the phosphomimetic Asp at the sites of C-terminal phosphorylation was an exceptionally strong transactivator of PRDI/PRDIII and ISRE containing promoters.
  • Phosphorylation was also required for the association of IRF-3 with the CBP co-activator protein.
  • Sendai virus infection resulted in IRF-3 degradation; again, phosphorylation was required as a signal for inducer mediated degradation since mutation of Ser/Thr cluster also blocked virus induced degradation.
  • IRF-3 In uninfected cells, IRF-3 was partitioned in both the nucleus and cytoplasm; virus infection changed the nuclear pattern of staining from extra-nucleolar homogeneous staining as observed for wtIRF-3 to an intense nuclear speckling. At this stage, the nature of the subnuclear changes in IRF-3 localization are not explained, although it is possible that IRF-3 (NES) translocates efficiently into the nucleus but becomes trapped in the nuclear pore complex during the export process .
  • IRF-3 NES
  • IRF-3 an exceptionally strong activator of IFN-/3 and ISG-15 gene expression.
  • the phosphomimetic form of IRF-3 alone was able to stimulate IFN-/3 expression as strongly as virus infection, a level of stimulation not previously observed in co-expression experiments (24,61).
  • IRF-3 was able to bind the ISRE element of ISG-15, as well as the PRDIII/PRDI and IE regions of the IFNB and IFNA promoters, respectively (2,56).
  • the recent crystal structure of the related IRF-1 protein bound to PRDI provides evidence for a novel helix-turn-helix motif that latches onto a GAAA core sequence via three of the five conserved tryptophan amino acids of the DNA binding domain (20) .
  • two GAAANN sequences present in PRDI11 of IFN-/3 and another GAAANN element present in PRDI may serve as DNA contacts for multiple IRF-3 (5D) proteins with strong activating potential.
  • the ISRE element of the ISG-15 promoter also contains several GAAANN anchors for potential IRF binding. Given the range of promoters that possess this hexameric sequence (48) , it will be of interest to determine the capacity of IRF-3 (5D) to stimulate expression of different cytokine and chemokine genes .
  • IRF-3 joins a growing list of cellular and viral proteins that functionally interact with CBP/p300 proteins, highly homologous proteins originally identified through their interactions with adenovirus E1A and CREB proteins (1,13) .
  • CBP/p300 possesses histone acetyltransferase activity (5,50). Acetylation of histones is involved in the destabilization and remodelling of nucleosomes, a crucial step in permitting the accessibility of transcriptional factors to DNA templates.
  • CBP/p300 participates in the transcriptional process by providing a scaffold for different classes of transcriptional regulators on specific chromatin domains (12,50) .
  • CBP/p300 A growing body of biochemical and genetic evidence also implicates CBP/p300 as a negative regulator of cell growth, based on its interactions with adenovirus Ela, SV40 large T antigen and the tumour suppressor p53, among others.
  • p53-CBP/p300 complex formation functional interaction between these two important growth regulatory proteins accounts for several of the known activities of p53 (3,28,40); interestingly, CBP/p300 was shown recently to acetylate p53 and stimulate its transactivation potential (27) .
  • Virus induced phosphorylation of IRF-3 also represents a signal for proteasome mediated degradation of IRF-3, since mutation of the Ser-396/Ser-398 or the use of proteasome inhibitors prevented the post infection degradation of IRF-3.
  • Virus induced degradation of IRF-3 is reminiscent of the virus-induced turnover of another member of the IRF family - IRF-2.
  • the 50 kD IRF-2 protein is proteolytically processed into a smaller, 24-27 kDa protein (51) comprising the 160 aa DBD of IRF-2, termed TH3 (14) or In4 (65) .
  • TH3 has been shown to bind DNA and repress transcription more efficiently than the full length IRF-2 protein (42) , its physiological role is not clear. Since the induction kinetics of TH3 are slower than that of IFN-j ⁇ in response to dsRNA or viral infection (14) , it has been suggested that the IRF-2 cleavage product may be a post-induction repressor of IFN-/3 gene expression (65) . Virus induced phosphorylation of IRF-3 at the
  • the amino-terminus of I ⁇ Ba represents a signal response domain for activation of NF-/_B and substitution of alanine for either Ser-32 or Ser-36 completely abolished the signal-induced phosphorylation and degradation of I ⁇ B , and blocked the activation of NF-/.B. These mutations also blocked in vi tro ubiquitination of the I/cBc. protein.
  • the amino-terminus of I ⁇ B is necessary for signal-induced phosphorylation and ubiquitination, but for degradation to occur, there is an absolute requirement for the C-terminal PEST domain (reviewed in 4,6).
  • IRF-3 turnover lies in the nature of the inducing stimuli.
  • IA.BC- phosphorylation/turnover and IRF-3 phosphorylation/degradation A significant temporal difference also exists between IA.BC- phosphorylation/turnover and IRF-3 phosphorylation/degradation. Many activators of NF- ⁇ B stimulate I/.Bo; phosphorylation within minutes and TNF induced degradation occurs within the first 15-30 minute after treatment. In the case of IRF-3, phosphorylation is not detected until 6-8 hours after infection and continues in a heterogenous manner over the next 10-12 hours. Previous experiments have, however, demonstrated that Sendai virus-induced turnover of I.BC. also occurs slowly over several hours (24) .
  • IRF-3 exists in a latent state in the cytoplasm of uninfected cells; the C-terminus may physically interact with the DNA binding domain in such a way as to obscure both the DBD and the IAD regions of the protein; the presence of an autoinhibitory domain within the C-terminal 20aa (407-427) would explain the activating effect of this deletion, as seen previously with IRF-4 (11,19).
  • Virus induced phosphorylation at the Ser/Thr at 396-405aa cluster leads to a conformational change in IRF-3, exposing both the DBD and IAD and relieving C-terminal autoinhibition.
  • Translocation to the nucleus occurring via an unidentified nuclear localization sequence or in conjunction with a transcriptional partner associating through the IAD region, leads to DNA binding at ISRE- and
  • PRDI/PRDIII-containing promoters Phosphorylation is also necessary for IRF-3 association with the chromatin remodelling activity of CBP/p300.
  • the presence of a NES element ultimately shuttles IRF-3 from the nucleus and terminates the initial activation of IFN responsive promoters.
  • the phosphorylated form of IRF-3 exported from the nucleus may now be susceptible to proteasome mediated degradation.
  • IRF-3 may represent a component of virus- or dsRNA-inducible complexes such as DRAF (16) or VIC (10,29) that could play a primary role in the induction of IFN- or IFN responsive genes.
  • IRF protein is useful as a tumour suppressor.
  • Example 1 Plasmid constructions and Mutagenesis.
  • the IRF-3 expression plasmid was prepared by cloning the EcoRI -XhoI fragment containing the IRF-3 cDNA from the pSKIRF-3 plasmid downstream of the CMV promoter of CMVBL vector.
  • CMVt-IRF-3 was constructed by cloning of IRF-3 cDNA downstream of the doxycycline-responsive promoter CMVt at the Ba-TiHI site of the neo CMVt BL vector (49) .
  • cDNAs encoding IRF-3 carboxyl terminal deletion mutations were generated by 28 cycles of PCR amplification with Vent DNA polymerase. DNA oligonucleotide primers were synthesized using an Applied
  • Biosystems DNA/RNA synthesizer The amino-terminal primer was synthesized with an EcoRI restriction enzyme site and the carboxyl-terminal primers were synthesized with Xbal restriction enzyme sites at their ends.
  • the PCR products were purified by phenol/chloroform extraction and ethanol precipitation, digested with .EcoRI and Xbal , and inserted into EcoRI /Xbal sites of CMVBL vector.
  • the point mutations of IRF-3 were generated by overlap PCR mutagenesis using Vent DNA polymerase. Mutations were confirmed by sequencing.
  • N-terminal deletion mutations ( ⁇ N, ⁇ N2A, ⁇ N3A and ⁇ N5A) of IRF-3 were generated by digestion of the related
  • GFP-IRF-3 expression plasmids were generated by cloning of cDNAs encoding wild type or mutated forms of IRF-3 into the downstream of EGFP in the pEGFP-Cl vector (Clonetech) .
  • the cDNAs coding for CBP were generated from the pRC-RSV/mCBP plasmid (provided by Dr. Dimitris Thanos) by PCR amplification.
  • the cDNA fragments were cloned in the downstream of myc-tag in 5' myc-PCDNA3 vector (provided by Dr. Stephane Richard) .
  • the IRF-7 cDNA was created by PCR and the resulting product was cloned into pFlag CMV-2 vector.
  • the cDNA encoding IRF-3 (5D) (aal32-427) was cut out from IRF-3 (5D) /CMVBL plasmid with Seal and Notl (blunted with Klenow enzyme) and was cloned into pFlag-IRF-7 (digested with Smal, which removed the C-terminal region of IRF-7 from 247-503) in frame with the IRF-7 N-termihal amino acid sequence (1-246) .
  • IRF-7 The point mutations of IRF-7 (D477- D479) were generated by overlap PCR mutagenesis essentially as described above for IRF-3 using Vent DNA polymerase. Codon AGC encoding residues Ser 477 and Ser 479 were mutated to GAC (Asp) . Mutations were confirmed by sequencing.
  • Example 2 Generation of IRF-3 cell lines. Plasmid CMVt-rtTA (49) was introduced into 293 cells by a calcium phosphate-based method. Cells were selected beginning at 48h after transfection for about one week in 0.MEM media (GIBCO-BRL) containing 10% heat-inactivated calf serum, glutamine, antibiotics and 2.5 ng/ ⁇ l puromycin (Sigma).
  • Resistant cells carrying the CMVt-rtTA plasmid were then transfected with the CMVt-IRF-3 plasmid.
  • Cells were selected beginning at 48h for a period of approximately 2 weeks in O.MEM containing 10% heat-inactivated calf serum, glutamine, antibiotics, 2.5 ng/ ⁇ l puromycin and 400 ⁇ g/ml G418 (Life Technologies, Inc.).
  • Example 3 Cell culture and transfections . All transfections for CAT assay were carried out in human embryonic kidney 293 cells or NIH3T3 cells grown in ⁇ MEM (293) or Dulbecco's MEM (NIH3T3) media (GIBCO-BRL) supplemented with 10% calf serum, glutamine and antibiotics. Subconfluent cells were transfected with 5 ⁇ g of CsCl purified chloramphenicol acetyltransferase (CAT) reporter and expression plasmids by calcium phosphate coprecipitation method (293 cells) or lipofectamine (NIH3T3 cells) .
  • CAT chloramphenicol acetyltransferase
  • the reporter plasmids were the SVo/3 CAT and ISG15 CAT reporter genes (56) ; also the transfection procedures were previously described (41,56) .
  • Example 4 Western blot analysis of IRF-3 modification and degradation.
  • IRF-3 protein To characterize the posttranslational regulation of IRF-3 protein, stable or transiently transfected IRF-3 expressing cells were infected with Sendai Virus (80 HAU/ml) or treated with 5 ng/ l TNF-c., either with or without addition of 50 ⁇ g/ml cycloheximide. In some experiments, cells were treated with either 100 ⁇ M calpain inhibitor I (ICN) , 40 ⁇ M MG132 proteasome inhibitor, or an equivalent volume of their respective solvent (ethanol) as control.
  • ICN calpain inhibitor I
  • MG132 proteasome inhibitor 40 ⁇ M MG132 proteasome inhibitor
  • Cells were washed with phosphate-buffered saline and lysed in 10 mm Tris-Cl pH 8.0, 200 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol (DTT), 0.5% Nonidet P-40 (NP-40) , 0.5 mm phenylmethysulfonyl fluoride (PMSF) , 5 ⁇ g/ml leupeptin, 5 ⁇ g/ml pepstatin, and 5 ⁇ g/ml aprotinin.
  • Equivalent amounts of whole cell extract (20 ⁇ g) were subject to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel.
  • Hybond transfer membrane (Amersham) in a buffer containing 30 mm This, 200 mm glycine and 20% methanol for lh.
  • the membrane was blocked by incubation in phosphate-buffered saline (PBS) containing 5% dried milk for lh and then probed with IRF-3 antibody in 5% milk/PBS, at a dilution of 1:3000. These incubations were done at 4°C overnight or at RT for l-3h. After four 10 minute washes with PBS, membranes were reacted with a peroxidase-conjugated secondary goat anti-rabbit antibody (Amersham) at a dilution of 1:2500. The reaction was then visualized with the enhanced chemiluminescence detection system (ECL) as recommended by the manufacturer (Amersham Corp.).
  • ECL enhanced chemiluminescence detection system
  • Example 5 Phosphatase treatment .
  • the phosphatase inhibitor mix contained 10 mm NaF, 1.5 mm Na 2 Mo0 4 , 1 mm -glycerophosphate, 0.4 mm Na 3 V0 4 and 0.1 ⁇ g/ml okadaic acid.
  • Example 6 Subcellular localization of GFP-IRF-3 proteins.
  • the GFP-IRF-3 expression plasmids (5 ⁇ g) were transiently transfected into COS-7 cells by the calcium phosphate coprecipitation method.
  • transfected cells were infected with Sendai virus (80 hemagglutinating units per mL for 2h) at 24h post transfection.
  • GFP fluorescence was analyzed in living cells with a Leica fluorescence microscope using a 4Ox objective.
  • Example 7 Electromobilitv Shift Assay.
  • Nuclear extracts were prepared from 293 cells at different times after infection with Sendai virus (80HAU/ml) . In some experiments, extracts were prepared from cells transfected with different IRF-3 expression plasmids, as indicated in individual experiments. Cells were washed in
  • Buffer A 10 mM HEPES, pH 7.9; 1.5 mm MgCl 2 ; 10 mM KCl; 0.5 mM dithiothreitol (DTT); and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] and were resuspended in Buffer A containing 0.1% NP-40. Cells were then chilled on ice for 10 minutes before centrifugation at 10,000 g.
  • Pellets were then resuspended in Buffer B (20mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl 2 ; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM PMSF; 5 ⁇ g/ml leupeptin; 5 ⁇ g/ml pepstatin; 0.5 mM spermidine; 0.15 mM spermine; and 5 ⁇ g/ml aprotinin) . Samples were incubated on ice for 15 minutes before being centrifuged at 10,000 g.
  • Buffer B 20mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl 2 ; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM PMSF; 5 ⁇ g/ml leupeptin; 5 ⁇ g/ml pepstatin; 0.5 mM
  • Nuclear extract supernatants were diluted with Buffer C (20 mM HEPES, pH 7.9; 20% glycerol; 0.2 mM EDTA; 50 mM KCl; 0.5 mM DTT; and 0.5 mM PMSF) .
  • Nuclear extracts were subjected to EMSA by using a 32P-labelled probe corresponding to the PRDIII region of the IFN-jS promoter (5' -GGAAAACTGAAAGGG-3 ' ) or the ISRE region of the ISG-15 promoter (5' -GATCGGGAAAGGGAAACCGAAACTGAAGCC-3 ' ) .
  • the resulting protein-DNA complexes were resolved by 5% polyacrylamide gel and exposed to X-ray film. To demonstrate the specificity of protein-DNA complex formation, 125-fold molar excess of unlabelled oligonucleotide was added to the nuclear extract before adding labelled probe.
  • Example 8 Immunoprecipitation and Western analysis of CBP associated proteins.
  • IRF-3 is expressed constitutively in various cells and its expression is not enhanced by viral infection or by IFN treatment.
  • IRF-3 protein is regulated by post-translational modification after virus infection.
  • 293 cells were transiently transfected with an IRF-3 expression plasmid and subsequently infected with Sendai virus 24h later.
  • endogenous IRF-3 protein was easily detected using a polyclonal IRF-3 antibody and in cells transfected with the IRF-3 expression plasmid, IRF-3 protein levels were significantly increased (Fig.l, lanes 1 and 3).
  • the kinetics of virus-induced modification of IRF-3 were characterized in a 293 cell line that expressed IRF-3 inducibly under the control of the tetracycline responsive promoter CMVt (25,26). Infection of this cell line (designated rtTA-IRF-3) with Sendai virus resulted in a decrease in the amount of IRF-3 between 12 and 24h after infection (Fig. 2A) .
  • Two forms of IRF-3 protein (designated I and II) were detected in uninfected cells (Fig. 2A, lane 1) and following virus infection, a third slowly migrating form of IRF-3 was also detected (Fig.2A, lanes 4-7) .
  • IRF-3 virus-induced phosphorylation
  • PPA potato acidic phosphatase
  • CIP calf intestine alkaline phosphatase
  • Fig. 2B phosphatase inhibitors
  • IRF-3 A series of deletions of IRF-3 were generated to identify the virus-induced phosphorylation site(s) of IRF-3 (Fig. 3A) .
  • 293 cells were transiently transfected with IRF-3 deletion mutants and the virus mediated phosphorylation was measured by immunoblotting (Fig. 3B) .
  • Full length and 407aa forms of IRF-3 were phosphorylated as a consequence of virus infection (Fig. 3B, lanes 1-4) .
  • a phosphorylation domain of IRF-3 protein was localized to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407.
  • Point mutations in the several putative Ser and Thr phosphorylation residues within this region were generated in the full length protein and the ⁇ 9-133 ( ⁇ N) protein (Fig. 4A) .
  • the Ser-396/Ser398/Ser-402/Thr-404/Ser-405 residues were replaced by alanine (5A) , as were the three residues Ser-402/Thr-404/Ser-405 (3A) and the two residues Ser-396/Ser-398 (2A) .
  • IRF-3 was localized in the cytoplasm of uninfected cells (67) ; to investigate the role of phosphorylation on IRF-3 localization, wild type and point mutated forms of IRF-3 were linked to green fluorescent protein (GFP) , transfected into COS-7 cells and examined for Sendai virus induced changes in subcellular localization (Fig. 5) .
  • GFP-IRF-3 localized exclusively to the cytoplasm; Sendai virus infection resulted in translocation of IRF-3 to the nucleus within 8h in 90-95% of the cells (Fig. 5A and B) .
  • GFP-IRF-3 (NES) localized to the nucleus with an intense speckled pattern of nuclear fluorescence in greater than 95% of the cells, suggesting that IRF-3 (NES) may be trapped in the nucleus associated with the nuclear pore complex.
  • IRF-3 the capacity of IRF-3 to regulate gene expression was analysed by transient transfection in human 293 and murine NIH3T3 cells using the IFN ⁇ and ISG-15 promoters in reporter gene assays.
  • Introduction of the C-terminal point mutants - IRF-3 (2A) , IRF-3 (3A) IRF-3 (5A) - reduced the low transactivation capacity of IRF-3 to control levels (Fig. 6A) .
  • deletion of the C-terminal 20aa of IRF-3 to IRF-3 stimulated IFN/3 activity about 6 fold, indicative of the removal of an inhibitory domain in IRF-3.
  • further deletion to 394, 357 or 240 abrogated transactivation potential (Fig. 6A) .
  • Mutation of the NES element was not sufficient to stimulate IFN/3 activity.
  • substitution of the Ser/Thr cluster at aa397-405 in IRF-3 with the phosphomimetic Asp generated a very strong, constitutive transactivator protein that alone stimulated the IFN/3 promoter 90 fold.
  • IRF-3 (407) and RelA(p65) stimulated IFN/3 activity about 40 fold, supporting the idea of the removal of an inhibitory domain in IRF-3, whereas both the IRF-3 (394) and the IRF-3 (NES) failed to synergise with RelA in the activation of the IFN/3 promoter.
  • RelA and IRF-3 (NES) produced a relatively weak 8 fold induction of IFN/3 expression, indicating that nuclear localization is not sufficient for IRF-3 activation.
  • the combination of RelA and IRF-3 (5D) produced an 80 fold stimulation of IFN/3 promoter activity (Fig.
  • IRF-3 (5D) alone appears to be capable of full stimulation of the IFN/3 promoter and further synergy with RelA is not observed (compare Fig. 6A and B) .
  • IRF-3 (5A) and RelA produced a 30 fold stimulation, suggesting that 5A can still synergise with RelA, despite mutation of the Ser/Thr cluster.
  • the transactivation potential of IRF-3 was also analysed using the ISG-15 promoter, an ISRE containing regulatory element (Fig. 6C) .
  • IRF-3 alone weakly activated the ISG-15 promoter; in the context of this regulatory element, IRF-3 was weaker than IRF-1, which produced a 9 fold stimulation. Again deletion of the C-terminal 20aa of IRF-3 generated a protein that stimulated gene expression; with the ISG-15 promoter, a 12 fold induction was observed; IRF-3 (394) and IRF-3 (357) did not stimulate gene expression but rather slightly repressed ISG-15. Again remarkably, IRF-3 (5D) produced a 50 fold induction of the ISG-15 promoter (Fig.
  • IRF-3-inducible cells were used to determine whether other cytokine-chemokine genes may be regulated by IRF-3; an (Rnase Protection Analysis (RPA) with multiple human cytokine-chemokine probes (Pharmingen) was used to examine RNA derived from rtTA-IRF-3 or rtTA-IRF-3 (5D) cells. Strikingly, the RANTES gene was highly expressed in the IRF- 3 (5D) -inducible cells, as well as in virus-infected cells (Fig. 7A, lanes 3, 5, and 7) but not in uninfected rtTA- or wt IRF-3 - expressing cells (Fig. 7A, lanes 1 and 4).
  • IRF-3 (5D) was a strong transactivator of the IFN-/3 promoter in transient transfection assays, the possibility of an autoregulatory effect of IFN-C.//3 expression on transcription of RANTES promoter via JAK-STAT activation was considered. Activation of RANTES did not occur secondary to the production of IFN- ⁇ .//?, since RANTES mRNA was not detected in control rtTA-expressing cells treated directly with IFN- ⁇ //3 (Fig. 7A, lane 2) ; furthermore, addition of neuralizing antibody directed against type I IFN did not block the stimulation of RANTES gene expression by IRF-3 (5D) (Fig. 7A, lane 8).
  • IRF-3 transgene induction and RANTES mRNA expression were characterized at various times following Dox induction.
  • IRF-3 (5D) was detected at 8 to 12 hours with peak levels at 24 hours following Dox addition.
  • RANTES mRNA was first detectable at 18 hours after Dox induction with peak levels at 40 hours (Fig. 7C, lanes 5 to 10) .
  • Induction of RANTES protein expression as detected by ELISA (Fig. 7D) was first observed at 12 hours after Dox induction of IRF-3, in good agreement with the mRNA levels, and accumulated thereafter with a dramatic increase between 24 and 32 hours after stimulation, also in agreement with mRNA levels.
  • IRF-3 (5D) may be directly activating another transcription factor such as NF-KB, which in turn would stimulte RANTES transciption, was also considered. No evidence for IRF-3 (5D) -mediated activation of NF-A.B DNA binding activigy was observed. Similarly, IRF-3 (5D) expression did not activate the human immunodeficiency virus (HIV) -long terminal repeat, a complex promoter controlled by NF-/.B and other transcription factors (data not shown) . Inhibition of IRF-3 degradation.
  • HAV human immunodeficiency virus
  • IRF-3 Another consequence of virus infection is the degradation of the IRF-3. Since phosphorylation of proteins is functionally associated with the process of protein degradation via the ubiquitin-dependent proteasome pathway (53,57,60), the effect of proteasome inhibitors on virus-induced turnover of IRF-3 was examined. In cells transfected with the ⁇ N and ⁇ N5A forms of IRF-3, virus induced degradation of full length
  • the endogenous IRF-3 also co-precipitated from virus-infected cells (Fig. 9B, lane 1) .
  • mutation of the Ser/Thr residues identified as the virus inducible phosphorylation sites abrogated the association of IRF-3 with CBP.
  • IRF-3 (2A) and IRF-3 (5A) were detected in whole cell extract immunoblot but not in the CBP immunoprecipitate (Fig. 9B, compare lanes 4 and 6 with lanes 11 and 13).
  • IRF-3 (3A) mutant interaction with CBP was still observed (Fig. 9B, lane 5) .
  • the high background in all lanes represents secondary antibody reactivity with rabbit IgG from the immunoprecipitation.
  • Immunoblot analysis of the whole cell extracts revealed that phosphorylated IRF-3, as well as forms I and II were present in virus infected cells (Fig. 9B, lane 10) and in cells transfected with 2A, 3A and 5A the forms I and II were observed but not the phosphorylated form of IRF-3 (Fig. 9B, lanes 11-13) .
  • CBP has several domains that bind transcription factors, designated CBP1, CBP2, and CBP3 respectively (Fig. 9A, reviewed in (28) ) .
  • CBP1, CBP2, and CBP3 bind transcription factors
  • Fig. 9A reviewed in (28)
  • the three specific subdomains were myc-tagged at the 5' end by subcloning into the pCDNA3 vector (Fig. 9A) .
  • 293 cells were co-transfected with these myc-tagged CBP expression plasmids together with the IRF-3 ⁇ N ( ⁇ 9-133) expression plasmid.
  • IRF-3 ⁇ N ⁇ 9-133 expression plasmid.
  • cells were infected with Sendai virus, co-immunopreciptated with anti-myc antibody 16h later (21) and then immunoblotted for IRF-3.
  • Figure 11 shows the relative activity of various forms of IRF-3 and IRF-7, and binary mixtures thereof, in transactivation studies. Both the IRF-3 (5D) and IRF-7 (2D) mutants show increased activity relative to their corresponding wild-type proteins. There is a synegistic effect present when both proteins are present, and this effect is most pronounced in a mixture of the IRF-3 (5D) and IRF-7 (2D) (D477/479) mutants.
  • Figure 14 shows that the chimeric protein IRF-7 (1-246) /IRF-3 (5D) (132-427) has a markedly increased activity over the mixture of the IRF-3 (5D) and IRF-7 (2D) (D477/479) mutants.
  • a pharmaceutical composition may be prepared, with a protein of the invention as active ingredient, for the treatment of a viral infection, such as an influenza infection, a herpes infection or an HIV infection.
  • a viral infection such as an influenza infection, a herpes infection or an HIV infection.
  • compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers.
  • the active compounds of the invention may be formulated for oral, buccal, transdermal (e.g., patch), intranasal, parenteral (e.g., intravenous, intramuscular or subcutaneous) or rectal administration or in a form suitable for administration by inhalation or insufflation.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate) ; lubricants (e.g., magnesium stearate, talc or silica); disintegrants
  • binding agents e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., magnesium stearate, talc or silica
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats) ; emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxybenzoates or sorbic acid
  • buccal administration the composition may take the form of tablets or lozenges formulated
  • the active compounds of the invention may be formulated for parenteral administration by injection, including using conventional catherization techniques or infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • a suitable vehicle e.g., sterile pyrogen- free water
  • the active compounds of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides .
  • the active compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurized container or nebulizer may contain a solution or suspension of the active compound.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
  • the protein of the invention can also be made available using gene therapy.
  • the DNA encoding the protein can be introduced to cells of an organism at a target site, for example, by a viral vector, by electroporation, by co- transfection with a selectable marker, or by DNA vaccine.
  • the CBP coactivator is a histone acetyltransferase. Nature 384:641-643.
  • Nuclear receptor coactivator ACTR is a noval histone acetyltransferases and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569-580.
  • Double-stranded RNA activates novel factors that bind to the interferon stimulated response element. Mol .Cell .Biol . 13:3756-3764.
  • Pip a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 9:1377-1387.
  • the transcriptional coactivators p300 and CBP are histone acetyltransferases . Cell 87:953-959. 51. Palombella, V. and Maniatis, T. 1992. Inducible processing of interferon regulatory factor-2. Mol .Cell .Biol .
  • Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA. J. Biol. Chem. 272:9785-9792.
  • NF-A.B a lesson in family values. Cell 80:529-532.

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