U.S. patent application number 10/531543 was filed with the patent office on 2008-04-24 for composition for treating virus infection disease comprising jab1.
This patent application is currently assigned to SUNGKYUNKWAN UNIVERSITY. Invention is credited to Han-Woong Lee, Sung Ryul Lee, Wonkyung Oh, Suhk Neung Pyo, Jaewhan Hwan Song, Young Hoon Sung, Joo-Sung Yang.
Application Number | 20080095742 10/531543 |
Document ID | / |
Family ID | 36000244 |
Filed Date | 2008-04-24 |
United States Patent
Application |
20080095742 |
Kind Code |
A1 |
Song; Jaewhan Hwan ; et
al. |
April 24, 2008 |
Composition for Treating Virus Infection Disease Comprising
Jab1
Abstract
Disclosed is a composition for treating or preventing a viral
infection or associated disease comprising a Jab1 protein, a
nucleic acid having a nucleotide sequence coding for a Jab1 protein
or a recombinant virus expressing a jab1 protein.
Inventors: |
Song; Jaewhan Hwan;
(Seongnam-si, KR) ; Oh; Wonkyung; (Gwanglu,
KR) ; Sung; Young Hoon; (Suwon-si, KR) ; Lee;
Sung Ryul; (Yongin-si, KR) ; Lee; Han-Woong;
(Seongnam-si, KR) ; Pyo; Suhk Neung; (Seoul,
KR) ; Yang; Joo-Sung; (Seoul, KR) |
Correspondence
Address: |
OHLANDT, GREELEY, RUGGIERO & PERLE, LLP
ONE LANDMARK SQUARE, 10TH FLOOR
STAMFORD
CT
06901
US
|
Assignee: |
SUNGKYUNKWAN UNIVERSITY
SEOUL
KR
|
Family ID: |
36000244 |
Appl. No.: |
10/531543 |
Filed: |
August 31, 2004 |
PCT Filed: |
August 31, 2004 |
PCT NO: |
PCT/KR04/02190 |
371 Date: |
October 28, 2005 |
Current U.S.
Class: |
424/93.2 ;
435/29; 435/6.16; 435/7.1; 514/20.2; 514/3.7; 514/4.2; 514/44R |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 38/1709 20130101; G01N 33/5023 20130101; A61P 31/12 20180101;
G01N 33/5041 20130101; Y02A 50/30 20180101; A61P 31/14 20180101;
G01N 33/5011 20130101; G01N 33/5008 20130101; A61P 19/02 20180101;
A61P 21/00 20180101; Y02A 50/393 20180101; G01N 2333/18 20130101;
G01N 2500/10 20130101; C07K 14/47 20130101 |
Class at
Publication: |
424/93.2 ;
435/29; 435/6; 435/7.1; 514/12; 514/44 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 31/7088 20060101 A61K031/7088; A61K 48/00 20060101
A61K048/00; A61P 31/12 20060101 A61P031/12; C12Q 1/02 20060101
C12Q001/02; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53 |
Claims
1. A composition for treating or preventing a flavivirus or
pestivirus infection, comprising a Jab1 (Jun-activation binding
protein 1) protein.
2. The composition as set forth in claim 1, wherein the Jab1
protein has an amino acid sequence designated as SEQ ID No. 2.
3. The composition as set forth in claim 1, wherein the Jab1
protein is encoded by a nucleotide sequence designated as SEQ ID
No. 1.
4. A composition for treating or preventing a flavivirus or
pestivirus infection, comprising a nucleic acid having a nucleotide
sequence coding for a Jab1 protein.
5. The composition as set forth in claim 4, wherein the nucleic
acid having the nucleotide sequence coding for the Jab1 protein is
a recombinant vector having a nucleotide sequence coding for an
amino acid sequence designated as SEQ ID No. 2.
6. The composition as set forth in claim 4, wherein the nucleic
acid having the nucleotide sequence coding for the Jab1 protein is
a recombinant vector having a nucleotide sequence designated as SEQ
ID No. 1.
7. The composition as set forth in claim 5 or 6, wherein the
recombinant vector is a recombinant viral vector.
8. The composition as set forth in claim 7, wherein the recombinant
viral vector is selected from among recombinant retrovirus,
adenovirus, adeno-associated virus and herpes simplex virus.
9. A composition for treating or preventing a flavivirus or
pestivirus infection, comprising a recombinant virus expressing a
Jab1 protein.
10. The composition as set forth in claim 9, wherein the
recombinant vector expressing the Jab1 protein is a recombinant
virus expressing a Jab1 protein having an amino acid sequence
designated as SEQ ID No. 2.
11. The composition as set forth in claim 9, wherein the
recombinant vector expressing the Jab1 protein is a recombinant
virus expressing a Jab1 protein encoded by a nucleotide sequence
designated as SEQ ID No. 1.
12. The composition as set forth in claim 9, wherein the
recombinant vector is selected from among adenovirus,
adeno-associated virus and herpes simplex virus.
13. The composition as set forth in claim 12, wherein the
recombinant vector is selected from among retrovirus and
adenovirus.
14. The composition as set forth in any one of claims 1, 4 and 9,
wherein the infection is a flavivirus infection.
15. The composition as set forth in claim 14, wherein the
flavivirus is West Nile virus.
16. The composition as set forth in any one of claims 1, 4 and 9,
wherein the infection is associated with fever, rash, bleeding,
jaundice, arthralgia, myalgia, encephalitis or meningitis.
17. A method of screening a compound stimulating expression of a
Jab1 protein, comprising: (a) culturing a cell expressing the Jab1
protein; (b) contacting the cell cultured at (a) with candidate
compounds for stimulating expression of the Jab1 protein; (c)
comparing an expression level of the Jab1 protein at (b) with that
in a control not contacted with the candidate compounds; and (d)
identifying a compound increasing expression levels of the Jab1
protein.
18. A method of screening a compound stimulating interaction
between a Jab1 protein and a capsid (Cp) protein, comprising: (a)
culturing a cell transformed with both a recombinant vector
expressing the Jab1 protein and another recombinant vector
expressing the Cp protein of flavivirus or pestivirus; (b)
contacting the cell cultured at (a) with candidate compounds for
stimulating interaction between the Jab1 protein and the Cp
protein; (c) comparing an expression level of the Cp protein at (b)
with that in a control not contacted with the candidate compounds;
and (d) identifying a compound reducing expression levels of the Cp
protein.
19. The method as set forth in claim 17 or 18, wherein the
comparison of expression levels at (c) is carried out in protein or
mRNA levels.
20. The method as set forth in claim 19, wherein the comparison of
expression levels is carried out by an immunoassay method.
21. The method as set forth in claim 19, wherein the comparison of
expression levels is carried out in mRNA levels by RT-PCT (Reverse
Transcription-Polymerization Chain Reaction).
Description
TECHNICAL FIELD
[0001] The present invention relates, in general, to a composition
for treating a viral infection comprising Jab1. More particularly,
the present invention relates to a composition for treating or
preventing a viral infection comprising a Jab1(Jun-activation
binding protein 1) protein, a nucleic acid having a nucleotide
sequence coding for a Jab1 protein or a recombinant virus
expressing a Jab1 protein.
BACKGROUND ART
[0002] Flavivirus and pestivirus belong to the Flaviviridae family
which possesses a single-stranded positive sense RNA genome and
causes various diseases in vertebrate hosts. West Nile virus (WNV)
(Burt et al., Emerg Infect Dis., 8(8):820-826, 2002; Asnis et al.,
Clin Imfect Dis 30(3): 413-418, 2000) causes diseases including
fever, rash, arthralgia and myalgia when infecting susceptible
hosts. Apoptosis in wild-type WNV-infected brain cells is induced
in a Bax-dependent manner (Parquet et al., FEBS Lett.,
500(1-2):17-24. 2001), and the apoptosis is induced by the capsid
protein of WNV through the mitochondrial/caspase-9 pathway (Yang et
al., Emerg Infect Dis., 8(12):1379-1384, 2002). However, the
intracellular pathological mechanism of West Nile virus infection
has not been completely understood.
[0003] Immunoglobulins and antiviral agents such as interferon
alpha-2b and ribavirin were conventionally used for preventing and
treating West Nile virus infection (Agrawal and Petersen., J Infect
Dis, 188(1):1-4, 2003; Morrey et al., Antiviral Res.,
55(1):107-116, 2002; Anderson et al., Emerg Infect Dis.,
8(1):107-108, 2002), but they have low therapeutic effects. At
present, there is no effective drug for treating or preventing West
Nile virus infection. Thus, there is a need for the development of
such effective drugs.
[0004] On the other hand, Jab1 (Jun-activation binding protein 1)
was initially known as a coactivator of AP-1 (Jun/Fos
proto-oncogene) protein and has the following, various functions.
Jab1 is a component (CSN5) of the COP9 signalosome (CSN) (Wei et
al., Annu Rev Cell Dev Biol., 19:261-286, 2003), and Jab1/CSN5
exists in a wide spectrum of organisms, ranging from yeasts to
plants and animals. Overexpression of Jab1 causes the translocation
of cyclin dependent kinase inhibitor p27/Kip1 from the nucleus to
the cytoplasm, accelerates the Ub-26S proteasome-dependent
degradation, and participates in the G1-S transition of the cell
cycle, mediated by p27/Kip1 (Tomoda et al., Nature,
398(6723):160-165, 1999). In addition, Jab1 involves the nuclear
translocation of PGP9.5 that is overexpressed in primary lung
cancer cells (Caballero et al., Oncogene, 21(19):3003-3010, 2002).
Jab1 interacts with p53, Smad4 and lutropin/choriogonadotropin
receptor and stimulates degradation of these proteins (Bech-Otschir
et al., EMBO J., 20(6):1630-1639, 2001; Li et al. J Biol Chem.,
275(18):13386-13393, 2000; Wan et al., EMBO J., 3(2):171-176,
2002). Taken together, Jab1 translocates proteins from the nucleus
to the cytoplasm by interaction with intracellular proteins and
thus stimulates protein degradation in a proteasome-dependent
manner.
[0005] However, there is no report for interaction between Jab1 and
viral proteins upon flavivirus infection.
[0006] Based on this background, the present inventors identified
Jab1 as a protein interacting with the capsid protein of
flavivirus, and found that Jab1 inhibits apotosis by accelerating
degradation of the capsid protein and that Jab1 is useful for
treating or preventing a viral infection thereby leading to the
present invention.
DISCLOSURE OF THE INVENTION
[0007] It is therefore an object of the present invention to
provide a composition for treating or preventing a flavivirus or
pestivirus infection, which comprises a Jab1 (Jun-activation
binding protein 1) protein.
[0008] It is another object of the present invention to provide a
composition for treating or preventing a flavivirus or pestivirus
infection, which comprises a nucleic acid having a nucleotide
sequence coding for a Jab1 protein.
[0009] It is a further object of the present invention to provide a
composition for treating or preventing a flavivirus or pestivirus
infection, which comprises a recombinant virus expressing a Jab1
protein.
[0010] It is yet another object of the present invention to provide
a method of treating or preventing a flavivirus or pestivirus
infection, which is based on administering a pharmaceutically
effective amount of a Jab1 protein to a subject requiring treatment
or prevention of a viral infection.
[0011] It is still another object of the present invention to
provide a method of treating or preventing a flavivirus or
pestivirus infection, which is based on administering a
pharmaceutically effective amount of a nucleic acid having a
nucleotide sequence coding for a Jab1 protein to a subject
requiring treatment or prevention of a viral infection.
[0012] It is still another object of the present invention to
provide a method of treating or preventing a flavivirus or
pestivirus infection, which is based on administering a
pharmaceutically effective amount of a recombinant virus expressing
a Jab1 protein to a subject requiring treatment or prevention of a
viral infection.
[0013] It is still another object of the present invention to
provide a method of assaying a substance stimulating expression of
a Jab1 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0015] FIG. 1 shows the results of immunofluorescence analysis for
expression patterns of the capsid (Cp) protein of West Nile virus
(WNV) in three tumor cell lines;
[0016] FIG. 2 shows the results of immunofluorescence analysis for
expression patterns of WNV-Cp in SK-N-SH cells;
[0017] FIG. 3 shows the results of an annexin assay, displaying
apoptosis induction by WNV-Cp in two tumor cell lines;
[0018] FIG. 4 shows the results of FACS analysis, displaying
apoptosis induction by WNV-Cp in 293T cells;
[0019] FIG. 5 shows the procedure of a yeast two hybrid assay
resulting in obtainment of clones expressing proteins interacting
with WNV-Cp;
[0020] FIG. 6 shows the results of immunofluorescence analysis,
revealing that Jab1 translocates WNV-Cp from the nucleolus to the
cytoplasm;
[0021] FIG. 7 shows the results of immunoprecipitation, revealing
that Jab1 interacts with WNV-Cp;
[0022] FIG. 8 shows that co-expression of Jab1 and WNV-Cp leads to
a decrease in caspase activity;
[0023] FIG. 9 shows the results of Western blotting, displaying
that degradation of WNV-Cp by Jab1 is remarkably suppressed in the
presence of a 26S proteasome inhibitor LLnL;
[0024] FIG. 10 shows the results of FACS analysis, displaying an
apoptosis rate of normal cells not transfected with a C2-Cp
gene;
[0025] FIG. 11 shows the results of FACS analysis, displaying an
apoptosis rate of cells transfected with a pEGFP-C2-Cp plasmid;
[0026] FIG. 12 shows the results of FACS analysis, displaying an
apoptosis rate of cells transfected with a pEGFP-N1 plasmid;
[0027] FIG. 13 shows the results of FACS analysis, displaying an
apoptosis rate of cells transfected with a pEGFP-C2-Cp plasmid and
treated with 200 nM of a PI3K inhibitor;
[0028] FIG. 14 shows the results of FACS analysis, displaying an
apoptosis rate of cells transfected with a pEGFP-C2-Cp plasmid and
treated with 5 .mu.M of an Akt inhibitor;
[0029] FIG. 15 shows the results of FACS analysis, displaying an
apoptosis rate of cells transfected with a pEGFP-C2-Cp plasmid and
treated with 50 .mu.M of an Akt inhibitor;
[0030] FIG. 16 shows the results of Western blotting; displaying
that p53 expression decreases with increasing concentrations of
Jab1;
[0031] FIG. 17 shows a process of constructing a vector system for
establishing a Jab1 adenovirus stable cell line; and
[0032] FIG. 18 shows the results of Western blotting, demonstrating
that a NIH3T3 Jab1 retrovirus stable cell line is successfully
established.
BEST MODE FOR CARRYING OUT THE INVENTION
[0033] In one aspect, the present invention provides a composition
for treating or preventing a flavivirus or pestivirus infection
comprising a Jab1 protein.
[0034] Viral infections and associated diseases intended to be
treated or prevented according to the present invention are
flavivirus and pestivirus infections. Flavivirus and pestivirus
according to the classification of International Committee on
Taxonomy of viruses, belong to the Flaviviridae family, which
possesses a positive-stranded single strand RNA genome and has a
natural host range including vertebrates and arthropods. Flavivirus
and pestivirus virions consist of an envelope and a nucleocapsid.
Flavivirus virions are spherical and 40-50 nm in diameter, and
pestivirus virions are spherical to pleomorphic and 40-60 nm in
diameter. Flavivirus and pestivirus have a very similar structure
and infection mechanism and induce apoptosis of infected cells.
[0035] Flavivirus includes the mammalian tick-borne virus group,
seabird tick-borne virus group, Aroa virus group, Dengue virus
group, Japanese encephalitis virus group, Ntaya virus group,
Kokobera virus group, Spondweni virus group, Yellow fever virus
group, Entebbe virus group, Modac virus group and Rio Bravo virus
group. The present composition may be preferably applied to an
infection with the Japanese encephalitis virus group. The Japanese
encephalitis virus group includes Cacipacore virus, koutango virus,
Japanese encephalitis virus, Murray Valley encephalitis virus, St.
Louis encephalitis virus, Usutu virus, West Nile virus, and Yaounde
virus.
[0036] Pestivirus includes Border disease virus, bovine viral
diarrhea virus 1, Bovine viral diarrhea virus 2, and Classical
swine fever virus.
[0037] The present inventors, via a yeast two hybrid assay, found
that Jab1 is a protein directly interacting with the capsid (Cp)
protein of West Nile virus, which induces apoptosis in WNV-infected
cells, and investigated the effect of Jab1 on the capsid protein.
As a result, Jab1 was found to directly interact with the capsid
protein, translocate the capsid protein from the nucleus to the
cytoplasm and stimulate degradation of the capsid protein, thereby
remarkably inhibiting apoptosis mediated by the viral capsid
protein.
[0038] The Capsid denotes the protein shell that encloses the viral
nucleic acid and is formed by multiple copies of a single major
structural subunit protein. The structural subunit protein forming
the capsid is called the capsid protein. With respect to the
objects of the present invention, the capsid protein is the
flavivirus or pestivirus capsid protein to which the Jab1 protein
binds. The complete genome sequence of West Nile virus including
the nucleic acid sequence of the capsid protein of West Nile virus,
a member of flavivirus, is available from GenBank under accession
numbers AF206518, AF196835, AF202541 and M12294. The nucleic acid
sequences of capsid proteins of other members of flavivirus and
pestivirus are also available from GenBank, for example, for JEV,
under accession numbers M18370, D90194 and D90195; for SLEV, under
accession number M16614; for YFV, under accession numbers AF094612,
U17067, U17066, U54798, U21055, U21056 and X03700; for DENV,
accession numbers M23027, U88535, U88536 and U88537); and for BVDV,
accession number M31182.
[0039] The homology of the capsid protein between flavivirus and
pestivirus, which possess the capsid protein capable of binding the
Jab1 protein, is about 90%.
[0040] The Jab1 protein used in the present composition includes
all Jab1 proteins derived from yeasts, plants and animals, which
include a wild-type Jab1 protein and, as long as the function of
binding to the flavivirus or pestivirus capsid protein and
stimulating degradation of the capsid protein is retained, variants
of the Jab1 protein made by deletions, insertions,
non-conserveative or conservative substitutions, or combinations
thereof. In one embodiment, the Jab1 protein may have an amino acid
sequence designated as SEQ ID No. 2, and substitution, insertion
and deletion variants of this amino acid sequence may be useful in
the present composition.
[0041] The variant of Jab1 means the protein that has a sequence in
which one or more amino acid residues differ from a wild-type amino
acid sequence. An insertion is typically made by the addition of a
consecutive amino acid sequence of about 1 to 20 amino acids, or
may be made with a longer sequence. A deletion is typically in the
range of about 1 to 30 amino acid residues, or may be in part made
in a longer sequence such as the absence of one domain. Such
variants may be prepared by a chemical peptide synthesis method or
a DNA sequence-based recombinant method, which are known in the art
(Sambrook et al., Molecular Cloning, Cold Spring Harbour Laboratory
Press, New York, USA, 2d Ed., 1989). Amino acid exchanges in
proteins and peptides which do not generally alter the activity of
the protein or peptide are known in the art (H. Neurath, R. L.
Hill, The Proteins, Academic Press, New York, 1979). The most
commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,
Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,
Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly,
in both directions.
[0042] In addition, the Jab1 protein, if desired, may be modified
by phosphorylation, sulfation, acrylation, glycosylation,
methylation, farnesylation, and the like.
[0043] The variant or modified product may have the biological
activity functionally identical to its natural form, or, if
desired, may be made by altering the property of the natural form.
It is preferably a protein that is improved in enhanced structural
stability against heat, pH, etc., and protein activity by
alteration and modification of its amino acid sequence.
[0044] The Jab1 protein may be obtained by extraction and
purification from nature according to a method well known in the
art (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963), or may be
obtained using a genetic recombination technique.
[0045] When the protein is prepared by chemical synthesis, a
polypeptide synthesis method well known in the art may be used.
[0046] In the case of using the genetic recombination technique,
the Jab1 protein may be obtained by a process including inserting a
nucleic acid coding for Jab1 into a suitable expression vector,
transforming a host cell with the vector, cultivating the host cell
to allow Jab1 to express and recovering expressed Jab1 from the
cultured host cell.
[0047] As the expression vector for expressing the Jab1 protein,
all common expression vectors may be used. Since expression levels
and modification of proteins differ according to host cells, the
most suitable host cell may be selected according to the intended
use. Available host cells include, but are not limited to,
prokaryotic cells such as Escherichia coli, Bacillus subtilis,
Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus.
Among them, E. coli is most commonly used. In addition, useful as
host cells are lower eukaryotic cells, such as fungi (e.g.,
Aspergillus) and yeasts (e.g., Pichia pastoris, Saccharomyces
cerevisiae, Schizosaccharomyces, Neurospora crassa), insect cells,
plant cells, and cells derived from higher eukaryotes including
mammals.
[0048] After a protein is expressed in a selected host cell, it may
be isolated and purified by a general biochemical isolation
technique, for example, treatment with a protein precipitating
agent (salting out), centrifugation, ultrasonic disruption,
ultrafiltration, dialysis, and various chromatographies, such as
molecular sieve chromatography (gel filtration), adsorption
chromatography, ion exchange chromatography and affinity
chromatography. Typically, these techniques are used in
combinations of two or more to obtain highly pure isolation of a
protein (Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982);
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,
Cold Spring Harbor Laboratory Press(1989); Deutscher, M., Guide to
Protein Purification Methods Enzymology, vol. 182. Academic Press.
Inc., San Diego, Calif. (1990)).
[0049] In another aspect, the present invention provides a
composition for treating or preventing a flavivirus or pestivirus
infection comprising a nucleic acid having a nucleotide sequence
coding for a Jab1 protein.
[0050] The Jab1-encoding nucleotide sequence in the present
composition, which encodes the Jab1 protein in the form of a wild
type or a variant as described above, may be altered by
substitutions, deletions, insertions or combinations thereof of one
or more bases, and may be naturally occurring or chemically
synthesized.
[0051] The chemical synthesis of the Jab1-encoding nucleotide
sequence may be carried out by a synthesis method well known in the
art, for example, as described in the literature: Engels and
Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988. Examples of the
synthesis method include triester, phosphite, phosphoramidate and
H-phosphate methods, PCR and other autoprimer methods, and
oligonucleotide synthesis methods on solid phase supports.
[0052] In an embodiment, the Jab1-encoding nucleotide sequence is
exemplified as a nucleotide sequence encoding the amino acid
sequence of SEQ ID No. 2, preferably a nucleotide sequence
designated as SEQ ID No. 1.
[0053] A nucleic acid having the aforementioned nucleotide sequence
may be single-stranded or double-stranded, and may be DNA (genome,
cDNA or synthetic) or RNA molecules.
[0054] In a preferred aspect, the Jab1-encoding nucleotide sequence
is operably linked to a vector to provide a recombinant expression
vector expressing the nucleotide sequence.
[0055] The term "vector", as used herein, means a vehicle for
introducing a nucleic acid sequence (e.g., DNA, RNA, etc.) coding
for a target gene into a host cell. Also, the term "expression
vector", as used herein, which is a vector capable of expressing a
target protein or target RNA in a suitable host cell, refers to a
genetic construct that comprises essential regulatory elements to
which a gene insert is operably linked thereto in such a manner as
to be expressed in a host cell.
[0056] The term "operably linked", as used herein, refers to a
functional linkage between a nucleic acid expression control
sequence (such as a promoter) and a second nucleic acid sequence
coding for a target protein or RNA in a manner that allows general
functions. For example, when a nucleic acid sequence coding for a
protein or RNA is operably linked to a promoter, the promoter may
affect the expression of a coding sequence. The operable linkage to
a recombinant vector may be prepared using a genetic recombinant
technique well known in the art, and site-specific DNA cleavage and
ligation may be carried out using enzymes generally known in the
art.
[0057] The vector useful in the present invention includes plasmid
vectors, cosmid vectors and viral vectors. A suitable expression
vector includes expression regulatory elements, such as a promoter,
an operator, an initiation codon, a stop codon, a polyadenylation
signal and an enhancer, and a signal sequence or leader sequence
for membrane targeting or secretion, and may be prepared in various
constructs according to the intended use. The initiation and stop
codons are generally considered to be a portion of a nucleotide
sequence encoding an immunogenic target protein. Also, the
initiation and stop codons are necessary to be functional in an
individual to whom a genetic construct has been administered, and
must be in frame with the coding sequence. The promoter of the
vector may be constitutive or inducible. Also, the expression
vector includes a selectable marker for selecting a host cell
containing a vector, and, in the case of being replicable, includes
a replication origin. The vector may be self-replicated or
integrated into host DNA.
[0058] In a more preferred aspect, the present invention provides a
composition comprising a recombinant viral vector carrying a
nucleotide sequence coding for a Jab1 protein.
[0059] The term "recombinant viral vector", as used herein,
typically denotes a viral vector that contains one or more
exogenous genes, and, in the present invention, means a viral
vector carrying a Jab1 gene. The viral vector is preferably a
replication-defective vector that lacks a replicon.
[0060] Non-limiting examples of the recombinant vector include
retrovirus, which is exemplified by HIV (Human Immunodeficiency
Virus) MLV (Murine Leukemia Virus), ASLV (Avian Sarcoma/Leukosis
Virus), SNV (Spleen Necrosis Virus), RSV (Rous Sarcoma Virus) and
MMTV (Mouse mammary tumor virus), and recombinant viral vectors,
which are exemplified by adenovirus, adeno-associated virus and
herpes simplex virus.
[0061] The nucleic acid having a nucleotide sequence coding for a
Jab1 protein may be delivered into target cells of a patient for
treating or preventing a viral infection by a method known in the
art, for example, direct injection of a vector in naked DNA form
(Wolff et al., Science, 247:1465-8, 1990: Wolff et al., J Cell Sci.
103:1249-59, 1992), or using liposomes, cationic polymers, and the
like. Liposomes are phospholipid membranes made by mixing cationic
phospholipids such as DOTMA or DOTAP for gene delivery. When
cationic liposomes are mixed with anionic nucleic acids in a
predetermined ratio, nucleic acid-liposome complexes are formed.
These complexes are internalized into cells by endocytosis and stay
in the endosome (Schaefer-Ridder M et al., Sceience.
215(4529):166-168, 1982; Hodgson et al., Nat Biotechnol.,
14(3):339-342, 1996). Release of an internalized gene from the
endosome into the cytoplasm and transport of the endosomally
released gene from the cytoplasm to the nucleus determine the
efficiency for gene transfer and therapy. This gene transfer allows
repeated administration and ensures high safety due to low
immunogenicity, but has a disadvantage of providing low efficiency
in gene expression. Cationic polymers used in gene transport
include poly-L-lysine, spermine, polyethylenimine (PEI) and
chitosan (Hashida, Br J Cancer., 90(6):1252-1258, 2004; Wiseman,
Gene Ther., 10(19):1654-1662, 2003; Koping-Hoggard, Gene Ther.,
8(14):1108-1121, 2001). When a gene is administered into the body
in a complex form with a cationic polymer, in vivo detention time
and expression duration of the gene remarkably increased in
comparison with the case of being administered in naked DNA
form.
[0062] In a further aspect, the present invention provides a
composition for treating or preventing a flavivirus or pestivirus
infection comprising a recombinant virus expressing a Jab1
protein.
[0063] Since the infection of cells of a patient with infective
viral particles manipulated to express Jab1 in infected cells
results in an increase in the expression efficiency of Jab1, this
method provides a highly therapeutic effect.
[0064] Non-limiting examples of recombinant viruses useful in the
present composition comprising a recombinant virus include
retroviruses, adenoviruses, adeno-associated viruses and herpes
simplex virus. Preferred are retroviruses and adenoviruses, and
more preferred are adenoviruses.
[0065] Retroviruses have an advantage of providing long-lasting
gene expression because they are irreversibly fused to the host
chromosome. Adenoviruses, which are the most frequently used system
in general gene therapy studies, are applicable to a wide spectrum
of mammalian cells. Adeno-associated viruses have advantages of
having a broad range of host cells where a therapeutic gene is
delivered, fewer side effects on the immune system upon repeated
administration and a long duration of gene expression. Herpes
simplex virus is a highly neurotropic virus, which infects neural
cells where its genome remains as a stable episomal element within
the nucleus of neural cells without disturbing normal function of
neural cells. When a replication-deficient herpes simplex virus was
used for gene delivery, expression of a reporter gene in the
nervous system was found to be sustained for a period of over one
year.
[0066] In yet another aspect, the present invention provides a
method of treating or preventing a flavivirus or pestivirus
infection, which is base on administering, to a subject requiring
treatment or prevention of a viral infection, a pharmaceutically
effective amount of a Jab1 protein, a nucleic acid having a
nucleotide sequence coding for a Jab1 protein or a recombinant
virus expressing a Jab1 protein.
[0067] The Jab1 protein, nucleic acid having a nucleotide sequence
coding for a Jab1 protein or recombinant virus expressing a Jab1
protein, used in the treatment method of the present invention, is
the same as described above.
[0068] The treatment method of the present invention is provided
for preventing or treating a viral infection in vertebrates, which
includes mammals such as humans and livestock.
[0069] The pharmaceutical composition of the present invention,
comprising the aforementioned Jab1 protein, nucleic acid having a
nucleotide sequence coding for a Jab1 protein or recombinant virus
expressing a Jab1 protein, is used for treating or preventing an
infection of a virus belonging to the Flaviviridae family,
preferably flavivirus or pestivirus. In particular, the present
composition may be preferably used for treating or preventing a
flavivirus infection. The aforementioned viruses are known to cause
fever, rash, bleeding, jaundice, arthralgia, myalgia, encephalitis
and meningitis (Watt et al., Am J Trop Med Hyg., 68(6):704-706,
2003; Anninger et al., Clin Infect Dis., 38(7):55-56, 2004). The
pharmaceutical composition of the present invention may be used for
suppressing or treating the incidence of the aforementioned
diseases.
[0070] The present composition may include a pharmaceutically
acceptable carrier. Examples of the pharmaceutically acceptable
carrier may include, for oral administration, binders, lubricants,
disintegrators, excipients, solubilizing agents, dispersing agents,
stabilizing agents, suspending agents, pigments and aromatics; for
injectable preparations, buffering agents, preservatives,
analgesics, solubilizing agents, tonic adjusting agents and
stabilizing agents; and for topical administration, bases,
excipients, lubricants and preservatives. The pharmaceutical
composition of the present invention may be formulated into a
variety of dosage forms in combination with the aforementioned
pharmaceutically acceptable carrier. For example, for oral
administration, the pharmaceutical composition may be formulated
into tablets, troches, capsules, elixirs, suspensions, syrups or
wafers. For injectable preparations, the pharmaceutical composition
may be formulated into a unit dosage form, such as a multidose
container or an ampoule as a single-dose dosage form.
[0071] The pharmaceutical composition of the present invention may
be administered via any of the common routes, if it is able to
reach a desired tissue. Therefore, the present composition may be
administered topically, orally, parenterally, intranasally,
intravenously, intramuscularly, subcutaneously, intraocularly and
intradermally, and may be formulated into solutions, suspensions,
tablets, pills, capsules and sustained release preparations.
Injectable preparations are preferred. Injection may be carried cut
subcutaneously, intramuscularly and intravenously.
[0072] The present composition may be administered in a
therapeutically or preventively effective amount. The dosage may
vary according to the patient's age and sex, type and severity of
the illness, administration routes, target cells and expression
levels, and may be easily determined by an expert in the art.
[0073] In still another aspect, the present invention relates to a
method of screening a compound stimulating expression of a Jab1
protein, comprising: (a) culturing a cell expressing the Jab1
protein; (b) contacting the cell cultured at (a) with candidate
compounds for stimulating expression of the Jab1 protein; (c)
comparing an expression level of the Jab1 protein at (b) with that
in a control not contacted with the candidate compounds; and (d)
identifying a compound increasing expression levels of the Jab1
protein.
[0074] In still another aspect, the present invention relates to a
method of screening a compound stimulating interaction between a
Jab1 protein and a capsid (Cp) protein, comprising: (a) culturing a
cell transformed with both a recombinant vector expressing the Jab1
protein and another recombinant vector expressing the Cp protein of
flavivirus or pestivirus; (b) contacting the cell cultured at (a)
with candidate compounds for stimulating interaction between the
Jab1 protein and the Cp protein; (c) comparing an expression level
of the Cp protein at (b) with that in a control not contacted with
the candidate compounds; and (d) identifying a compound reducing
expression levels of the Cp protein.
[0075] In the above screening method, the Cp protein of flavivirus
or pestivirus, and preferably the Cp protein of West Nile virus,
may be used.
[0076] Decreased or increased expression levels of the Jab1 and Cp
proteins may be detected in protein or mRNA levels.
[0077] Protein expression levels may be detected by electorphoresis
where each protein is loaded onto a gel, and preferably by
immunoassay where the amount of formed antigen-antibody complexes
are assayed using an antibody to the Jab1 or Cp protein. Examples
of these analysis methods include Western blotting, RIA and
immunoprecipitation assay.
[0078] In the above detection method, the amount of
antigen-antibody complexes formed may be quantitatively analyzed
based on the size of signals of a detection label. The detection
label may be selected from the group consisting of enzymes,
fluorescent materials, ligands, luminescent materials,
microparticles, redox molecules and radioisotopes, but the present
invention is not limited to these examples.
[0079] The antigen-antibody complex formation may be detected using
one selected from the group consisting of a calorimetric method, an
electrochemical method, a fluorimetric method, luminometry, a
particle counting method, visual assessment and a scintillation
counting method, but the present invention is not limited to the
examples.
[0080] mRNA expression levels may be detected by a method using
primers specific for the Jab1 or Cp protein. Examples of the method
include RT-PCR and Northern blotting. Preferred is RT-PCR, a simple
analysis method that allows quantitative analysis of transcription
of Jab1 or Cp to mRNA by analysis of band patterns and
intensity.
[0081] The present invention will be explained in more detail with
reference to the following examples in conjunction with the
accompanying drawings. However, the following examples are provided
only to illustrate the present invention, and the present invention
is not limited to the examples.
EXAMPLE 1
Expression of the WNV-Cp Protein in Human Cells
[0082] To investigate the expression patterns of the WNV capsid
(WNV-Cp) protein in various human cell lines, human kidney 293T
cells (ATCC), osteosarcoma U2OS cells (ATCC), HeLa cells (ATCC) and
human neuroblastma SK-N-SH cells (ATCC) were transfected with a
vector carrying a WNV-Cp gene using a Lipofectamine reagent, and
were subjected to immunofluorescent staining.
[0083] Primarily, a WNV-Cp gene was amplified by PCR using
pcDNA3.1WNV-Cp as a template and primers designated as SEQ ID Nos.
3 and 4, and the WNV-Cp DNA was digested with EcoRI and XhoI and
inserted into a pcDNA3HA plasmid, thus generating pcDNA3-HA/WNV-Cp.
293T, U2OS, HeLa and SK-N-SH cells were transfected with the
pcDNA3-HA/WNV-Cp. After 24 hrs, the transfected cells were fixed
and subjected to immunofluorescent staining using a primary
HA-mouse monoclonal antibody (1:100 diluted; Santa Cruz) and a
secondary fluorescein isothiocyanate (FITC)-conjugated antibody
(1:100 diluted; Sigma). Then, the expression of WNV-Cp (green) was
observed using a UV confocal microscope. Nucleus was stained with
DAPI (blue). The WNV-Cp protein was found to exist in the nucleolus
in 293T, U20S and HeLa cells (FIG. 1) and in the cytoplasm in
SK-N-SH cells (FIG. 2).
[0084] The existence in the cytoplasm of WNV-Cp present mainly in
the nucleolus indicates that WNV-Cp interacts with some
intracellular proteins.
EXAMPLE 2
Apoptosis Induction by WNV-Cp in Human Cells
[0085] The WNV-Cp protein is known to induce apoptosis by previous
studies revealing that the WNV-Cp protein, in HeLa cells, induces
nuclear condensation that is a typical feature of cells undergoing
apoptosis, and such apoptosis occurs via the capase-9 pathway. In
this test, these facts were confirmed by annexin-V staining and PI
staining.
[0086] Primarily, a WNV-Cp gene was amplified by PCR using
pcDNA3.1WNV-Cp as a template and primers designated as SEQ ID Nos.
3 and 7. The amplified WNV-Cp DNA was digested with EcoRI and BamHI
and inserted into a pEGFP-C2plasmid, thus generating pEGFP-WNV-Cp.
Then, 293T and U20S cells were individually transfected with a GFP
expression vector, pEGFP-C2(control vector; CLONTECH) and the
pEGFP-WNV-Cp. After 24 hrs, the cells were stained with annexin-V
(red) to bind annexin-V to an apoptosis indicator, phosphatidyl
serine that is externalized upon apoptosis, and were observed under
a Carl Zeiss vision microscope. As a result, apoptosis occurred in
the cells with transfected the pEGFP-WNV-Cp (FIG. 3).
[0087] Separately, 293T cells were transfected with
pEGFP-C2(control vector) and pEGFP-WNV-Cp. After 48 hrs, cell
lysates were collected, stained with PI (propidium iodide) to
measure apoptosis, and subjected to FACS analysis. The transfection
with the control vector pEGFP resulted in an apoptosis rate of
15.97%, and the transfection with the pEGFP-WNV-Cp resulted in an
apoptosis rate of 27.03% (FIG. 4).
EXAMPLE 3
Screening for Proteins Interacting with WNV-Cp
[0088] A possible mechanism of the apoptosis induction by WNV-Cp
involves direct or indirect interaction of the capsid protein with
regulators capable of causing apoptosis. In this regard, to better
understand the apoptosis induction by the capsid protein, the
regulators interacting with WNV-Cp need to be screened. For
screening the regulators, a yeast two hybrid assay was performed
using a cDNA library from human brain tissue that is a major
infection site of West Nile virus.
[0089] Primarily, a WNV-Cp gene (450 bp) was amplified by PCR using
pcDNA3.1 WNV-Cp as a template and primers designated as SEQ ID Nos.
3 and 4, below. The amplified WNV-Cp gene was cloned into
EcoRI/SalI sites of a pGBK-T7 vector containing a TRP1 marker and a
Gal4-DNA binding domain, thus generating a pGBK-T7 WNV-Cp
construct.
TABLE-US-00001 Forward primer (SEQ ID No. 3): 5'-CCG GAA TTC TCT
AAA AAA CCA GGT GGC CCC GG-3' Reverse primer (SEQ ID No. 4): 3'-CCG
CTC GAG CTA CGC GCC CAC GCT GGC GAT CAG-5'
[0090] The yeast two hybrid assay was carried out using the pGBK-T7
WNV-Cp plasmid as a bait and, as a prey, a human brain cDNA library
(Clontech) carrying the LEU2 marker and fused to the downstream of
the Gal4 activation domain. A yeast strain AH109 was transfected
with the bait plasmid pGBK-T7 WNV-Cp by a lithium acetate method
(Gietz et al. 1995), mixed for mating with another yeast strain
Y187 transfected with 1 ml of the human brain cDNA library, and
smeared onto fifty 150-mm SD plates lacking adenine, leucine,
histidine and tryptophan. 945 colonies were obtained (the a of FIG.
5), and candidates to have the potential to interact with the
capsid protein were selected on the same plate (the b of FIG. 5).
For second screening, replica plating was carried out on the
selection medium, SD/-Ade-Leu-His-Trp, and blue colonies were
obtained (the c of FIG. 5). The blue colonies were tested again,
and eventually, eighty clones were obtained (the d of FIG. 5).
Yeast plasmid was isolated from the clones by lyticase-based cell
disruption and subjected to DNA sequencing using primers designated
as SEQ ID Nos. 5 and 6, below, followed by blast searching for
identifying corresponding proteins.
TABLE-US-00002 Forward primer (SEQ ID No. 5) 5'-CTA TTC GAT GAT GAA
GAT ACC CCA CCA AAC CC-3' Reverse primer (SEQ ID No. 6) 3'-AGT GAA
CTT GCG GGG TTT TTC AGT ATC TAC GAT-5'
[0091] Eight proteins were identified, which were Jab1, TPR1,
RanBPM (RanBP9), PAP-1BP, Snapin (Synaptosomal-associated protein),
Bassoon protein, a likely ortholog of mouse rabphilin3A and
CG13214-PA.
EXAMPLE 4
Translocation of WNV-Cp by Jab1
[0092] To evaluate the effect of Jab1, identified to interact with
WNV-Cp, on the intracellular location of WNV-cp, 293T, U2OS and
HeLa cells were cotransfected with the WNV-Cp protein and Jab1.
293T, U2OS and HeLa cells were cotransfected with HA-tagged
pcDNA-HA/WNV-Cp and Flag-tagged pCMV Tag2B-Jab1. After 24 hrs, the
cells were stained using an anti-HA antibody (green) and an
anti-Flag antibody (red) and observed under a confocal microscope.
As shown in FIG. 6, like Jab1, the immunofluorescence signal for
WNV-Cp appeared in the cytoplasm. A merge of two confocal images
shows that WNV-Cp and Jab1 are expressed in the same site,
cytoplasm, and PC (phase contrast) displays the whole cell
morphology.
[0093] Separately, immunoprecipitation (IP) was performed to
confirm the interaction between WNV-Cp and Jab1. 293T cells were
cotransfected with Flag-Jab1 and HA-WNV-Cp plasmids, and the whole
cell lysates were immunoprecipitated with an anti-HA mouse
antibody. As a control, IP was carried out with an anti-Myc
antibody. Immunoprecipited proteins were run on a 12% SDS-PAGE gel,
transferred to a nitrocellulose membrane, and detected with an
anti-Flag mouse antibody to visualize immunoprecipitated Jab1 along
with WNV-Cp (FIG. 7).
[0094] Jab1 was found to be co-immunoprecipitated with WNV-Cp. This
result indicates that Jab1 interacts with WNV-Cp in 293T cells and
translocates WNV-Cp from the nucleolus to the cytoplasm.
EXAMPLE 5
Inhibition of WNV-Induced Apoptosis by Jab1
[0095] WVP-Cp, which is a pathogenic protein, is known to induce
apoptosis via the mitochondrial/caspase-9 pathway. In this regard,
a caspase activity assay was performed to evaluate the effect of
Jab1 on WVP-Cp-induced apoptosis.
[0096] 293T cells were plated onto 60-mm plates at a density of
3.times.10.sup.5 cells, cultured, and transfected with pcDNA3-HA,
pcDNA3-HA/Cp, pCMV-tag2B-Jab1, both pcDNA3-HA/Cp and
pCMV-tag2B-Jab1, and pcDNA3-Bax, respectively. After 24 hrs, the
cells were washed with 1.times.PBS twice, transferred to 1.5-ml
tubes, and lysed with 20 .mu.l of buffer C (25% glycerol, 0.42 M
NaCl, 1.5 M MgCl.sub.2, 0.2 mM EDTA, 20 mM HEPES, 1 mM DTT, 0.5 mM
PMSF, pH7.9). After being incubated for 10 min on ice, the lysed
cells were centrifuged. The total protein concentration in each
supernatant was measured, and samples of zero to 300 .mu.g of
proteins were placed into a 96-well plate. To the 96-well plate, 50
.mu.l of 2.times. reaction buffer and 5 .mu.l of 4 mM
DNA-conjugated substrate, provided in a caspase calorimetric
substrate set II plus kit (Biovision), were added. After a 1-hr
incubation at 37.degree. C., the activity of caspase-3 and
caspase-9 was measured at 410 nm using a microtiter plate reader.
The results are given in the A panel of FIG. 8. In cells expressing
WNV-Cp, the activity of capase-3 and caspase-9 was similar to that
in cells expressing Bax, a member of the pro-apoptotic Bcl-2
family. When cells co-expressed WNV-Cp and Jab1, the caspase
activity was remarkably reduced. These results indicate that Jab1
suppresses the activation of caspase-3 and caspase-9 by WNV-Cp. The
expression of the proteins used in this caspase activity assay was
detected by Western blotting, and the results are given in the B
panel of FIG. 8.
[0097] Taken together, these results indicate that Jab1 expressed
in the cell suppresses WNV-Cp-induced apoptosis through the
mitochondrial/caspase-9 pathway by translocating WNV-Cp from the
nucleolus to the cytoplasm.
EXAMPLE 6
Degradation of WNV-Cp by Jab1
[0098] To determine whether Jab1 stimulates degradation of WNV-Cp,
protein levels of WNV-Cp were assessed in cells treated with a 26S
proteasome inhibitor, LLnL (Sigma). 293T cells were plated onto
60-mm plates at a density of 3.times.10.sup.5 cells, cultured, and
transfected with pcDAN3-HA/WNV-Cp alone, and pcDAN3-HA/WNV-Cp and
pCMV-tag2B-Jab1 together using an Effectene transfection reagent
(Qiagen). In the case of the co-transfection, to equalize the
levels of expressed DNA, 12 hrs after transfetion, cells were
divided into two plates by pipetting. 20 hrs after transfection,
the cells were treated for 4 hrs with 20 .mu.M/ml of the proteasome
inhibitor LLnL (N-acetyl-L-luecinyl-norleucinal, Sigma). Then, the
cells were washed with 1.times. PBS and collected. Samples of 50
.mu.g protein were loaded onto a SDS-PAGE gel and analyzed by
Western blotting using an anti-HA antibody (Santa Cruz) for the
detection of WNV-Cp expression and an anti-Flag M2 antibody (Sigma)
for the detection of Jab1 expression. Actin was used as a loading
control.
[0099] Compared to the single expression of WNV-Cp, the
co-expression with Jab1 resulted in a large decrease in protein
levels of WNV-Cp (lanes 1 and 2 of FIG. 9). In contrast, upon the
treatment with LLnL, the cotransfected cells exhibited increased
protein levels of WNV-Cp (lane 3 of FIG. 9). These results indicate
that Jab1 accelerates the degradation of WNV-Cp.
[0100] Taken together, these results indicate that Jab1 suppresses
the function of WNV-Cp by inducing the degradation of WNV-Cp
through the ubiquitin proteasome pathway.
EXAMPLE 7
Inhibition of Apoptosis Using Signal Transduction Pathway
Regulators
[0101] Human neuroblastoma SH-SY5Y cells were transfected with a
WNV-Cp gene. To determine an apoptosis rate in cells expressing the
capsid protein of WNV, the cells were stained with annexin V-PE and
subjected to FACS analysis (BioRAD, Win BRYTE). As a result, the
cells were fractionated into four fractions: A, B, C and D. The A
fraction indicates a cell population that was not injected with the
WNV-Cp gene and stained with annexin V-PE. The B fraction displays
a cell population that was injected with the WNV-Cp gene and
stained with annexin V-PE. The C fraction displays a cell
population that was not injected with the WNV-Cp gene and not
stained with annexin V-PE. The D fraction displays a cell
population that was injected with the WNV-Cp gene and not stained
with annexin V-PE.
[0102] Normal cells not injected with a C2-Cp gene displayed an
apoptosis rate of 14.0% (FIG. 10), and cells expressing WNV-Cp
exhibited an apoptosis rate of 69.6% (FIG. 11). These results
confirmed that WNV-Cp greatly increases apoptosis of cells. As a
negative control, pEGFP-N1-injected cells showed an apoptosis rate
of 23.8% (FIG. 12), which was higher than as expected. This high
apoptosis rate in the negative control is believed to be due to an
EGFP signal being very strong and thus cross-linked with a FL2
signal, and may therefore be substantially lower.
[0103] Separately, SH-SY5Y cells were transfected with a WNV-Cp
gene. After 6 hrs, the cells were treated with 200 nM of a PI3K
inhibitor, wortmanin (Sigma), and 5 .mu.M and 50 .mu.M of an Akt
inhibitor, calbiochem (CN Biosciences). After 24 hrs, the cells
were stained with annexin V-PE that is capable of detecting a step
of apoptosis and subjected to FACS analysis (BioRAD, WinBryte) for
measuring an apoptosis rate. About 10-30% of the cells were found
to be successfully transfected with the WNV-Cp gene. The
transfected cells were analyzed on a FL3 channel (green
fluorescence) and annexin V-PE binding was analyzed on a FL2
channel. An apoptosis rate in capsid-expressing cells was
calculated according to Reaction 1, below. Cells transfected with a
pEGFP-N1 plasmid were used as a negative control, and cells
transfected with a C2-Cp plasmid and not treated with the inhibitor
were used as a positive control. [0104] [Reaction 1] [0105]
[Annexin V-PE-positive cells/(all cells expressing
C2-Cp)].times.100
[0106] Cells expressing WNV-Cp displayed an apoptosis rate of
69.6%. In contrast, when treated with 50 .mu.M of the Akt inhibitor
calbiochem and 200 nM of the PI3K inhibitor wortmanin, these cells
exhibited apoptosis rates of 15.9% and 22.8%, respectively. That
is, the treatment of the inhibitors resulted in suppression of
apoptosis induced by WMV-Cp (FIGS. 13, 14 and 15). These results
indicate that the PI3K inhibitor and Akt inhibitor suppress the
apoptosis induced by the expression of WNV-Cp.
EXAMPLE 8
Decreased Expression of Endogenous p53 by Jab1
[0107] The COP9 signalosome-specific phosphorylation targets the
tumor suppressor gene p53 to degradation by the ubiquitin-26S
proteasome-dependent pathway (Bech-Otschir et al., EMBO J.,
20(7):1630-1639, 2001). On the assumption that Jab1 interacts with
p53 because it is a member of the COP9 signalosome, Jab1 was
evaluated for its effect on p53 expression.
[0108] U2OS cells were transfected with Flag/mdm2 (control) and
Flag/Jab1 with various concentrations of 1, 3 and 5 .mu.g using a
Lipofectamin/plus reagent (Invitrogen). After 48 hrs, cell lysates
were collected, and total protein concentrations were measured by a
BSA (PIERCE) method. Samples of 100 .mu.g/ml protein were separated
on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane.
The blot was blocked with 5% skim milk for 30 min, and treated with
a rabbit anti-HA antibody (Santa Cruz) and a mouse anti-Flag
antibody (Sigma) to examine expression levels of p53 according to
increased expression of mdm2 and Jab1. Expression of p53 was rarely
affected by the control mdm2, but remarkably decreased with
increasing concentrations of Jab1 (FIG. 16).
EXAMPLE 9
Establishment of Jab1 Adenovirus Stable Cell Line
[0109] A stable cell line producing an adenovirus inducing
overexpression of Jab1 was established using an AdEasy XL
adenoviral vector system (Stratagene). Jab1 was cloned into
BglII/PvuI sites of a pShuttle-IRES-hrGFP vector (the A and B of
FIG. 17). The resulting pShuttle-IRES-hrGFP/Jab1 vector was
purified by maxi preparation (maxi-prep), digested with PmeI, and
transformed into anE. coli strain BJ5183 which contains AD1
(Stratagene) to produce homologous recombinant adenovirus plasmid.
Emerged colonies were picked and grown in a culture broth, and
plasmid DNA was isolated from the culture. Cloning was found to be
successful by restriction mapping with PacI (the C of FIG. 17). The
plasmid was then amplified by being transfected into mammalian
AD293 cells (Stratagene) using a Lipofectamin/plus reagent
(Invitrogen). Produced adenovirus was transfected again into AD293
cells, thus generating a stable cell line producing a recombinant
adenovirus carrying a Jab1 gene, that is, a Jab1 adenovirus stable
cell line.
[0110] The Ad1-Jab1 plasmid used in the production of the
adenovirus stable cell line was deposited at an international
depositary authority, KCCM (Korean Culture Center of
Microorganisms; 2nd Floor, Yourim Building, 361-221, Hongje 1-dong
Seodaemun-gu, Seoul, Korea) on Aug. 31, 2004, and assigned
accession number KCCM 10593.
EXAMPLE 10
Establishment of NIH3T3 Jab1 Stable Retrovirus Cell Line
[0111] A HA/Jab1 fragment excised from the pcDNA3-HA/Jab1 plasmid
was subcloned into an EcoRI site of the PLPCX retroviral vector (BD
Bioscience) capable of producing retrovirus, thus generating
pLPC/HA-Jab1. The pLPC/HA-Jab1 construct carrying a puromycin
resistant gene was cotransfected with the pCL packaging plasmid (BD
Bioscience) into 293T cells using a Lipofectamine reagent
(Invitrogen). After two days, viral particles were purified with a
0.45-.mu.m filter. 1 ml of the viral particles was diluted in 2 ml
of medium and supplemented with 4 .mu.g/ml polybren (Sigma) helping
viral infection, and infected NIH3T3 cells. After 24 hrs, the cells
were selected in a medium containing 2 .mu.g/ml puromycin (Sigma),
thereby generating a stable cell line producing a recombinant
retrovirus carrying a Jab1 gene, that is, a Jab1 retrovirus stable
cell line. A recombinant retrovirus produced by the stable cell
line, Retro-Jab1, was deposited at an international depositary
authority, KCCM (Korean Culture Center of Microorganisms; 2nd
Floor, Yourim Building, 361-221, Hongje 1-dong Seodaemun-gu, Seoul,
Korea) on Aug. 31, 2004, and assigned assess number KCCM 10592. In
a control cell line not carrying an exogenous Jab1 gene and the
retrovirus stable cell line highly expressing Jab1, expression
levels of Jab1 and p53 were examined. The retrovirus stable cell
line displayed high expression of Jab1 and decreased expression of
p53 (FIG. 18).
[0112] The decreased expression of p53, shown in FIG. 18,
correlated with the results of Example 8. These results indicate
that stable gene transfer using a recombinant retrovirus
overexpressing Jab1 leads to degradation of a viral capsid
protein.
INDUSTRIAL APPLICABILITY
[0113] As described hereinbefore, the present composition for
treating a viral infection comprising Jab1 is capable of
effectively treating diseases caused by flavivirus or pestivirus
infections, including fever, rash, bleeding, jaundice, arthralgia,
myalgia, encephalitis and meningitis.
Sequence CWU 1
1
611262DNAHomo sapiensgene(1)..(1262)Jab1 1ctggtgggga aggtccaaag
cccgcacgct gaggcccagt agaagaaagt tgcatcttga 60ttgtggagcg acagcttctc
cggtgcctcg gcc atg gca gct tcc ggg 108 Met Ala Ala Ser Gly 1 5agt
ggt atg gcc cag aaa acc tgg gaa ttg gcc aac aac atg cag gaa 156Ser
Gly Met Ala Gln Lys Thr Trp Glu Leu Ala Asn Asn Met Gln Glu 10 15
20gcg cag agt atc gat gaa atc tac aaa tat gac aaa aaa caa caa caa
204Ala Gln Ser Ile Asp Glu Ile Tyr Lys Tyr Asp Lys Lys Gln Gln Gln
25 30 35gaa atc ctg gcg gcg aaa ccc tgg act aag gat cac cac tac ttt
aaa 252Glu Ile Leu Ala Ala Lys Pro Trp Thr Lys Asp His His Tyr Phe
Lys 40 45 50tac tgc aaa atc tca gca ttg gct cta ctg aaa atg gtg atg
cat gcc 300Tyr Cys Lys Ile Ser Ala Leu Ala Leu Leu Lys Met Val Met
His Ala 55 60 65agg tca gga ggc aac ttg gaa gtg atg ggt ttg atg ctc
ggg aaa gtc 348Arg Ser Gly Gly Asn Leu Glu Val Met Gly Leu Met Leu
Gly Lys Val 70 75 80 85gac ggc gag acc atg atc atc atg gac agt ttc
gct ttg cct gta gag 396Asp Gly Glu Thr Met Ile Ile Met Asp Ser Phe
Ala Leu Pro Val Glu 90 95 100ggc aca gaa act cga gta aat gct caa
gct gct gcg tat gag tat atg 444Gly Thr Glu Thr Arg Val Asn Ala Gln
Ala Ala Ala Tyr Glu Tyr Met 105 110 115gct gca tac ata gaa aat gcc
aaa cag gtt ggc cgc ctt gag aat gca 492Ala Ala Tyr Ile Glu Asn Ala
Lys Gln Val Gly Arg Leu Glu Asn Ala 120 125 130atc ggt tgg tat cat
agc cac cct ggt tat ggc tgc tgg ctc tcc ggg 540Ile Gly Trp Tyr His
Ser His Pro Gly Tyr Gly Cys Trp Leu Ser Gly 135 140 145att gat gtt
agt aca cag atg ctg aac cag cag ttt caa gaa cca ttt 588Ile Asp Val
Ser Thr Gln Met Leu Asn Gln Gln Phe Gln Glu Pro Phe150 155 160
165gta gca gtg gtg att gat cca acc aga aca atc tct gca gga aaa gtg
636Val Ala Val Val Ile Asp Pro Thr Arg Thr Ile Ser Ala Gly Lys Val
170 175 180aat ctt ggc gcc ttt agg aca tat cca aag ggc tac aaa cct
cct gat 684Asn Leu Gly Ala Phe Arg Thr Tyr Pro Lys Gly Tyr Lys Pro
Pro Asp 185 190 195gaa gga cct tct gag tac cag act atc cca ctt aat
aaa ata gaa gat 732Glu Gly Pro Ser Glu Tyr Gln Thr Ile Pro Leu Asn
Lys Ile Glu Asp 200 205 210ttt ggc gtg cac tgc aaa caa tat tat gcc
tta gaa gtc tca tat ttc 780Phe Gly Val His Cys Lys Gln Tyr Tyr Ala
Leu Glu Val Ser Tyr Phe 215 220 225aaa tca tct ttg gat cgt aaa cta
ctt gag ctt ttg tgg aat aaa tac 828Lys Ser Ser Leu Asp Arg Lys Leu
Leu Glu Leu Leu Trp Asn Lys Tyr230 235 240 245tgg gtg aat acc ctg
agt tcc tct agc ttg ctt act aat gca gac tac 876Trp Val Asn Thr Leu
Ser Ser Ser Ser Leu Leu Thr Asn Ala Asp Tyr 250 255 260acc aca ggc
cag gtg ttt gat ttg tct gag aag tta gag cag tcg gaa 924Thr Thr Gly
Gln Val Phe Asp Leu Ser Glu Lys Leu Glu Gln Ser Glu 265 270 275gcc
caa ctg gga cgt ggc agt ttc atg ttg ggc tta gaa aca cat gac 972Ala
Gln Leu Gly Arg Gly Ser Phe Met Leu Gly Leu Glu Thr His Asp 280 285
290cgc aag tcg gaa gac aaa ctt gcc aaa gct act aga gac agc tgt aaa
1020Arg Lys Ser Glu Asp Lys Leu Ala Lys Ala Thr Arg Asp Ser Cys Lys
295 300 305acc acc ata gaa gcc atc cat gga ctg atg tct cag gtt att
aag gat 1068Thr Thr Ile Glu Ala Ile His Gly Leu Met Ser Gln Val Ile
Lys Asp310 315 320 325aaa ctg ttt aat cag att aac gtt gct tagtt
accaccaagt acttctcaaa 1120Lys Leu Phe Asn Gln Ile Asn Val Ala
330gctggtgtgt ggaaggaaaa gaagctcaag taacactttt aacccagtta
ccaaaactca 1180gattagaaga ctaaggtgct gtgtggtgtc ctgagtatta
gcactgtaat aaaactatca 1240cgtgaaaaaa aaaaaaaaaa aa 12622334PRTHomo
sapiens 2Met Ala Ala Ser Gly Ser Gly Met Ala Gln Lys Thr Trp Glu
Leu Ala 1 5 10 15Asn Asn Met Gln Glu Ala Gln Ser Ile Asp Glu Ile
Tyr Lys Tyr Asp 20 25 30Lys Lys Gln Gln Gln Glu Ile Leu Ala Ala Lys
Pro Trp Thr Lys Asp 35 40 45His His Tyr Phe Lys Tyr Cys Lys Ile Ser
Ala Leu Ala Leu Leu Lys 50 55 60Met Val Met His Ala Arg Ser Gly Gly
Asn Leu Glu Val Met Gly Leu 65 70 75 80Met Leu Gly Lys Val Asp Gly
Glu Thr Met Ile Ile Met Asp Ser Phe 85 90 95Ala Leu Pro Val Glu Gly
Thr Glu Thr Arg Val Asn Ala Gln Ala Ala 100 105 110Ala Tyr Glu Tyr
Met Ala Ala Tyr Ile Glu Asn Ala Lys Gln Val Gly 115 120 125Arg Leu
Glu Asn Ala Ile Gly Trp Tyr His Ser His Pro Gly Tyr Gly 130 135
140Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln Met Leu Asn Gln
Gln145 150 155 160Phe Gln Glu Pro Phe Val Ala Val Val Ile Asp Pro
Thr Arg Thr Ile 165 170 175Ser Ala Gly Lys Val Asn Leu Gly Ala Phe
Arg Thr Tyr Pro Lys Gly 180 185 190Tyr Lys Pro Pro Asp Glu Gly Pro
Ser Glu Tyr Gln Thr Ile Pro Leu 195 200 205Asn Lys Ile Glu Asp Phe
Gly Val His Cys Lys Gln Tyr Tyr Ala Leu 210 215 220Glu Val Ser Tyr
Phe Lys Ser Ser Leu Asp Arg Lys Leu Leu Glu Leu225 230 235 240Leu
Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser Ser Ser Leu Leu 245 250
255Thr Asn Ala Asp Tyr Thr Thr Gly Gln Val Phe Asp Leu Ser Glu Lys
260 265 270Leu Glu Gln Ser Glu Ala Gln Leu Gly Arg Gly Ser Phe Met
Leu Gly 275 280 285Leu Glu Thr His Asp Arg Lys Ser Glu Asp Lys Leu
Ala Lys Ala Thr 290 295 300Arg Asp Ser Cys Lys Thr Thr Ile Glu Ala
Ile His Gly Leu Met Ser305 310 315 320Gln Val Ile Lys Asp Lys Leu
Phe Asn Gln Ile Asn Val Ala 325 330332DNAArtificial Sequenceprimer
for WNV-Cp amplification 3ccggaattct ctaaaaaacc aggtggcccc gg
32433DNAArtificial Sequenceprimer for WNV-Cp amplification
4gactagcggt cgcacccgcg catcgagctc gcc 33532DNAArtificial
Sequenceprimer for DNA sequencing 5ctattcgatg atgaagatac cccaccaaac
cc 32633DNAArtificial Sequenceprimer for DNA sequencing 6tagcatctat
gactttttgg ggcgttcaag tga 33
* * * * *