Note: Descriptions are shown in the official language in which they were submitted.
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1
HIGHLY ACTIVE FORMS OF INTERFERON REGULATORY FACTOR PROTEINS
BACKGROUND OF THE INVENTION
Interferons (IFNs) 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). Among the many virus- and
IFN-inducible proteins are the growing family of IRF
transcription factors, the Interferon Regulatory Factors
(IRFs) .
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-Q 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~y/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 (HHVB) (55).
The presence of IRF-like binding sites in the
promoter region of the IFNA and IFNB genes implicated the IRF
factors as essential mediators of the induction of IFN genes.
The original results of Harada et al. (30,32) indicated that
IFN gene induction was activated by IRF-1, while the related
IRF-2 factor suppressed IFN expression. However, the essential
role of IRF-1 and IRF-2 in the regulation of IFNA and IFNB gene
expression has become controversial with the observation that
mice containing homozygous deletion of IRF-1 or IRF-2, or
fibroblasts derived from these mice, induced IFNA and IFNB gene
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expression after virus infection to the same level as the
wild-type mice or cells (44).
On the other hand, 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~y/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).
Several other members of the IRF family have been
identified. The ICSBP gene is expressed exclusively in the
cells of the immune system (18,64) and its expression can be
enhanced by IFN~y. 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 lymphoid specific Pip/LSIRF/IRF-4 was identified
(19,43,66) that interacts with phosphorylated PU.1, a member of
the Ets family of transcription factors (15). The Pip/PU.1
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).
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
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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. In previous studies, it has been
shown that 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).
Most of the IRF family members so far identified
appear to have specific and critical functions in lymphoid
cells and/or their action is related to the signalling pathway
induced by IFN or viruses. Interestingly, there is recent
evidence indicating that the IRF(s) may also play a role in the
transcriptional activation of viral promoters. 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-a. Using 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). By homology search of the HGF ETS cDNA library
the Pitha group has also identified a novel IRF whose sequence
is identical to that of IRF-7. At the amino acid level, IRF-7
shows highest homology to IRF-3. Several open reading frames
(ORFs) of IRF-7 have been identified. Pagano's group found
three shorter ORFs, listed in the database as IRF-7A, B and C
((69), accession nos. U53830, U53831 and U53832, respectively).
A new IRF-7 isoform, IRF-7H, was recently identified by Pitha's
group ((70), accession number AF076494).
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In vitro translated IRF-7 encodes a protein of 68 kDa
(69, 72). Interestingly, while in vitro 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). In contrast to IRF-3, IRF-
7 expression is not generally constitutive but can be
effectively induced by IFN-a in fibroblast cells, B-cells and
other cells of lymphoid origin (70, 71). Like 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 IFNa4 and IFN~i gene transcription in
murine cells. Once produced and secreted from the infected
cell, IFNa4 and IFN~i 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-IFNa4 gene expression (71).
SUI~iARY OF THE INVENTION
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. Such 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.
More specifically, the present invention provides a
modified interferon regulatory factor (IRF) protein, the
protein comprising at least one modified serine or threonine
CA 02325354 2000-10-10
05-OE-2000 CA 009900314
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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.
5 The present invention also provides nucleotide
sequences which encode a protein of the invention as described
above. Such nucleotide sequences may, for example, be used to
modify a target cell of an organism.
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, a hepatitis infection or an HIv
infection. ~ '
The present invention also provides a commercial
package containing the IRF protein or pharmaceutical
composition according to the invention, together with
instructions for its use for the treatment of cancer or of a.
viral infection, for example, an influenza infection, a herpes
infection, a hepatitis 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.
DESCRIPTION OF THE FIGURES
Figure 1. Sendai virus infection induces IRF-3
degradation. IRF-3 expression plasmid CMVBL-IRF3
(lanes 1 and 2) or CMVHL vector alone (lanes 3 and 4), both at
5 ~g were transiently transfected into 293 cells by the calcium
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phosphate method. At ~24h post. transfection, . cel.ls were
infected with Sendai virus for 16h (lanes 2 and- 4). or left
uninfected (lanes 1 and 3) . Whole cell extracts (20 .fig) were
'prepared and analyzed by immunoblotting with anti-IRF-3'
antibody. .
- Figure 2. .Sendai virus induced phosphorylat~ion and
degradation of IRF-3 protein. A) rtTA-IRF-3 cells, selected as
described in 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 fig) ~ by .
.- immunoblot.~ Two forms of IRF-3 were detected, designated as
- form I and form II. B)-At 24h post ~Dox induction, 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 ~ . .
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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). Phosphorylated IRF-3 protein appears as a distinct band in
immunoblots, migrating more slowly than IRF-3 forms I and II.
Figure 3. Analysis of IRF-3 deletion mutants in
Sendai virus induced phosphorylation.
(A) Schematic representation of four IRF-3 deletions.
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). Whola cell extracts (20 fig) 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.
Figure 4. Analysis of IRF-3 point mutations in Sendai
virus induced phosphorylation.
(A) Schematic representation of IRF-3 point mutations.
Thick solid lines and thin dashed lines indicate included and
excluded sequences, respectively. The N-terminal IRF homology
domain, the Nes element and C-terminal IRF association domain
are indicated. Amino acids residues from 382 to 414 and from
141 to 147 are shown. The amino acids targeted for alanine or
aspartic acid substitution are shown in large print. The point
mutations are indicated below the sequence: (2A: 5396A/S398A;
3A: S402A/T404A/S405A; 5A: S396A/S398A/S402A/T404A/S405A); 5D
S396D/S398D/S402D/T404D/S405D; J2A: S385A/S386A; NES:
S145A/S146A).
(B) Expression plasmids (5 ~.g each) encoding wild type and
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point 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, 10, 12, 14, 16 and 18) or left uninfected
(lanes 1, 3, 5, 7, 9, 11, 13, 15 and 17). 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.
Figure 5. Virus dependent cytoplasmic-nuclear
translocation of 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 40x objective.
Figure 6. Transactivation of PRDI/PRDIII and ISRE
containing promoters by IRF-3.
293 cells were transfected with IFN~i-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 (IFN~i-CAT) or 10 ~g
{ISG15-CAT) of total protein extract for 1-2h 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 ~i-Gal
activity); the values represent the average of three
experiments with variability shown in the error bar.
Figure 7. IRF-3 inducible expression of RANTES gene.
(A) Stimulation of RANTES gene transcription in
virus-infected and IRF-3{5D)-expressing cells. 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//3 (100 IU/ml). The neutralizing antibody
for type I IFN {Sigma) was added at the time of Dox addition.
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Total RNA was isolated from each sample and analyzed by RPA
using the hCK5 kit (Pharmingen).
(B) Repression of virus-induced RANTES gene transcription
by a dominant-negative form of IRF-3. The rtTA- and
IRF-3(~N)-expressing cells were either left untrated or
infected with Sendai virus (80 HAU/ml) for 16 hours. Total RNA
was isolated from each sample and analyzed by RPA.
(C) The kinetics of RANTES expression induced by IRF-3
(5D). Total RNA from IRF-3(5D)-expressing cells was isolated
from each sample after Dox addition and analyzed by RPA.
(D) Cell culture supernatants were analyzed for the
presence of RANTES protein by an ELISA performed as specified
by the manufacturer (Biosource International).
Figure 8. Stabilization of IRF-3 by proteasome
inhibitors.
IRF-3 t1N (09-133) (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 CHP in virus infected
cells.
(A) Schematic representation of CBP, illustrating the
domains, involved in interaction with host or viral proteins
(modified from (28)) and the myc-tagged CBP proteins (CBP1,
CBP2, CBP3) used for immunoprecipitation.
(B) 293 cells were transfected with wild type and deletion
mutants of iRF-3 expression plasmid (5 ug, as indicated above
the lanes) or left untransfected (lanes 1 and 8). At 24h after
transfection, 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 fig, except lane 1, which was 600 fig)
were immunoprecipitated with anti-CBP antibody A22 (lanes 1-6)
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or with preimmune serum (lane 7). The immunoprecipitated
complexes (lanes 1-7) or 30 ~g whole cell extracts (lanes 8-13)
were run on 5~ SDS-PAGE and subsequently probed with anti-IRF-3
antibody.
(C) 293 cells were co-transfected with myc-tagged CBP
expression plasmids (as indicated above the lanes) and IRF-3 ON
(D9-133) expression plasmid. At 24h after transfection, cells
were infected with Sendai virus (lanes 2, 4 and 6) or left
uninfected (lanes 1, 3 and 5). Whole cell extracts (300
were immunoprecipitated with monoclonal anti-myc-tag antibody
9E10. 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.
(D) Whole cell extracts (30 fig) from (C) were also
analyzed directly for the expression of myc-tagged CBP proteins
by immunoblotting using anti-myc antibody 9E10.
Figure 10. The cDNA sequence encoding IRF-3(5D),
together with the amino acid sequence of IRF-3(5D).
Figure 11. Transactivation study as described in
Figure 6, using the IFN~i-CAT reporter plasmid to indicate the
activity of various forms of IRF-3 and IRF-7 and binary
mixtures thereof.
Figure 12. The cDNA sequence encoding IRF-7A(2D),
together with the amino acid sequence of IRF-7A(2D).
Figure 13. The cDNA sequence encoding the
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.
Figure 14. Transactivation study as described in
Figure 6, using the IFN/3-CAT reporter plasmid to indicate the
relative activity of various forms of IRF-3 and IRF-7, binary
mixtures thereof and the chimeric protein
IRF-7(1-246)/IRF-3(132-427) (IRF-7N-IRF-3(5D}C in Figure 14).
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "nucleotide sequence" means
a DNA or RNA molecule or sequence, and can include, for
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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.
Two DNA, RNA or polypeptide sequences are
5 "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
10 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) .
The 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.
Furthermore, while 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
CA 02325354 2000-10-10
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11
having aspartic acid residues in at least one of positions 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 asparatic 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). ,; -
Also within the scope of the invention are chirneric
proteins comprising a carboxy-terminus domain of one modified
IRF protein, modified as discussed above, and an amino-terminal
domain of another IRF protein. Preferably, 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). Even more preferred is a chimeric
protein comprising residues 1 to 246 of IRF-7 and residues 132
to 427 of IRF-3(5D) (Figure 13). ;
Also within the scope of the invention are proteins
which are substantially homologous to the above proteins and
which retain the function of those proteins. This includes ;~ -
proteins based on human IRF-3 and IRF-7, as well as
corresponding IRF-3 and IRF-7 proteins of other~species.
Nucleotide sequences within the scope of the
invention are those which encode a protein of the invention.
Preferably, 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). Also,
preferably, 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 in Figure 12, which DNA encodes IRF-7(2D). Also ,
-~, , _
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preferably, the nucleotide sequence is a coding DNA sequence as
defined in Figure 13 or a DNA sequence which is ; ,
hybridizableunder 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. ._
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
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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
S 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.
Interestingly, 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. These results are discussed in terms of a
model in which virus-inducible C-terminal phosphorylation of
IRF-3 alters protein conformation to permit nuclear
translocation, association with transcriptional partners and
primary activation of IFN- and IFN-responsive genes.
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. 2) 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. 3) Sendai virus
infection induced the DNA binding and transactivation potential
of IRF-3. Furthermore, 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. 4) Phosphorylation was also required for the
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13
association of IRF-3 with the CBP co-activator protein. 5)
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.
Cytoplasmic to nuclear translocation of IRF-3 as a
consequence of virus infection was inhibited by mutation of the
Ser/Thr cluster, indicating an important regulatory role for
C-terminal phosphorylation in the activation of IRF-3. Also
strikingly, the conversion of the phosphorylation sites to the
phosphomimetic Asp altered the subcellular localization of
IRF-3 in uninfected cells. A proportion of IRF-3(5D) was
localized to the nucleus of uninfected cells, suggesting that
some IRF-3 may shuttle to and from the nucleus constitutively;
this observation is consistent with the identification of a
nuclear export signal in IRF-3. Mutation of L144A/L145A in the
NES element produced the most impressive alterations in
subcellular localization. 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.
One of the striking results of the mutagenesis of the
C-terminal domain of IRF-3 was the generation of IRF-3(5D), an
exceptionally strong activator of IFN-~i and ISG-15 gene
expression. The phosphomimetic form of IRF-3 alone was able to
stimulate IFN-~i expression as strongly as virus infection, a
level of stimulation not previously observed in co-expression
experiments (24,61). In previous experiments, it has been
demonstrated that 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). Virus induction
results in the appearance of two new protein-DNA complexes;
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supershift experiments confirmed that both complexes contain
IRF-3; it is not clear at this stage whether the upper complex
also contains other proteins such as in the VIC (10,29) and
DRAF (16) complexes or whether the lower complex represents a
breakdown product of IRF-3. Strikingly, the same complexes
appeared following co-transfection of IRF-3(5D) expression
plasmid in the absence of virus induction, indicating that
IRF-3(5D) represented a constitutive DNA binding form of IRF-3.
Thus, in uninfected cells, IRF-3(5D) localized in part to the
nucleus (Fig. 5), interacted with DNA constitutively and was a
strong activator of gene expression (Fig. 6).
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). By analogy with IRF-3, two GAAANN
sequences present in PRDIII of IFN-~i and another GAAANN element
present in PRDI may serve as DNA contacts for multiple
IRF-3(5D) proteins with strong activating potential.
Similarly, 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 ElA and CREB proteins (1,13). As
a critical determinant of its global transcriptional
coactivator activity, 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. Several studies have
now demonstrated that CBP/p300 participates in the
transcriptional process by providing a scaffold for different
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classes of transcriptional regulators on specific chromatin
domains (12,50}. 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,
5 SV40 large T antigen and the tumour suppressor p53, among
others. With regard to 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
10 recently to acetylate p53 and stimulate its transactivation
potential (27) .
It will be of interest to determine whether IRF-3 is
similarly modified by CBP association. The functional
consequences of IRF-3 interaction with CBP/p300 remain to be
15 elucidated, although recent studies demonstrated that CBP/p300
also functionally interacts with STAT 1 (68) and STAT 2 (7) and
may contribute to IFNa and IFN~y nuclear signalling. Recently
published studies have demonstrated that synergistic activation
of the IFNR promoter requires recruitment of CBP/p300 to the
enhanceosome, via a new activating surface assembled from the
activation domains of all the transcription factors in the
enhanceosome (37,45). Alterations in any of the activation
domains decreased both CBP recruitment and transcriptional
synergy. By analogy, recruitment of CBP/p300 to DNA bound
IRF-3 is likely required for maximal transcriptional
activation. Association requires the interaction of the
C-terminal domain of IRF-3 and the C-terminal interaction
domain of CBP, a region previously shown to associate with the
p53 tumour suppressor, whereas STAT1 and STAT2 associate with
different regions of CBP (7, 68} .
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. In response to dsRNA or viral induction, the 50 kD
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IRF-2 protein is proteolytically processed into a smaller,
24-27 kDa protein (51) comprising the 160 as DBD of IRF-2,
termed TH3 (14) or In4 (65). Although 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-~i 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
C-terminal Ser/Thr residues and its subsequent degradation by a
proteasome dependent pathway are also similar to the well
studied phosphorylation and degradation of IrcBa which leads to
activation of NF-xB binding activity (reviewed in 4,6). In
unstimulated cells, NF-KB heterodimers are retained in the
cytoplasm by inhibitory IrcB proteins. Upon stimulation by many
activating agents, including cytokines, viruses and dSRNA, IKBa
is rapidly phosphorylated and degraded, resulting in the
release and nuclear translocation of NF-xB. The amino-terminus
of IrcBa represents a signal response domain for activation of
NF-~cB and substitution of alanine for either Ser-32 or Ser-36
completely abolished the signal-induced phosphorylation and
degradation of IrcBa, and blocked the activation of NF-KH.
These mutations also blocked in vitro ubiquitination of the
IKBa protein. The amino-terminus of IKBcx 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).
Similarities and differences exist between the
observed degradation of IRF-3 and the mechanism of IKBa
degradation. The C-terminal phosphorylation of IRF-3 as a
consequence of virus infection is required for its subsequent
degradation based on the deletion and point mutation analysis
of the region -ISNSHPLSLTSDQ- between amino acids 395 and 407.
Minimally, phosphorylation of Ser-396 and Ser-398 are required
for subsequent degradation, although Ser-402, Ser-404 and
Ser-405 may represent secondary phosphorylation sites.
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Likewise, in the case of IKBa, phosphorylation and Ser-32 and
Ser-36 are required for inducer mediated degradation.
Furthermore, the protease inhibitor calpain inhibitor I and the
more specific proteasome inhibitor MG132 block IRF-3 turnover.
S A major difference in the mechanisms of IKBa and
IRF-3 turnover lies in the nature of the inducing stimuli.
Multiple inducers - cytokines such as TNF and IL-1, viruses,
LPS, oxidative stress, etc (6) - all lead to the induction of
IkBa phosphorylation and degradation whereas IRF-3
phosphorylation appears to be induced only by virus infection
and dsRNA addition; other inducers have not resulted in IRF-3
turnover.
A significant temporal difference also exists between
IrcBa phosphorylation/turnover and IRF-3
phosphorylation/degradation. Many activators of NF-rcB
stimulate IrcBa 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 IKBa also occurs slowly over several
hours (24) .
Based on the data presented herein and by analogy
with the properties of other IRF family members (48), the
following model is proposed to explain several observations.
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
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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. This scenario shares
several features with the protein synthesis independent
activation of NF-KB, and further suggests that 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.
In view of the above-mentioned properties, and in
particular its ability to stimulate an immune response, IRF
protein is useful as a tumour suppresser.
The invention is described in more detail in the
following examples.
Example 1: Plasmid constructions and Mutacrenesis.
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
BamHI site of the nee 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 XbaI
restriction enzyme sites at their ends. The PCR products were
purified by phenol/chloroform extraction and ethanol
precipitation, digested with EcoRI and XbaI, and inserted into
EcoRI/XbaI sites of CMVBL vector.
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The point mutations of IRF-3 were generated by
overlap PCR mutagenesis using Vent DNA polymerase. Mutations
were confirmed by sequencing.
The N-terminal deletion mutations (~N, ~N2A, AN3A and
S ~NSA) of IRF-3 were generated by digestion of the related
IRF-3/CMVBL plasmid with BamHI (filled in with Klenow enzyme),
partial digestion with ScaI, and re-ligation. 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-C1 vector (Clonetech). For construction of
plasmids encoding myc-tagged CBP truncated proteins, 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).
For the construction of pFlag-IRF-7, the IRF-7 cDNA
was created by PCR and the resulting product was cloned into
pFlag CMV-2 vector. To generate the IRF-7(aal-246)-IRF-3(5D)
(aa132-427) chimera, the cDNA encoding IRF-3 (5D) {aa132-427)
was cut out from IRF-3 (5D)/CMVBL plasmid with ScaI and NotI
{blunted with Klenow enzyme) and was cloned into pFlag-IRF-7
(digested with SmaI, which removed the C-terminal region of
IRF-7 from 247-503) in frame with the IRF-7 N-terminal amino
acid sequence (1-246). 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 aMEM
media (GIBCO-BRL) containing 10~ heat-inactivated calf serum,
glutamine, antibiotics and 2.5 ng/~.1 puromycin (Sigma).
Resistant cells carrying the CMVt-rtTA plasmid (rtTA-293 cells)
were then transfected with the CMVt-IRF-3 plasmid. Cells were
selected beginning at 48h for a period of approximately 2 weeks
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in aMEM containing 10~ heat-inactivated calf serum, glutamine,
antibiotics, 2.5 ng/~1 puromycin and 400 ~g/ml 6418 (Life
Technologies, Inc.).
Example 3: Cell culture and transfections.
S All transfections for CAT assay were carried out in
human embryonic kidney 293 cells or NIH3T3 cells grown in aMEM
(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
10 chloramphenicol acetyltransferase (CAT) reporter and expression
plasmids by calcium phosphate coprecipitation method (293
cells) or lipofectamine (NIH3T3 cells). The reporter plasmids
were the SVo,Q CAT and ISG15 CAT reporter genes (56); also the
transfection procedures were previously described (41,56). For
15 individual transfections, 100 ~,g (SVo~i CAT) or 10 ~,g (ISG15
CAT) of total protein extract was assayed for 1-2h at 37°C. The
CAT activity was normalized with ~i-Gal assay. All
transfections were performed 3-6 times.
Example 4: Western blot analysis of IRF-3 modification and
20 degradation.
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/ml TNF-a, 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. Cells were washed
with phosphate-buffered saline and lysed in 10 mm Tris-C1 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/mI
aprotinin. Equivalent amounts of whole cell extract (20 ~.g)
were subject to SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) in a lOg polyacrylamide gel. After electrophoresis,
the proteins were transferred to Hybond transfer membrane
(Amersham) in a buffer containing 30 mm This, 200 mm glycine
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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/PHS, at a dilution of 1:3000. These incubations were done
at 4°C overnight or at RT for 1-3h. After four 10 minute washes
with PHS, 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.).
Example 5: Phosphatase treatment.
Twenty to sixty ~g of whole cell extract were treated
with 0.3 units of potato acidic phosphatase (Sigma) in a final
volume of 30 ~,1 PIPES buffer (10 mm PIPES pH 6.0, 0.5 mm PMSF,
5 ~,g/ml aprotinin, 1 ~g/ml leupeptin, and 1 ~g/ml pepstatin) or
5 units of calf intestine alkaline phosphatase (Pharmacia) in
30 ~,l CIP buffer. The phosphatase inhibitor mix contained 10
mm NaF, 1.5 mm Na2Mo04, 1 mm (3-glycerophosphate, 0.4 mm Na3V04
and 0.1 ~.g/ml okadaic acid.
Example 6: Subcellular localization of GFP-IRF-3 t~roteins.
To analyse the subcellular localization of wild type
and mutated forms of IRF-3 proteins in uninfected and virus
infected cells, the GFP-IRF-3 expression plasmids (5 fig) were
transiently transfected into COS-7 cells by the calcium
phosphate coprecipitation method. For virus infection,
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 40x 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 MgCl2; 10 mM KC1; 0.5 mM
dithiothreitol (DTT); and 0.5 mM phenylmethylsulfonyl fluoride
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(PMSF)3 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
MgClz; 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. Nuclear extract
supernatants were diluted with Buffer C (20 mM HEPES, pH 7.9;
20% glycerol; 0.2 mM EDTA; 50 mM KC1; 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-~i 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.
Whole cell extract (300 fig) were prepared from either
transfected or untransfected cells and precleared with 5 ~1 of
preimmune rabbit serum and 20 ~cl of protein A-Sepharose beads
(Pharmacia) for 1 hour at 4°C. The extract was incubated with
10 ~,1 of anti-CBP antibody A-22 (Santa Cruz) or 2 ~,l anti-myc
antibody 9E10 (21) and 30 ~1 of protein A-Sepharose beads for
2-3 hours at 4°C. Precipitates were washed 5 times with lysis
buffer, eluted by boiling the beads 3 minutes in lx SDS sample
buffer. Eluted proteins were separated by SDS PAGE,
transferred to Hybond transfer membrane. Membranes were
incubated with anti-IRF-3 (1:3000) or anti-myc antibody 9E10
(1:1000). Immunocomplexes were detected by using a
chemiluminescence-based system.
The results of the above examples are summarized
below.
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Virus induced phosphorvlation of IRF-3 protein.
IRF-3 is expressed constitutively in various cells
and its expression is not enhanced by viral infection or by IFN
treatment. To investigate whether the 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. In cells transfected with CMVBL vector alone,
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.i, lanes 1 and 3). Interestingly, Sendai virus
infection resulted in two alterations in the expression of
IRF-3: 1) an overall decrease in the amount of IRF-3 in
transfected and control cells (Fig. 1, lanes 2 and 4) and the
generation of a more slowly migrating form of IRF-3 (Fig. 1,
compare lanes 1 and 2). In all experiments, the turnover of
IRF-3 after virus infection was more pronounced with the
endogenous protein than with the transfected proteins (see
Fig. l, as well as others). Because the transfected proteins
were driven by the CMV promoter, ongoing synthesis of
transfected IRF-3 may partially obscure the turnover of IRF-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). To determine whether the slowest
form of IRF-3 was due to virus-induced phosphorylation
(P-IRF-3), the different forms of IRF-3 were subjected to
treatment in vitro with potato acidic phosphatase {PPA) or calf
intestine alkaline phosphatase (CIP) and/or phosphatase
inhibitors (Fig. 2B). These treatments did not affect the
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mobilities of forms I and II in uninfected cells (Fig. 2B,
lanes 1-3). However, in rtTA-IRF-3 expressing 293 cells
infected with Sendai virus for 12h, an additional slowly
migrating, presumably phosphorylated form of IRF-3 was also
detected (Fig. 2B, lane 6); this form of IRF-3 completely
disappeared following CIP or PPA treatment (Fig.2B, lanes 6 and
7) but was maintained in the presence of CIP/PPA when
phosphatase inhibitors were also added to the reaction (Fig.
2B, lanes 5 and 8).
Mapping the IRF-3 phosphorylation sites.
A series of deletions of IRF-3 were generated to
identify the virus-induced phosphorylation sites) 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). The results indicated
that a virus-induced phosphorylation of IRF-3 occurs at the
C-terminal end of IRF-3 since the mutations that contained only
the N-terminal part of IRF-3 protein (133, 240, 328, 357 or
394aa) were not phosphorylated (Fig. 3B). Full length and
407aa forms of IRF-3 were phosphorylated as a consequence of
virus infection (Fig. 3B, lanes 1-4). C-terminal truncation of
IRF-3 to a protein of 394 or 357aa removed the sites) of
inducible phosphorylation (Fig. 3B, lanes 5-8), although the
shortened versions of forms I and II were still observed. Also
in the IRF-3 D9-133 mutation (AN) which had the DNA binding,
N-terminal amino acids (aa9 to aa133) removed, both virus
induced phosphorylation of IRF-3 and the differential migration
of the shortened forms I and II were easily detected (Fig. 3B,
lanes 9 and 10). Degradation of the endogenous forms of IRF-3
by virus infection was also detected in this experiment
(compare Fig. 3B, lanes 7 and 9 with lanes 8 and 10).
Thus, by deletion analysis, 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
D9-133 (ON} protein (Fig. 4A). In the IRF-3 cDNA encoding
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these proteins, 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). Transfection of these plasmids into 293
5 cells and subsequent virus infection revealed that full length
wild type IRF-3 was phosphorylated (Fig. 4B, lanes 4 and 8),
whereas the IRF-3 proteins containing 2A and 5A mutations were
no longer phosphorylated in virus infected cells (Fig. 4B,
lanes 6 and 10). Interestingly, IRF-3-3A was also very weakly
10 phosphorylated as a consequence of virus infection, thus
implicating Ser-402/Thr-404/Ser-405 as potential secondary
sites of phosphorylation. Using the ON IRF-3 protein and the
relevant point mutations, phosphorylation was detected with ~N
(Fig. 4B, lane 12) but not with ~N-2A and ~N-5A (Fig. 4B,
15 lanes 14 and 18); likewise, 0N-3A displayed very weak
phosphorylation (Fig. 4B, lane 16).
These experiments thus implicate Ser-396 and Ser-398
as critical sites of virus-induced phosphorylation of IRF-3;
however, Ser-402/Thr-404/Ser-405 residues also contribute to
20 the observed phosphorylation, since the migration of
phosphorylated ~N-3A is significantly faster than ON and the
phosphorylation level is decreased (Fig. 4B, lanes 12 and 16).
Another study suggested the involvement of the Ser residues at
aa385 and 386 as potential phosphoacceptor sites (67).
25 However, in studies with the S385A/S386A mutation, no evidence
was found for inducible phosphorylation at these sites.
Nevertheless, since these sites represent consensus sites for
CKI and CKII, constitutive phosphorylation is a possibility.
IRF-3 phosphorylation induces cytoplasmic to nuclear
translocation of IRF-3.
Initial studies indicated that 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). In
uninfected cells, GFP-IRF-3 localized exclusively to the
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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). Mutation of the Ser/Thr cluster in GFP-IRF-3(5A)
completely abrogated virus-induced cytoplasmic to nuclear
translocation (Fig. 5, C and D). Interestingly, the
substitution of the Ser/Thr cluster with the phosphomimetic Asp
in GFP-IRF-3(5D) likewise altered subcellular localization.
IRF-3(5D) localized both to the nucleus and cytoplasm in
uninfected cells (Fig. 5E), while virus infection resulted in
an intense nuclear pattern of IRF-3(5D) fluorescence (Fig. 5F).
Point mutation of a putative nuclear export signal in IRF-3,
the L145A/L146A modification - termed IRF-3(NES) - also changed
subcellular localization of IRF-3. In uninfected cells,
GFP-IRF-3(NES) was localized to the nucleus and cytoplasm, with
a homogeneous, extra-nucleolar pattern of nuclear staining.
After virus infection, 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.
Transactivation of PRDI/PRDIII and ISRE promoters by IRF-3.
Next, the capacity of IRF-3 to regulate gene
expression was analysed by transient transfection in human 293
and murine NIH3T3 cells using the IFN~i and ISG-15 promoters in
reporter gene assays. Expression of NF-rcB RelA(p65), IRF-1 and
IRF-3 alone minimally induced IFN~3 promoter activity between 3
to 4 fold (Fig. 6A and B), as shown previously (24,56,61).
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). Interestingly, deletion
of the C-terminal 20aa of IRF-3 to IRF-3(407) stimulated IFNQ
activity about 6 fold, indicative of the removal of an
inhibitory domain in IRF-3. However, further deletion to 394,
357 or 240 abrogated transactivation potential (Fig. 6A).
Mutation of the NES element was not sufficient to stimulate
IFN~i activity. Strikingly, the substitution of the Ser/Thr
cluster at aa397-405 in IRF-3 with the phosphomimetic Asp
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27
generated a very strong, constitutive transactivator protein
that alone stimulated the IFN~i promoter 90 fold.
As shown previously, high level induction of the IFN~i
promoter requires synergistic activation by NF-xB and IRF
proteins (24,61). To analyse the properties of IRF-3 in
synergistic activation of the IFN/3 promoter, co-expression
studies were performed using RelA(p65) expression plasmid and
different wild type and mutant forms of IRF-3 (Fig. 6B).
Co-expression of RelA and IRF-1 or RelA and IRF-3 stimulated
IFN~i-CAT activity by 20-25 fold. IRF-3 (407) and RelA(p65)
stimulated IFN~i 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~i promoter. RelA and IRF-3{NES)
produced a relatively weak 8 fold induction of IFN~i 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~i promoter activity (Fig.
6B); together with the above data, IRF-3(5D) alone appears to
be capable of full stimulation of the IFN~i promoter and further
synergy with RelA is not observed (compare Fig. 6A and B).
Surprisingly, 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). As shown previously (2), and
above for the IFN~i promoter, 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. 6C),
thus demonstrating that substitution of the Ser/Thr sites with
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28
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.
Activation of RANTES Transcription by IRF-3 and Virus
Chemokine expression is demonstrated in Figure 7, the
chemokine being RANTES (Regulated on Activation Normal T-cell
Expressed and Secreted) protein. 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). Since IRF-3(5D) was
a strong transactivator of the IFN-~i promoter in transient
transfection assays, the possibility of an autoregulatory
effect of IFN-a/,~ expression on transcription of RANTES
promoter via JAK-STAT activation was considered. Activation of
RANTES did not occur secondary to the production of IFN-a/~i,
since RANTES mRNA was not detected in control rtTA-expressing
cells treated directly with IFN-a/(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). Other experiments
also demonstraed that IRF-3 itself was not activated by IFN
treatment (13a). Inducible expression of RANTES in cells
stably expressing a dominant-negative form of IRF-3 which lacks
the N-terminal amino acids 9 to 133 and does not bind to DNA
was also examined. As shown in Fig. 7B, RANTES gene
transcription was indcued by Sendai virus in control (rtTA)
cells (Fig. 7H) but not in IRF-3 (oN)-expressing cells (Fig.
7B). This experiment indicates that a non-DNA binding,
dominant-negative mutant of IRF-3 is able to block completely
virus-induced activation of RANTES transcription.
The kinetics of IRF-3 transgene induction and RANTES
mRNA expression were characterized at various times following
CA 02325354 2000-10-10
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29
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. The
possibility that IRF-3(5D) may be directly activating another
transcription factor such as NF-xB, which in turn would
stimulte RANTES transciption, was also considered. No evidence
for IRF-3(5D)-mediated activation of NF-rcB 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-xB and other transcription
factors (data not shown) .
Inhibition of IRF-3 degradation.
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 ON and ONSA
forms of IRF-3, virus induced degradation of full length
(endogenous) forms of IRF-3 (Fig. 8A, lanes 1 and 4) and the
truncated ~N (Fig. 8B, lanes 1 and 4) was detected. Addition
of the protease inhibitor calpain inhibitor I or the proteasome
inhibitor MG132 blocked virus-induced IRF-3 degradation (Fig.
8A and 8B, lanes 4-6). Particularly with the ~N protein, the
accumulation of the phosphorylated form of ~N was also detected
in virus infected cells (Fig. 8B, lanes 5 and 6), suggesting
that phosphorylation of IRF-3 may represent a signal for
subsequent degradation by the proteasome pathway. To confirm
this idea, the 5A point mutated form of IRF-3 was analysed; the
IRF-3-~NSA protein was resistant to virus induced degradation
(Fig. 8C, lanes 1 and 4); no further stabilization of
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
IRF-3-ANSA occurred with calpain inhibitor I or MG132 addition
and no phosphorylated IRF-3 was detected (Fig. 8C, lanes 4-6).
These experiments demonstrate that virus dependent
phosphorylation of the C-terminal of IRF-3 represents a signal
5 for subsequent proteasome mediated degradation.
Interaction between IRF-3 and CBP in virus infected cells.
To examine the possibility that IRF-3 associated with
the co-activator CBP/p300 (Fig. 9A) following Sendai virus
infection, CBP was immunoprecipitated from virus-infected cells
10 with anti-CBP antibody; an immunoblot for IRF-3 revealed that
IRF-3 was co-precipitated from virus-infected cells but not
from uninfected cells (Fig. 9B, lanes 2 and 3). This
interaction was observed clearly in cells co-transfected with
the IRF-3 expression plasmid (Fig. 9B, lane 3 ) but was not
15 seen when the immunoprecipitation was performed with pre-immune
serum (Fig. 9B, lane 7). The endogenous IRF-3 also
co-precipitated from virus-infected cells (Fig. 9B, lane 1).
However, mutation of the Ser/Thr residues identified as the
virus inducible phosphorylation sites abrogated the association
20 of IRF-3 with CBP. In particular, 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). With the IRF-3(3A) mutant, interaction with CHP was
still observed (Fig. 9B, lane 5). The high background in all
25 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
30 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 CBPl, CBP2, and CBP3 respectively (Fig. 9A,
reviewed in (28)). To determine which domain of CBP interacts
with IRF-3, 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
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99100314
31
plasmids together with the IRF-3 ON (D9-133) expression
plasmid. At 24h after transfection, cells were infected with
Sendai virus, co-immunopreciptated with anti-myc antibody 16h
later (21) and then immunoblotted for IRF-3. Endogenous IRF-3
and transfected IRF-3 ~N proteins co-precipitated with CBP-3
from virus-infected cells but not from uninfected cells (Fig.
9C, lane 6). In cells co-transfected with CBP-1 and CBP-2, no
endogenous or transfected ~N IRF-3 was detected (Fig. 9C, lanes
1-4). Immunoblot analysis of the whole cell extracts revealed
that all three myc-tagged CBP proteins were efficiently
expressed in uninfected and virus infected cells (Fig. 9D).
These results demonstrate that IRF-3 binds to the C-terminal
domain of CBP in virus infected cells and interaction with CBP
requires Ser-396/Ser-398 phosphorylation of IRF-3 but not at
Ser-402/Thr-404/Ser-405.
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.
The pharmaceutical compositions of the present
invention may be formulated in a conventional manner using one
or more pharmaceutically acceptable carriers. Thus, 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
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
32
administration or in a form suitable for administration by
inhalation or insufflation.
For oral administration, the pharmaceutical
compositions may take the form of, for example, tablets or
S 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
(e. g., potato starch or sodium starch glycollate); or wetting
agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. 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).
For buccal administration the composition may take
the form of tablets or lozenges formulated in conventional
manner.
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.
Alternatively, the active ingredient may be in powder form for
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
33
reconstitution with a suitable vehicle, e.g., sterile pyrogen-
free water, before use.
The active compounds of the invention may also be
formulated in rectal compositions such as suppositories or
S retention enemas, e.g., containing conventional suppository
bases such as cocoa butter or other glycerides.
For intranasal administration or administration by
inhalation, 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. In the case of a pressurized
aerosol, 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 (made, for example, from
gelatin) 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.
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49. Nguyen, H., Lin, R. and Hiscott, J. 1997. Activation of
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K., Petricoin, E.F., Larner, A.C., Schaper, F., Hauser, H. and
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P.H., Ozato, K. and Levi, B.-Z. 1992. Human interferon
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41
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CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
1/13
SEQUENCE LISTING
<110> THE SIR MORTIMER B. DAVIS-JEWISH GENERAL HOSPITAL
<120> HIGHLY ACTIVE FORMS OF INTERFERON REGULATORY FACTOR
PROTEINS
<130> IRF-3
<140>
<141>
<150> CA 2,234,588
<151> 1998-04-07
<160> 11
<170> PatentIn Ver. 2.0
<210> 1
<211> 1284
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(1281)
<400> 1
atg gga acc cca aag cca cgg atc ctg ccc tgg ctg gtg tcg cag ctg 48
Met Gly Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu
1 5 10 15
gac ctg ggg caa ctg gag ggc gtg gcc tgg gtg aac aag agc cgc acg 96
Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser Arg Thr
20 25 30
cgc ttc cgc atc cct tgg aag cac ggc cta cgg cag gat gca cag cag 144
Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Gln
35 40 45
gag gat ttc gga atc ttc cag gcc tgg gcc gag gcc act ggt gca tat 192
Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr
50 55 60
gtt ccc ggg agg gat aag cca gac ctg cca acc tgg aag agg aat ttc 240
Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe
65 70 75 80
05-06-2000 CA 02325354 2000-io-io.i i~ .; ;, CA 009900314
i "~'' . : . . r .
i : ? . i v.~i . . v . i . f i
i _... . i . . ~. . i . G
. i . . ~ ' . . . . . . i :
2/13 ~.. ._.. .. .. . .:- .:
cgc tct gcc ctc aac cgc aaa gaa ggg ttg cgt tta gca gag gac cgg 288
Arg Ser Ala Leu Asn Arg Lys Glu Gly Leu Arg Leu Ala Glu Asp Arg
85 90 95
agc aag gac cct cac gac cca cat aaa atc tac gag ttt gtg aac tca 336
Ser Lys Asp Pro His Asp Pro His Lys Ile Tyr Glu Phe Val Asn Ser
100 ~ 105 110
gga gtt ggg gac ttt tcc cag cca gac acc tct ccg gac acc aat ggt 384
1 0 Gly Val Gly Asp Phe Ser Gln Pro Asp Thr Ser Pro Asp Thr Asn Gly
115 120 125
gga ggc agt act tct gat acc cag gaa gac att ctg gat gag tta ctg 932
Gly Gly Ser Thr Ser Asp Thr Gln Glu Asp Ile Leu Asp Glu Leu Leu '
130 135 140
ggt aac atg gtg ttg gcc cca ctc cca gat ccg gga ccc cca agc ctg 480
Gly Asn Met Val Leu Ala Pro Leu Pro Asp Pro Gly Pro Pro 5er Leu
14s 150 ls5 160
get gta gcc cct gag ccc tgc cct cag ccc ctg cgg agc ccc agc ttg 528
Ala Val Ala Pro Glu Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu
165 170 175
gac aat ccc act ccc ttc cca aac ctg ggg ccc tct gag aac cca ctg 576
Asp Asn Pro Thr Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro Leu
180 185 190
aag cgg ctg ttg gtg ccg ggg gaa gag tgg gag ttc gag gtg aca gcc 624
3 0 Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala
195 200 205
ttc tac cgg ggc cgc caa gtc ttc cag cag acc atc tcc tgc ccg gag 672
Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser Cys Pro Glu
210 215 22D
ggc ctg cgg ctg gtg ggg tcc gaa gtg gga gac agg acg ctg cct gga 720
Gly Leu Arg Leu Val Gly 5er Glu Val Gly Asp Arg Thr Leu Pro Gly
225 23D 235 290
tgg cca gtc aca ctg cca gac cct ggc atg tcc ctg aca gac agg gga 768
Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr Asp Arg Gly
245 250 255
gtg atg agc tac gtg agg cat gtg ctg agc tgc ctg ggt ggg gga ctg 816
Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu Gly Gly Gly Leu
260 265 270
get ctc tgg cgg gcc ggg cag tgg ctc tgg gcc cag cgg ctg ggg cac 864
5 0 Ala Leu Trp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg Leu Gly His
275 280 285
tgc cac aca tac tgg gca gtg agc gag gag ctg ctc ccc aac agc ggg 912
Cys His Thz Tyr Trp Ala Val Ser Glu Glu Leu Leu Pro Asn Ser Gly
290 295 300
cat ggg cct gat ggc gag gtc ccc aag gac aag gaa gga ggc gtg ttt 960
His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys G1u Gly Gly Val Phe
305 31D 315 320
gac ctg ggg ccc ttc att gta gat ctg att acc ttc acg gaa gga agc 1008
Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr Phe Thr Glu Gly Ser
325 330 335
gga cgc tca cca cgc tat gcc ctc tgg ttc tgt gtg ggg gag tca tgg 1056
Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys Val Gly Glu Ser Trp
340 395 350
-..
AMENDED SHEET
05-06-2000 CA 02325354 2000-10-10
.: ~. i 1 . i i i i s , , i-1 CA 009900314
i.~ .. a . 1 . : : . r a
y : -i i ~~ii ~ i~~v 1 1~ i
i 1 ~ ~~~ . i . i~ v 1 i i i
1 i ~ 1 ~ 1 . . i 1 1 ~ i
~ 313 ~. ... .. .. . ..
ccc cag gac cag ccg tgg acc aag agg ctc gtg atg gtc aag gtt gtg 1104
Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val Met Val Lys Val Val
355 360 365
ccc acg tgc ctc agg gcc ttg gta gaa atg gcc cgg gta ggg ggt gcc 1152
Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val Gly Gly Ala
37D 375 380
tcc tcc ctg gag aat act gtg gac ctg cac att gac aac gac cac cca 1200
1 0 Ser Ser Leu Glu Asn Thr Val Asp Leu His Ile Asp Asn Asp His Pro
385 390 395 400
ctc gac ctc gac gac gac cag tac aag gcc tac ctg cag gac ttg gtg 1298
Leu Asp Leu Asp Asp Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val
405 910 415
gag ggc atg gat ttc cag ggc cct ggg gag agc tga 1284
Glu Gly Met Asp Phe Gln Gly Pro Gly Glu Ser
420 925
<210> 2
<211> 427
<212> PRT
<213> Homo sapiens
<400> 2
Met Gly Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu
1 5 10 15
Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser Arg Thr
zo z5 30
Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Gln
40 95
Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr
50 55 60
Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe
65 70 75 BO
Arg SerAlaLeuAsnArgLysGluGlyLeuArgLeuAlaGluAspArg
85 90 95
Ser LysAspProHisAspProHisLysIleTyrGluPheValAsnSer
100 105 110
Gly ValGlyAspPheSerGlnProAspThrSerProAspThrAsnGly
115 120 125
5 Gly GlySerThrSerAspThrGlnGluAspIleLeuAspGluLeuLeu
0
130 135 140
Gly AsnMetValLeuAlaProLeuProAspProGlyProProSerLeu
195 150 155 160
Ala ValAlaProGluProCysProGlnProLeuArgSerProSerLeu
165 170 175
Asp AsnProThrProPheProAsnLeuGlyProSerGluAsnProLeu
60 180 1B5 190
AMENDED SHEET
CA 02325354 2000-10-10
0~-06-2000 ;. ; ~,. ",.; " :, , r CA 009900314
, is .. . . ,-. . ~ ..: : . r ~
~ f . i 1 . ..i . i a:v i . I i
t . i i 1 . . . . - .. . . . i
. i . . . . . i . . ~ i i
x/13 ~~ ... .. .. . ..
Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala
195 200 205
Phe ArgGlyArgGlnValPheGlnGlnThrIleSerCysProGlu
Tyr
210 21s 220
Gly ArgLeuValGlySerGluValGlyAspArgThrLeuProGly
Leu
225 230 235 240
1 Trp ValThrLeuProAspProGlyMetSerLeuThrAspArgGly
0 Pro
245 250 255
Val SerTyrValArgHisValLeuSerCysLeuGlyGlyGlyLeu
Met
260 265 270
Ala TrpArgAlaGlyGlnTrpLeuTrpAlaGlnArgLeuGlyHis
Leu
275 280 285
Cys ThrTyrTrpAlaValSerGluGluLeuLeuProAsnSerGly
His
20 290 295 300
His ProAspGlyGluValProLysAspLysGluGlyGlyValPhe
Gly
305 310 315 320
Asp GlyProPheIleValAspLeuIleThrPheThrGluGlySer
Leu
325 330 335
Gly SerProArgTyrAlaLeuTrpPheCysValGlyGlu5erTrp
Arg
390 345 350
30
Pro AspGlnProTrpThrLysArgLeuValMetValLysValVal
Gln
355 360 365
Pro CysLeuArgAlaLeuValGluMetAlaArgValGlyGlyAla
Thr
370 375 3B0
Ser LeuGluAsnThrValAspLeuHisIleAspAsnAspHisPro
5er
385 390 395 400
4 Leu LeuAspAspAspGlnTyrLysAlaTyrLeuGlnAspLeuVal
0 Asp
905 910 415
Glu MetAspPheGlnGlyProGlyGluSer
Gly
420 425
<210>
3
<211>
13
<212>
PRT
<213>
Homo
sapiens
50 <900>
3
Ile AsnSerHisProLeuSerLeuThr5erAspGln
5er
1 5 10
<210>
4
<211>
9
<212>
PRT
<213> 444
Homo
Sapiens
<400>
4
-.~
AMENDED SHEET
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
5/13
Gly Ala Ala Ala
1
<210> 5
<211> 6
<212> PRT
<213> Homo sapiens
<400> 5
Gly Ala Ala Ala Asn Asn
1 5
<210>6
<211>15
<2I2>DNA
<213>Homo sapiens
<400> 6
ggaaaactga aaggg 15
<210> 7
<211> 30
<212> DNA
<213> Homo sapiens
<400> 7
gatcgggaaa gggaaaccga aactgaagcc 30
<210> 8
<211> 1512
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(1509)
<400> 8
atg gcc ttg get cct gag agg gca gcc cca cgc gtg ctg ttc gga gag 48
Met Ala Leu Ala Pro Glu Arg Ala Ala Pro Arg Val Leu Phe Gly Glu
1 5 10 15
tgg ctc ctt gga gag atc agc agc ggc tgc tat gag ggg ctg cag tgg 96
Trp Leu Leu Gly Glu Ile Ser Ser Gly Cys Tyr Glu Gly Leu Gln Trp
20 25 30
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
6/13
ctg gac gag gcc cgc acc tgt ttc cgc gtg ccc tgg aag cac ttc gcg 144
Leu Asp Glu Ala Arg Thr Cys Phe Arg Val Pro Trp Lys His Phe Ala
35 40 45
cgc aag gac ctg agc gag gcc gac gcg cgc atc ttc aag gcc tgg get 192
Arg Lys Asp Leu Ser Glu Ala Asp Ala Arg Ile Phe Lys Ala Trp Ala
50 55 60
gtg gcc cgc ggc agg tgg ccg cct agc agc agg gga ggt ggc ccg ccc 240
Val Ala Arg Gly Arg Trp Pro Pro Ser Ser Arg Gly Gly Gly Pro Pro
65 70 75 80
ccc gag get gag act gcg gag cgc gcc ggc tgg aaa acc aac ttc cgc 288
Pro Glu Ala Glu Thr Ala Glu Arg Ala Gly Trp Lys Thr Asn Phe Arg
85 90 95
tgc gca ctg cgc agc acg cgt cgc ttc gtg atg ctg cgg gat aac tcg 336
Cys Ala Leu Arg Ser Thr Arg Arg Phe Val Met Leu Arg Asp Asn Ser
100 105 110
ggg gac ccg gcc gac ccg cac aag gtg tac gcg ctc agc cgg gag ctg 384
Gly Asp Pro Ala Asp Pro His Lys Val Tyr Ala Leu Ser Arg Glu Leu
115 120 125
tgc tgg cga gaa ggc cca ggc acg gac cag act gag gca gag gcc ccc 432
Cys Trp Arg Glu Gly Pro Gly Thr Asp Gln Thr Glu Ala Glu Ala Pro
130 135 140
gca get gtc cca cca cca cag ggt ggg ccc cca ggg cca ttc ttg gca 480
Ala Ala Val Pro Pro Pro Gln Gly Gly Pro Pro Gly Pro Phe Leu Ala
145 150 155 160
cac aca cat get gga ctc caa gcc cca ggc ccc ctc cct gcc cca get 528
His Thr His Ala Gly Leu Gln Ala Pro Gly Pro Leu Pro Ala Pro Ala
165 170 175
ggt gac aag ggg gac ctc ctg ctc cag gca gtg caa cag agc tgc ctg 576
Gly Asp Lys Gly Asp Leu Leu Leu Gln Ala Val Gln Gln Ser Cys Leu
180 185 190
gca gac cat ctg ctg aca gcg tca tgg ggg gca gat cca gtc cca acc 624
Ala Asp His Leu Leu Thr Ala Ser Trp Gly Ala Asp Pro Val Pro Thr
195 200 205
aag get cct gga gag gga caa gaa ggg ctt ccc ctg act ggg gcc tgt 672
Lys Ala Pro Gly Glu Gly Gln Glu Gly Leu Pro Leu Thr Gly Ala Cys
210 215 220
get gga ggc cca ggg ctc cct get ggg gag ctg tac ggg tgg gca gta 720
Ala Gly Gly Pro Gly Leu Pro Ala Gly Glu Leu Tyr Gly Trp Ala Val
225 230 235 240
gag acg acc ccc agc ccc ggg ccc cag ccc gcg gca cta acg aca ggc 768
Glu Thr Thr Pro Ser Pro Gly Pro Gln Pro Ala Ala Leu Thr Thr Gly
245 250 255
gag gcc gcg gcc cca gag tcc ccg cac cag gca gag ccg tac ctg tca 816
Glu Ala Ala Ala Pro Glu Ser Pro His Gln Ala Glu Pro Tyr Leu Ser
260 265 270
ccc tcc cca agc gcc tgc acc gcg gtg caa gag ccc agc cca ggg gcg 864
Pro Ser Pro Ser Ala Cys Thr Ala Val Gln Glu Pro Ser Pro Gly Ala
275 280 285
ctg gac gtg acc atc atg tac aag ggc cgc acg gtg ctg cag aag gtg 912
Leu Asp Val Thr Ile Met Tyr Lys Gly Arg Thr Val Leu Gln Lys Val
290 295 300
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
7/13
gtgggacac ccgagctgcacg ttcctatac ggcccccca gacccaget 960
ValGlyHis ProSerCysThr PheLeuTyr GlyProPro AspProAla
305 310 315 320
gtccgggcc acagacccccag caggtagca ttccccagc cctgccgag 1008
ValArgAla ThrAspProGln GlnValAla PheProSer ProAlaGlu
325 330 335
ctcccggac cagaagcagctg cgctacacg gaggaactg ctgcggcac 1056
LeuProAsp GlnLysGlnLeu ArgTyrThr GluGluLeu LeuArgHis
340 345 350
gtggcccct gggttgcacctg gagcttcgg gggccacag ctgtgggcc 1104
ValAlaPro GlyLeuHisLeu GluLeuArg GlyProGln LeuTrpAla
355 360 365
cggcgcatg ggcaagtgcaag gtgtactgg gaggtgggc ggaccccca 1152
ArgArgMet GlyLysCysLys ValTyrTrp GluValGly GlyProPro
370 375 380
ggctccgcc agcccctccacc ccagcctgc ctgctgcct cggaactgt 1200
GlySerAla SerProSerThr ProAlaCys LeuLeuPro ArgAsnCys
385 390 395 400
gacaccccc atcttcgacttc agagtcttc ttccaagag ctggtggaa 1248
AspThrPro IlePheAspPhe ArgValPhe PheGlnGlu LeuValGlu
405 410 415
ttccgggca cggcagcgccgt ggctcccca cgctatacc atctacctg 1296
PheArgAla ArgGlnArgArg GlySerPro ArgTyrThr IleTyrLeu
420 425 430
ggcttcggg caggacctgtca getgggagg cccaaggag aagagcctg 1344
GlyPheGly GlnAspLeuSer AlaGlyArg ProLysGlu LysSerLeu
435 440 445
gtcctggtg aagctggaaccc tggctgtgc cgagtgcac ctagagggc 1392
ValLeuVal LysLeuGluPro TrpLeuCys ArgValHis LeuGluGly
450 455 460
acgcagcgt gagggtgtgtct tccctggat agcagcgac ctcgacctc 1440
ThrGlnArg GluGlyValSer SerLeuAsp SerSerAsp LeuAspLeu
465 470 475 480
tgcctgtcc agcgccaacagc ctctatgac gacatcgag tgcttcctt 1488
CysLeuSer SerAlaAsnSer LeuTyrAsp AspIleGlu CysPheLeu
485 490 495
atggagctg gagcagcccgcc tag 1512
MetGluLeu GluGlnProAla
500
<210> 9
<211> 503
<212 > PRT
<213> Homo sapiens
<400> 9
Met Ala Leu Ala Pro Glu Arg Ala Ala Pro Arg Val Leu Phe Gly Glu
1 5 10 15
Trp Leu Leu Gly Glu Ile Ser Ser Gly Cys Tyr Glu Gly Leu Gln Trp
20 25 30
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
8/13
Leu Asp Glu Ala Arg Thr Cys Phe Arg Val Pro Trp Lys His Phe Ala
35 40 45
Arg Lys Asp Leu Ser Glu Ala Asp Ala Arg Ile Phe Lys Ala Trp Ala
50 55 60
Val Ala Arg Gly Arg Trp Pro Pro Ser Ser Arg Gly Gly Gly Pro Pro
65 70 75 80
Pro Glu Ala Glu Thr Ala Glu Arg Ala Gly Trp Lys Thr Asn Phe Arg
85 90 95
Cys Ala Leu Arg Ser Thr Arg Arg Phe Val Met Leu Arg Asp Asn Ser
100 105 110
Gly Asp Pro Ala Asp Pro His Lys Val Tyr Ala Leu Ser Arg Glu Leu
115 120 125
Cys Trp Arg Glu Gly Pro Gly Thr Asp Gln Thr Glu Ala Glu Ala Pro
130 135 140
Ala Ala Val Pro Pro Pro Gln Gly Gly Pro Pro Gly Pro Phe Leu Ala
145 150 155 160
His Thr His Ala Gly Leu Gln Ala Pro Gly Pro Leu Pro Ala Pro Ala
165 170 175
Gly Asp Lys Gly Asp Leu Leu Leu Gln Ala Val Gln Gln Ser Cys Leu
180 185 190
Ala Asp His Leu Leu Thr Ala Ser Trp Gly Ala Asp Pro Val Pro Thr
195 200 205
Lys Ala Pro Gly Glu Gly Gln Glu Gly Leu Pro Leu Thr Gly Ala Cys
210 215 220
Ala Gly Gly Pro Gly Leu Pro Ala Gly Glu Leu Tyr Gly Trp Ala Val
225 230 235 240
Glu Thr Thr Pro Ser Pro Gly Pro Gln Pro Ala Ala Leu Thr Thr Gly
245 250 255
Glu Ala Ala Ala Pro Glu Ser Pro His Gln Ala Glu Pro Tyr Leu Ser
260 265 270
Pro Ser Pro Ser Ala Cys Thr Ala Val Gln Glu Pro Ser Pro Gly Ala
275 280 285
Leu Asp Val Thr Ile Met Tyr Lys Gly Arg Thr Val Leu Gln Lys Val
290 295 300
Val Gly His Pro Ser Cys Thr Phe Leu Tyr Gly Pro Pro Asp Pro Ala
305 310 315 320
Val Arg Ala Thr Asp Pro Gln Gln Val Ala Phe Pro Ser Pro Ala Glu
325 330 335
Leu Pro Asp Gln Lys Gln Leu Arg Tyr Thr Glu Glu Leu Leu Arg His
340 345 350
Val Ala Pro Gly Leu His Leu Glu Leu Arg Gly Pro Gln Leu Trp Ala
355 360 365
Arg Arg Met Gly Lys Cys Lys Val Tyr Trp Glu Val Giy Gly Pro Pro
370 375 380
Gly Ser Ala Ser Pro Ser Thr Pro Ala Cys Leu Leu Pro Arg Asn Cys
385 390 395 400
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
9/13
Asp Thr Pro Ile Phe Asp Phe Arg Val Phe Phe Gln Glu Leu Val Glu
405 410 415
Phe Arg Ala Arg Gln Arg Arg Gly Ser Pro Arg Tyr Thr Ile Tyr Leu
420 425 430
Gly Phe Gly Gln Asp Leu Ser Ala Gly Arg Pro Lys Glu Lys Ser Leu
435 440 445
Val Leu Val Lys Leu Glu Pro Trp Leu Cys Arg Val His Leu Glu Gly
450 455 460
Thr Gln Arg Glu Gly Val Ser Ser Leu Asp Ser Ser Asp Leu Asp Leu
465 470 475 480
Cys Leu Ser Ser Ala Asn Ser Leu Tyr Asp Asp Ile Glu Cys Phe Leu
485 490 495
Met Glu Leu Glu Gln Pro Ala
500
<210> 10
<211> 1629
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(1626)
<400> 10
atggccttggetcct gagagggca gccccacgc gtgctgttc ggagag 48
MetAlaLeuAlaPro GluArgAla AlaProArg ValLeuPhe GlyGlu
1 5 10 15
tggctccttggagag atcagcagc ggctgctat gaggggctg cagtgg 96
TrpLeuLeuGlyGlu IleSerSer GlyCysTyr GluGlyLeu GlnTrp
20 25 30
ctggacgaggcccgc acctgtttc cgcgtgccc tggaagcac ttcgcg 144
LeuAspGluAlaArg ThrCysPhe ArgValPro TrpLysHis PheAla
35 40 45
cgcaaggacctgagc gaggccgac gcgcgcatc ttcaaggcc tggget 192
ArgLysAspLeuSer GluAlaAsp AlaArgIle PheLysAla TrpAla
50 55 60
gtggcccgcggcagg tggccgcct agcagcagg ggaggtggc ccgccc 240
ValAlaArgGlyArg TrpProPro SerSerArg GlyGlyGly ProPro
65 70 75 80
cccgaggetgagact gcggagcgc gccggctgg aaaaccaac ttccgc 288
ProGluAlaGluThr AlaGluArg AlaGlyTrp LysThrAsn PheArg
85 90 95
tgcgcactgcgcagc acgcgtcgc ttcgtgatg ctgcgggat aactcg 336
CysAlaLeuArgSer ThrArgArg PheValMet LeuArgAsp AsnSer
100 105 110
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
10/13
ggg gacccggcc gacccgcacaag gtgtacgcg ctcagccgg gagctg 384
Gly AspProAla AspProHisLys ValTyrAla LeuSerArg GluLeu
115 120 125
tgc tggcgagaa ggcccaggcacg gaccagact gaggcagag gccccc 432
Cys TrpArgGlu GlyProGlyThr AspGlnThr GluAlaGlu AlaPro
130 135 140
gca getgtccca ccaccacagggt gggccccca gggccattc ttggca 480
Ala AlaValPro ProProGlnGly GlyProPro GlyProPhe LeuAla
145 150 155 160
cac acacatget ggactccaagcc ccaggcccc ctccctgcc ccaget 528
His ThrHisAla GlyLeuGlnAla ProGlyPro LeuProAla ProAla
165 170 175
ggt gacaagggg gacctcctgctc caggcagtg caacagagc tgcctg 576
Gly AspLysGly AspLeuLeuLeu GlnAlaVal GlnGlnSer CysLeu
180 185 190
gca gaccatctg ctgacagcgtca tggggggca gatccagtc ccaacc 624
Ala AspHisLeu LeuThrAlaSer TrpGlyAla AspProVal ProThr
195 200 205
aag getcctgga gagggacaagaa gggcttccc ctgactggg gcctgt 672
Lys AlaProGly GluGlyGlnGlu GlyLeuPro LeuThrGly AlaCys
210 215 220
get ggaggccca gggctccctget ggggagctg tacgggtgg gcagta 720
Ala GlyGlyPro GlyLeuProAla GlyGluLeu TyrGlyTrp AlaVal
225 230 235 240
gag acgaccccc agccccacttct gatacccag gaagacatt ctggat 768
Glu ThrThrPro SerProThrSer AspThrGln GluAspIle LeuAsp
245 250 255
gag ttactgggt aacatggtgttg gccccactc ccagatccg ggaccc 816
Glu LeuLeuGly AsnMetValLeu AlaProLeu ProAspPro GlyPro
260 265 270
cca agcctgget gtagcccctgag ccctgccct cagcccctg cggagc 864
Pro SerLeuAla ValAlaProGlu ProCysPro GlnProLeu ArgSer
275 280 285
ccc agcttggac aatcccactccc ttcccaaac ctggggccc tctgag 912
Pro SerLeuAsp AsnProThrPro PheProAsn LeuGlyPro SerGlu
290 295 300
aac ccactgaag cggctgttggtg ccgggggaa gagtgggag ttcgag 960
Asn ProLeuLys ArgLeuLeuVal ProGlyGlu GluTrpGlu PheGlu
305 310 315 320
gtg acagccttc taccggggccgc caagtcttc cagcagacc atctcc 1008
Val ThrAlaPhe TyrArgGlyArg GlnValPhe GlnGlnThr IleSer
325 330 335
tgc ccggagggc ctgcggctggtg gggtccgaa gtgggagac aggacg 1056
Cys ProGluGly LeuArgLeuVal GlySerGlu ValGlyAsp ArgThr
340 345 350
ctg cctggatgg ccagtcacactg ccagaccct ggcatgtcc ctgaca 1104
Leu ProGlyTrp ProValThrLeu ProAspPro GlyMetSer LeuThr
355 360 365
gac aggggagtg atgagctacgtg aggcatgtg ctgagctgc ctgggt 1152
Asp ArgGlyVal MetSerTyrVal ArgHisVal LeuSerCys LeuGly
370 375 380
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/003i4
11/13
gggggactgget ctctggcgg gccgggcag tggctctgg gcccagcgg 1200
GlyGlyLeuAla LeuTrpArg AlaGlyGln TrpLeuTrp AlaGlnArg
385 390 395 400
ctggggcactgc cacacatac tgggcagtg agcgaggag ctgctcccc 1248
LeuGlyHisCys HisThrTyr TrpAlaVal SerGluGlu LeuLeuPro
405 410 415
aacagcgggcat gggcctgat ggcgaggtc cccaaggac aaggaagga 1296
AsnSerGlyHis GlyProAsp GlyGluVal ProLysAsp LysGluGly
420 425 430
ggcgtgtttgac ctggggccc ttcattgta gatctgatt accttcacg 1344
GlyValPheAsp LeuGlyPro PheIleVal AspLeuIle ThrPheThr
435 440 445
gaaggaagcgga cgctcacca cgctatgcc ctctggttc tgtgtgggg 1392
GluGlySerGly ArgSerPro ArgTyrAla LeuTrpPhe CysValGly
450 455 460
gagtcatggccc caggaccag ccgtggacc aagaggctc gtgatggtc 1440
GluSerTrpPro GlnAspGln ProTrpThr LysArgLeu ValMetVal
465 470 475 480
aaggttgtgccc acgtgcctc agggccttg gtagaaatg gcccgggta 1488
LysValValPro ThrCysLeu ArgAlaLeu ValGluMet AlaArgVal
485 490 495
gggggtgcctcc tccctggag aatactgtg gacctgcac attgacaac 1536
GlyGlyAlaSer SerLeuGlu AsnThrVal AspLeuHis IleAspAsn
500 505 510
gaccacccactc gacctcgac gacgaccag tacaaggcc tacctgcag 1584
AspHisProLeu AspLeuAsp AspAspGln TyrLysAla TyrLeuGln
515 520 525
gacttggtggag ggcatggat ttccagggc cctggggag agctga 1629
AspLeuValGlu GlyMetAsp PheGlnGly ProGlyGlu Ser
530 535 540
<210> 11
<211> 542
<212> PRT
<213> Homo sapiens
<400> 11
Met Ala Leu Ala Pro Glu Arg Ala Ala Pro Arg Val Leu Phe Gly Glu
1 5 10 15
Trp Leu Leu Gly Glu Ile Ser Ser Gly Cys Tyr Glu Gly Leu Gln Trp
20 25 30
Leu Asp Glu Ala Arg Thr Cys Phe Arg Val Pro Trp Lys His Phe Ala
35 40 45
Arg Lys Asp Leu Ser Glu Ala Asp Ala Arg Ile Phe Lys Ala Trp Ala
50 55 60
Val Ala Arg Gly Arg Trp Pro Pro Ser Ser Arg Gly Gly Gly Pro Pro
65 70 75 80
Pro Glu Ala Glu Thr Ala Glu Arg Ala Gly Trp Lys Thr Asn Phe Arg
85 90 95
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
12/13
Cys Ala Leu Arg Ser Thr Arg Arg Phe Val Met Leu Arg Asp Asn Ser
100 105 110
Gly Asp Pro Ala Asp Pro His Lys Val Tyr Ala Leu Ser Arg Glu Leu
115 120 125
Cys Trp Arg Glu Gly Pro Gly Thr Asp Gln Thr Glu Ala Glu Ala Pro
130 135 140
Ala Ala Val Pro Pro Pro Gln Gly Gly Pro Pro Gly Pro Phe Leu Ala
145 150 155 160
His Thr His Ala Gly Leu Gln Ala Pro Gly Pro Leu Pro Ala Pro Ala
165 170 175
Gly Asp Lys Gly Asp Leu Leu Leu Gln Ala Val Gln Gln Ser Cys Leu
180 185 190
Ala Asp His Leu Leu Thr Ala Ser Trp Gly Ala Asp Pro Val Pro Thr
195 200 205
Lys Ala Pro Gly Glu Gly Gln Glu Gly Leu Pro Leu Thr Gly Ala Cys
210 215 220
Ala Gly Gly Pro Gly Leu Pro Ala Gly Glu Leu Tyr Gly Trp Ala Val
225 230 235 240
Glu Thr Thr Pro Ser Pro Thr Ser Asp Thr Gln Glu Asp Ile Leu Asp
245 250 255
Glu Leu Leu Gly Asn Met Val Leu Ala Pro Leu Pro Asp Pro Gly Pro
260 265 270
Pro Ser Leu Ala Val Ala Pro Glu Pro Cys Pro Gln Pro Leu Arg Ser
275 280 285
Pro Ser Leu Asp Asn Pro Thr Pro Phe Pro Asn Leu Gly Pro Ser Glu
290 295 300
Asn Pro Leu Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu
305 310 315 320
Val Thr Ala Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser
325 330 335
Cys Pro Glu Gly Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg Thr
340 345 350
Leu Pro Gly Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr
355 360 365
Asp Arg Gly Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu'Gly
370 375 380
Gly Gly Leu Ala Leu Txp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg
385 390 395 400
Leu Gly His Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu Leu Pro
405 410 415
Asn Ser Gly His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys Glu Gly
420 425 430
Gly Val Phe Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr Phe Thr
435 440 445
Glu Gly Ser Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys Val Gly
450 455 460
CA 02325354 2000-10-10
WO 99/51737 PCT/CA99/00314
13/13
Glu Ser Trp Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val Met Val
465 470 475 480
Lys Val Val Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val
485 490 495
Gly Gly Ala Ser Ser Leu Glu Asn Thr Val Asp Leu His Ile Asp Asn
500 505 510
Asp His Pro Leu Asp Leu Asp Asp Asp Gln Tyr Lys Ala Tyr Leu Gln
515 520 525
Asp Leu Val Glu Gly Met Asp Phe Gln Gly Pro Gly Glu Ser
530 535 540
