Note: Descriptions are shown in the official language in which they were submitted.
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COMPOUNDS AND METHODS FOR TREATING AND SCREENING
VIRAL REACTIVATION
FIELD OF THE INVENTION
' This invention relates to newly identified attributes of cellular and viral
polynucleotides and polypeptides, the uses of such polynucleotides and
polypeptides, as
well as the production of such polynucleotides and polypeptides. This
invention also
relates to inhibiting the biosynthesis, action or interaction of such
polynucleotides and/or
polypeptides and to the use of such inhibitors in therapy, particularly
therapy for viral
reactivation, especially Herpesvirus reactivation, as well as for prophylaxis.
BACKGROUND OF THE INVENTION
Following primary infection, latent Herpes simplex virus (herein "HSV")
persists in
sensory ganglia of the peripheral nervous system. The virus can undergo
sporadic
reactivation to produce recurrent mucocutaneous lesions at peripheral sites
innervated by
the infected ganglia (reviewed in Fraser et al., 1991, Roizman, 1991, Stevens,
1989).
Reactivation stimuli range from direct mechanical or pharmacological insults
to the neuron
and surrounding tissue to systemic changes in immune modulators and
neurotransmitters
(Fraser et al., 1991, Fraser & Valyi-Nagy, 1993, Hill, 1985). The earliest
molecular events
in neuronal cells that trigger reactivation of HSV remain unclear. It has been
suggested that
these events include altered expression of cellular factors such as induction
of
transcriptional activators and down-regulation of repressors (Sheng &
Greenberg, 1990).
Identification of cellular factors which are induced during the reactivation
process may lead
to better understanding of the cellular environment during viral induction and
should
facilitate development of an effective treatment or prophylaxis of
reactivation an/or
infection.
The present knowledge of the molecular pathogenesis of HSV latency and
reactivation was generated from studies in laboratory animals including mice,
guinea pigs
and rabbits (reviewed in Roizman & Sears, 1987). The Applicants and others
have found
current murine in vivo models to be inefficient in reactivation of the viral
genome (Fawl et
al., 1996, Fawl & Roizman, 1993, Harwick et al., 1987, Openshaw et al., 1979,
Sawtell &
' 35 Thompson, 1992, Willey et al., 1984). In contrast, the murine explant
reactivation model is
exceptionally useful for studying the molecular mechanisms of HSV
reactivation, because
infectious virus can be efficiently recovered upon explantation and culture of
latently
infected sensory ganglia (reviewed in Fraser & Valyi-Nagy, 1993). Using this
model, we
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have shown that cellular IE factors oct-1, fos, jun and myc are induced at
early times
following explantation of latently infected trigeminal ganglia (TG) (Tal-
Singer et al., 1997,
Valyi-Nagy et al., 1991).
The present invention provides cellular factors with a putative role in the
reactivation process of Herpesviruses, particularly HSV-l, HSV-2, varicella
zoster virus
(herein "VZV"), Epstein Barr virus (herein "EBV") and human cytomegalovirus
(herein
"HCMV"). This invention was made, in part, using differential display RT-PCR
(DDRT-
PCR), which allows the visualization and subsequent isolation of cDNAs
corresponding to
mRNAs displaying altered expression in different cell populations (Liang et
al., 1995,
Liang & Pardee, 1992). Using this method, levels of gene expression in TG
populations
derived from various time points following explantation were compared. Thus,
any factors
modulated during the first 4 hours, the period in which viral gene expression
is first
detected (Devi-Rao et al., 1994, Tal-Singer et al., 1997), are believed to be
important in the
initial stimulation of latent viral genomes.
Certain previous studies have used DDRT-PCR to identify genes that are
involved
in neural stress and injury (Inokuchi et al., 1996, Kiryu et al., 1995, Qu et
al., 1996). For
example, Kiryu et al. (Kiryu et al., 1995) demonstrated differential
expression of the rat
neuronal glutamate transporter in axotomized hypoglossal motor neurons. Since
glutamate
transporter expression was induced in response to neuronal injury, it may be
involved in the
regeneration process. Others used DDRT-PCR to isolate a novel neuropeptide,
called
melanin-concentrating hormone, in the hypothalamic response to starvation (Qu
et al.,
1996). DDRT-PCR has also been used to identify cellular genes modulated by
SV40 and
EBV transformation (Sompayrac et al., 1996, Yan et al., 1996). However, it is
a novel
approach to study viral reactivation.
This invention demonstrates the isolation of forty eight differentially-
displayed
cDNAs representing transcripts whose levels were altered within the first 4
hours following
explantation of latently infected TG. Five cDNAs were identical to marine
TIS7, whose
sequence has been shown to be related to interferons (IFNs) (Skup et al.,
1982, Tirone &
Shooter, 1989, Varnum et al., 1989). Rapid induction of IFNs a and ~i in
neuronal cells of
TG explants was also detected. In addition, other factors, such as the
transcriptional
activator IRF-1 (Interferon Regulatory Factor -1 ), and the mitogen TNF- a
(tumor necrosis
factor -a), were induced by explantation. The Applicants believe that HSV
reactivation
involves the induction of a regulatory pathway shared with IFN-related genes
and herein
provide certain invention embodiments based on this important discovery.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows PCR differential-display of cDNA derived from latently-infected
mouse TG following explantation. Autoradiograph of radiolabeled DDRT-PCR
products.
Arrows denote PCR products representing (Left Panel) band #64 amplified with
3' primer
#2 and 5' primer #7, (Right Panel) band #56 amplified with 3' primer #2 and 5'
primer #3.
Figure 2 shows sequences of DDRT-PCR products. cDNAs isolated from
differential-display gels were reamplified using primers that included the T7
promoter
sequence, and PCR products sequenced. Sequences were analyzed by BLAST
searches.
(Panel A) BLAST output using band #56 as query sequence (P value=7.7 X 10 ~'9)
demonstrates sequence identity with marine TIS7 . (Panel B) Alignment of each
DDRT
band with TIS7 mRNA. Five cDNA bands corresponded to mouse IFN-related gene
TIS7
mRNA. Band #56 (primers:#2, #7), band #64 (primers #2,#3), band #116 (#6,#2),
and band
#125 (#6, #7), band #201 (#8, #2).
Figure 3 shows confirmation of differential display. RNA was prepared from
uninfected TG explants at 0, 1, 2, and 4 h p.e. Complementary DNA from
differentially
displayed band #56 was reamplified by PCR using 3' primers that included the
T7
promoter. PCR products were used as templates to prepare riboprobes labeled
with 'Z-P-
UTP, and added to each RNA sample. Following hybridization at 37°C
and RNase
digestion, samples were separated by PAGE. (Panel A) The input probe (P) and
protected
fragments were visualized using phosphorimager screens. (Panel B) The
intensity of each
protected fragment was quantitated using Imagequant software. Fold induction
was
expressed as the ratio between each band to the 0 time point. (Panel C)
Complementary
DNA from latently-infected TG explants at 0-24 hours post explantation was
subjected to
PCR using primers specific for TIS7 (TIS7A set). Products were separated on
2.5% agarose
gels stained with ethidium bromide, and visualized by fluorimager analysis.
Figure 4 shows detection of IFN-(3, IRF-1, IFNa(3R, and IRF-2 transcripts in
marine TG following explantation. RT-PCR was used to detect (Panel A) IFN-(3,
(Panel B)
IRF-1, (Panel C) IFNa(3R, (Panel D) IRF-2, and each was compared to the level
of
cyclophilin mRNA. Duplicate samples of TG explant RNA from 0, 1, 2, and 4 h
post-
explantation were analysed. Products were separated by agarose gel
electrophoresis,
followed by fluorimager scanning and analysis using Imagequant software. The
relative
amount of cDNA is expressed in these graphs in arbitrary units representing
the ratio
between the intensity of the PCR-product band to the intensity of cyclophilin.
The ratio at
the 0 time point is designated as 1. L indicates latent.
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Figure 5 shows immunostaining of interferon protein in TG following. Latently-
infected or uninfected BALB/c mice were sacrificed, TG were excised, and
incubated in
culture media for 0-24 h p.e. Paraffin-embedded sections were processed as
described in
Methods and reacted with rabbit polyclonal antisera against IFN a and (3. (A)
0 h p.e. of
latently infected TG, (B) 4 h, (C) 8 h, (D) 24 h., (E) 0 h p.e. of uninfected
TG, (F) 4 h p.e.
of uninfected TG. The experiment was repeated twice, and duplicate slides were
screened.
Figure 6 shows RT-PCR detection of TNF-a, cyclophilin, and ~i-actin
transcripts in
murine TG cultured for varying times post explantation. RNA from latently-
infected TG
explants was prepared and analyzed by RT-PCR for (Panel A) TNF-a, and (Panel
B) ~3-
actin, and cyclophilin, as described in Materials and Methods. Products were
visualized by
ethidium bromide staining as shown in the inserts. The graphs represent the
ratio between
the PCR product band and the cyclophilin band. The ratio at the time of
explantation (0)
was determined as 1. Experiments were done in duplicate in four separate
experiments.
Figure 7 shows the locations and sequences of IRF-1 consensus binding sites in
the
genome of HSV-I, with reference to the nucleotide numbering system of GENEMBL
entry
HE1CG.
Figure 8 shows that IRF-I binds specifically to HSV-I LAT DNA consensus
sequence. In vitro translated human IRF-1 was incubated with 32-P-labeled LAT
probe in
the presence or absence of antisera specific for IRF-1 or IRF-2. Samples were
analyzed by
electromobility shift assay (EMSA). Probe alone was used as control.
Figure 9 shows that IRF-2 binds specifically to LAT DNA. Competition
experiments were carried out with mutant LAT oligonucleotides.
Figure 10 shows that TIS7 and IRF-1 are induced by hyperthermia measured by
heat shock protein 70 (HSP70) induction. Groups of latently infected mice
(n=4) were
subjected to 10 minutes transient hyperthermia and sacrificed at I, 2, 6, 15,
and 24 hours
post-treatment. RNA was prepared from TG and subjected to RT-PCR. Products
were
analyzed as described in Figure 6. Samples were obtained from individual mice
represented
in each time point by colored bars Untreated mice were used as controls.
SUMMARY OF THE INVENTION
This invention provides cellular proteins from murine trigeminal ganglia
induced
by the stress of injury in viral infection and/or reactivation.
In accordance with another aspect of the present invention, there are provided
polynucleotides (DNA or RNA) which bind such polypeptides, or which alter the
biological
activity of such polypeptides.
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In particular the invention provides polynucleotides having the DNA sequences
given herein.
The invention also relates to novel oligonucleotides derived from the
sequences
given herein which can act as PCR primers in the process herein described to
determine, for
' exaple, whether or not the genes identified herein in whole or in part are
expressed in an
infected tissue or individual. It is recognised that such sequences will also
have utility in
diagnosis of the stage of infection and type of infection the pathogen has
attained. The
proteins so identified are also useful as targets in screens designed to
identify antiviral
compounds.
In accordance with yet a further aspect of the present invention, there is
provided
the use of a polypeptide and/or polynucleotide of the invention for
therapeutic or
prophylactic purposes, for example, as an antiviral agent or a vaccine.
In accordance with another aspect of the present invention, there is provided
the
use of a polynucleotide and/or polypeptide of the invention for therapeutic or
prophylactic
purposes.
In accordance with yei another aspect of the present invention, there are
provided
inhibitors or activators to such poiypeptides and/or polynucleotides, useful
as antiviral
agents. In particular, there are provided antibodies against such polypeptides
and
polypeptide-polynucleotide complexes.
Another aspect of the invention is a pharmaceutical composition comprising the
above polypeptide, polynucleotide, inhibitor or activator of the invention and
a
pharmaceutically acceptable carrier.
In a particular aspect the invention provides the use of an activator or
inhibitor of
the invention as an antiviral agent.
The invention further relates to the manufacture of a medicament for such
uses,
particularly a medicament to treat HSV-1 and/or HSV-2 infection or
reactivation.
Further provided, is a composition comprising IRF-1, particularly used as a
screening target for the development of prophylactic and therapeutic
compounds, such as
compounds that interfere with HSV (herein "HSV" means HSV-1 and/or HSV-2) or
VZV
reactivahon.
Also provided by the invention is a method for inhibiting the binding of IRF-1
to a
polynucleotide, such as a DNA element (for example, ISRE's), particulary DNA
comprising
or near a viral origins of replication.
A further aspect of the invention is a composition comprising an IRF-1: IRF-BP
complex.
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A method for screening for a compound capable of interfering with HSV
reactivation agonizing or antagonizing binding of IRF-1 to an inhibitor
factor, such as IRF-
BP.
Also provided are compounds capable of agonizing or antagonizing any compound
in IRF-1 and/or interferon genetic regulatory pathway. Preferred embodiments
provide
such compounds to treat HSV infection and/or reactivation (herein
"reactivation" means
viral reactivation from latency}.
The invention also provides compositions comprising IRF-1 binding site
consensus
sequences in HSV oriL and oriS and HSV long terminal repeats.
GLOSSARY OF TERMS
The following definitions are provided to facilitate understanding of certain
terms
used frequently herein.
"Factor(s) of the invention" refers, among others, to a polypeptide comprising
the
amino acid sequence any or a combination of the factors involved in viral
infection and/or
reactivation disclosed herein, such as IRF-l, TIS7, IFN-a and IFN-(3, or an
allelic variant
thereof; also included are polynucleotides encoding any of these polypeptides.
"Biological Activity of the Receptor" refers to the metabolic or physiologic
function of said Factors of the invention including similar activities or
improved activities
or these activities with decreased undesirable side-effects. Also included are
antigenic and
immunogenic activities of said Factors of the invention.
"Factor gene(s)" refers to a polynucleotide comprising the nucleotide sequence
encoding a Factor of the invention or allelic variants thereof and/or their
complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies,
chimeric, single chain, and humanized antibodies, as well as Fab fragments,
including the
products of an Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from the natural state. If an
"isolated" composition or substance occurs in nature, it has been changed or
removed from
its original environment, or both. For example, a polynucleotide or a
polypeptide naturally
present in a living animal is not "isolated," but the same polynucleotide or
polypeptide
separated from the coexisting materials of its natural state is "isolated", as
the term is
employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA. "Polynucleotides" include, without limitation single- and double-stranded
DNA,
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DNA that is a mixture of single- and double-stranded regions, single- and
double-stranded
RNA, and RNA that is mixture of single- and double-stranded regions, hybrid
molecules
comprising DNA and RNA that may be single-stranded or, more typically, double-
stranded
or a mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to
' triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term
polynucleotide also includes DNAs or RNAs containing one or more modified
bases and
DNAs or RNAs with backbones modified for stability or for other reasons.
"Modified"
bases include, for example, tritylated bases and unusual bases such as
inosine. A variety of
modifications has been made to DNA and RNA; thus, "polynucleotide" embraces
chemically, enzymatically or metabolically modified forms of polynucleotides
as typically
found in nature, as well as the chemical forms of DNA and RNA characteristic
of viruses
and cells. "Polynucleotide" also embraces relatively short polynucleotides,
often referred
to as oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino
acids
joined to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres.
"Polypeptide" refers to both short chains, commonly referred to as peptides,
oligopeptides
or oligomers, and to longer chains, generally referred to as proteins.
Polypeptides may
contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides"
include
amino acid sequences modified either by natural processes, such as
posttranslational
processing, or by chemical modification techniques which are well known in the
art. Such
modifications are well described in basic texts and in more detailed
monographs, as well as
in a voluminous research literature. Modifications can occur anywhere in a
polypeptide,
including the peptide backbone, the amino acid side-chains and the amino or
carboxyl
termini. It will be appreciated that the same type of modification may be
present in the
same or varying degrees at several sites in a given polypeptide. Also, a given
polypeptide
may contain many types of modifications. Polypeptides may be branched as a
result of
ubiquitination, and they may be cyclic, with or without branching. Cyclic,
branched and
branched cyclic polypeptides may result from posttranslation natural processes
or may be
made by synthetic methods. Modifications include acetylation, acylation, ADP-
ribosylation, amidation, covalent attachment of flavin, covalent attachment of
a heme
' 35 moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment
of a lipid or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking,
cyclization, disulfide bond formation, demethylation, formation of covalent
cross-links,
formation of cystine, formation of pyrogIutamate, formylation, gamma-
carboxylation,
glycosylation, GPI anchor formation, hydroxylation, iodination, methylation,
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myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids to
proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS
-
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and - Company, New York, 1993 and Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,
New York, 1983; Seifter et al., "Analysis for protein modifications and
nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein
Synthesis:
Posttranslational Modifications and Aging", Ann NYAcad Sci (1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that
differs
from a reference polynucleotide or polypeptide respectively, but retains
essential
properties. A typical variant of a polynucleotide differs in nucleotide
sequence from
another, reference polynucleotide. Changes in the nucleotide sequence of the
variant may
or may not alter the amino acid sequence of a polypeptide encoded by the
reference
polynucleotide. Nucleotide changes may result in amino acid substitutions,
additions,
deletions, fusions and truncations in the polypeptide encoded by the reference
sequence, as
discussed below. A typical variant of a polypeptide differs in amino acid
sequence from
another, reference polypeptide. Generally, differences are limited so that the
sequences of
the reference polypeptide and the variant are closely similar overall and, in
many regions,
identical. A variant and reference polypeptide may differ in amino acid
sequence by one or
more substitutions, additions, deletions in any combination. A substituted or
inserted
amino acid residue may or may not be one encoded by the genetic code. A
variant of a
polynucleotide or polypeptide may be a naturally occurring such as an allelic
variant, or it
may be a variant that is not known to occur naturally. Non-naturally occurring
variants of
polynucleotides and polypeptides may be made by mutagenesis techniques or by
direct
synthesis.
"Identity" is a measure of the identity of nucleotide sequences or amino acid
sequences. In general, the sequences are aligned so that the highest order
match is
obtained. "Identity" per se has an art-recognized meaning and can be
calculated using
published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk,
A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS
AND GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, 1993;
COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin,
H.G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR
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BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS
PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,
1991).
While there exist a number of methods to measure identity between two
polynucleotide or
polypeptide sequences, the term "identity" is well known to skilled artisans
(Carillo, H.,
' and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly
employed to
determine identity or similarity between two sequences include, but are not
limited to, those
disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press,
San Diego,
1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073.
Methods to
determine identity and similarity are codified in computer programs. Preferred
computer
program methods to determine identity and similarity between two sequences
include, but
are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids
Research
( 1984) 12( 1 ):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J Molec
Biol ( 1990)
215:403).
As an illustration, by a polynucleotide having a nucleotide sequence having at
least,
for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: I
is
intended that the nucleotide sequence of the polynucleotide is identical to
the reference
sequence except that the polynucleotide sequence may include up to five point
mutations
per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
In other
words, to obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a
reference nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may
be deleted or substituted with another nucleotide, or a number of nucleotides
up to 5% of
the total nucleotides in the reference sequence may be inserted into the
reference sequence.
These mutations of the reference sequence may occur at the 5 or 3 terminal
positions of
the reference nucleotide sequence or anywhere between those terminal
positions,
interspersed either individually among nucleotides in the reference sequence
or in one or
more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for
example, 95% "identity" to a reference amino acid sequence of is intended that
the amino
acid sequence of the polypeptide is identical to the reference sequence except
that the
polypeptide sequence may include up to five amino acid alterations per each
100 amino
acids of the reference amino acid sequence. In other words, to obtain a
polypeptide having
an amino acid sequence at least 95% identical to a reference amino acid
sequence, up to 5%
of the amino acid residues in the reference sequence may be deleted or
substituted with
another amino acid, or a number of amino acids up to 5% of the total amino
acid residues in
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the reference sequence may be inserted into the reference sequence. These
alterations of
the reference sequence may occur at the amino
or carboxy terminal positions of the reference amino acid sequence or anywhere
between
those terminal positions, interspersed either individually among residues in
the reference
sequence or in one or more contiguous groups within the reference sequence.
"Individual(s)" means a eukaryote, particularly a mammal, and especially a
human,
particularly one infected with a virus or believed to be infected by a virus.
"Plasmids" are designated by a lower case p preceded and/or followed by
capital
letters and/or numbers. The starting plasmids herein are either commercially
available,
publicly available on an unrestricted basis, or can be constructed from
available plasmids in
accord with published procedures. In addition, equivalent plasmids to those
described are
known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction
enzyme that acts only at certain sequences in the DNA. The various restriction
enzymes
used herein are commercially available and their reaction conditions,
cofactors and other
requirements were used as would be known to the ordinarily skilled- artisan.
For analytical
purposes, typically 1 ~tg of plasmid or DNA fragment is used with about 2
units of enzyme
in about 20 Irl of buffer solution. For the purpose of isolating DNA fragments
for plasmid
construction, typically 5 to 50 pg of DNA are digested with 20 to 250 units of
enzyme in a
larger volume. Appropriate buffers and substrate amounts for particular
restriction
enzymes are specified by the manufacturer. Incubation times of about 1 hour at
37 C are
ordinarily used, but may vary in accordance with the supplier's instructions.
After digestion
the reaction is electrophoresed directly on a polyacrylamide gel to isolate
the desired
fragment.
Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel described by Goeddel, D. et al., (1980) Nucleic Acids Res.,
8:4057.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or
two
complementary polydeoxynucleotide strands which may be chemically synthesized.
Such
synthetic oligonucleotides have no 5' phosphate and thus will not ligate to
another
oligonucleotide without adding a phosphate with an ATP in the presence of a
kinase. A
synthetic oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
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"Ligation" refers to the process of forming phosphodiester bonds between two
double stranded nucleic acid fragments (Maniatis, T., et al., supra., p. 146).
Unless
otherwise provided, ligation may be accomplished using known buffers and
conditions with
units to T4 DNA ligase ("ligase") per 0.5 Ng of approximately equimolar
amounts of the
° DNA fragments to be ligated.
10 A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that
functions as an autonomous unit of DNA replication in vivo; i.e., capable of
replication
under its own control.
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which
another
DNA segment may be attached so as to bring about the replication of the
attached segment.
1 S A "double-stranded DNA molecule" refers to the polymeric form of
deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a
double-stranded
helix, both relaxed and supercoiled. This term refers only to the primary and
secondary
structure of the molecule, and does not limit it to any particular tertiary
forms. Thus, this
term includes double-stranded DNA found, inter alia, in linear DNA molecules
(e.g.,
restriction fragments), viruses, plasmids, and chromosomes. In discussing the
structure of
particular double-stranded DNA molecules, sequences may be described herein
according
to the normal convention of giving only the sequence in the 5' to 3' direction
along the
nontranscribed strand of DNA (i.e., the strand having the sequence homologous
to the
mRNA).
A DNA "coding sequence of" or a "nucleotide sequence encoding" a particular
protein, is a DNA sequence which is transcribed and translated into a
polypeptide when
placed underxhe control of appropriate regulatory sequences.
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream (3'
direction) coding
sequence. For purposes of defining the present invention, the promoter
sequence is bound
at the 3' terminus by a translation start codon (e.g., ATG) of a coding
sequence and extends
upstream (5' direction) to include the minimum number of bases or elements
necessary to
initiate transcription at levels detectable above background. Within the
promoter sequence
will be found a transcription initiation site (conveniently defined by mapping
with nuclease
Sl), as well as protein binding domains (consensus sequences) responsible for
the binding
of RNA polymerase. Eukaryotic promoters will often, but not always, contain
"TATA"
boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences
in
addition to the -10 and -35 consensus sequences.
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DNA "control sequences" refers collectively to promoter sequences, ribosome
binding sites, polyadenylation signals, transcription termination sequences,
upstream
regulatory domains, enhancers, and the like, which collectively provide for
the expression
(i.e., the transcription and translation) of a coding sequence in a host cell.
A control sequence "directs the expression" of a coding sequence in a cell
when
RNA polymerase will bind the promoter sequence and transcribe the coding
sequence into
mRNA, which is then translated into the polypeptide encoded by the coding
sequence.
A "host cell" is a cell which has been transformed or transfected, or is
capable of
transformation or transfection by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenous DNA has
I S been introduced inside the cell membrane. Exogenous DNA may or may not be
integrated
(covalently linked) into chromosomal DNA making up the genome of the cell. In
prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an
episomal element, such as a plasmid. With respect to eukaryotic cells, a
stably transformed
or transfected cell is one in which the exogenous DNA has become integrated
into the
chromosome so that it is inherited by daughter cells through chromosome
replication. This
stability is demonstrated by the ability of the eukaryotic cell to establish
cell lines or clones
comprised of a population of daughter cell containing the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common
ancestor by
mitosis. A "cell line" is a clone of a primary cell that is capable of stable
growth in vitro
for many generations.
A "heterologous" region of a DNA construct is an identifiable segment of DNA
within or attached to another DNA molecule that is not found in association
with the other
molecule in nature.
DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered that cellular interferon-related genes are induced
in
tissues in response to certain stimuli as described herein. The tissue studied
is primarily
nerve tissue (ganglia) where HSV and other Herpesviruses establish latent
infections.
These stimuli also induce reactivation of HSV and other Herpesviruses from
latency into
active replication. Applicants also discovered that IRF-1, an important
cellular regulatory
protein, is induced by the same stimuli, and connected this regulatory factor
to HSV and
other Herpesviruses replication through the identification of IRF-1 binding
sites in the
DNA sequence of HSV and other Herpesviruses DNA.
It is believed that IRF-1, and/or the IRF-1 regulatory pathway, are essential
for
reactivation of HSV and other Herpesviruses from latency, and that
pharmacological
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interference with this pathway will interfere with HSV and other Herpesvirus
reactivation
resulting in arrest of clinical disease.
In view of this the invention provides various targets for screening for
therapeutic
compounds using the methods described herein. Such targets include, for
example, IRF-1.
A compound interfering with HSV and other Herpesvirus reactivation by
inhibiting the
binding of IRF-1 to viral DNA elements (ISRE's) rnay be found using such
screens and are
useful in antiviral therapy, especially against HSV and other Herpesviruses.
Another target, an IRF-1: IRF-BP complex, comprising IRF-1 and IRF-BP, can be
used to screen for compounds interfering with HSV reactivation by, among other
modes of
action, freezing or mimicking the binding of IRF-1 to it's inhibitor factor
IRF-BP. Such
compounds are useful in antiviral therapy, especially against HSV.
Other target include any element in the IRF-1 and interferon genetic
regulatory
pathway which subsequently impacts HSV reactivation from latency. These
compounds
are useful to screen for antiviral compounds. Such compounds are useful in
antiviral
therapy, especially against HSV.
Yet another target are polynucleotides derived from or comrpising an IRF-1
binding site consensus sequences in HSV LAT/ICPO, oriL and oriS play a
significant role
in HSV replication. A preferred embodiment of this sequence is set forth in
SEQ ID NO:1.
Such compounds are useful in antiviral therpy, especially against HSV.
To demonstrate this reactivation, marine trigeminal ganglia explants by
differential
display RT-PCR (DDRT) were analyzed. Five of the DDRT hits mapped to marine
TIS7,
an interferon-related cellular Immediate Eariy gene which is activated in
several cell types
in response to stress (Guardavaccaro et al., 1995, Herschman et al., 1994,
Tirone &
Shooter, 1989, Varnum et al., 1989, Varnum et al., 1994). Using RT-PCR and in-
situ
hybridization it was shown that other IFN-related gene transcripts, including
IFN-[3, and the
interferon regulatory factor-1 (IRF-1) were induced following explantation of
marine
trigeminal ganglia (Tal-Singer et al. 1998).
IRF-1 is a transcriptional activator and tumor supressor protein which
regulates
interferon genes transcription such as IL-1 beta (via Interleukin Converting
Enzyme (ICE)
pathways) (Tamara et al., 1996), and MHC-I expression (Drew er al., 1995,
Hobart et al.,
1997). It is activated by interferons and the STATI pathway (Pine et al.,
1994). It binds to
cellular proteins such as TFIIB, and NFxB (Drew et al., 1995, Wang et al.,
1996). Its DNA
binding and transcriptional activation functions are inhibited when it is
bound to IRF-BP
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protein (Kondo, 1996). Its known functions involve the inflammatory response
and
regulation of the cell cycle via induction of p21 (Tanaka et al., 1996).
Other target include any element in the IRF-I and interferon genetic
regulatory
pathway which subsequently impacts HSV reactivation from latency. These
compounds
are useful to screen for antiviral compounds. Such compounds are useful in
antiviral
therapy, especially against HSV.
Yet another target are polynucleotides derived from or comrpising an IRF-1
binding site consensus sequences in HSV LAT/ICPO, oriL and oriS which play a
significant
role in HSV replication. A preferred embodiment of this sequence is set forth
in SEQ ID
NO:1. Such compounds are useful in antivirat therpy, especially against HSV.
To demonstrate this reactivation, murine trigeminal ganglia explants were
analyzed
by differential display RT-PCR (DDRT). Five of the DDRT hits mapped to murine
TIS7,
an interferon-related cellular Immediate Early gene which is activated in
several cell types
in response to stress (Guardavaccaro et al., 1995, Herschman et al., 1994,
Tirone &
Shooter, 1989, Varnum et al., 1989, Varnum et al., 1994). Using RT-PCR and
immunostaining it was shown that other IFN-related gene transcripts, including
IFN-beta,
and the interferon regulatory factor-1 (IRF-1) were induced following
explantation of
murine trigeminal ganglia.
IRF-I is a transcriptional activator and tumor supressor protein which
regulates
interferon genes transcription such as IL-1 beta (via Interleukin Converting
Enzyme (ICE)
pathways) (Tamura et al., 1996), and MHC-I expression (Drew et al., 1995,
Hobart et al.,
1997)]. It is activated by interferons and the STAT1 pathway (Pine et al.,
1994). It binds to
cellular proteins such as TFIIB, and NFkB (Drew et al., 1995, Wang et al.,
1996). Its DNA
binding and transcriptional activation functions are inhibited when it is
bound to IRF-BP
protein (Kondo, 1996). Its known functions involve the inflammatory response
and
regulation of the cell cycle via induction of p21 (Tanaka et al., 1996).
IRF-1 is a member of a multigene family which recognizes the same promoter
consensus sequence (herein referred to as "ISRE" or "ICS"). Sequence analysis
using
Findpatterns function of the GCG software (Genetics Computer Group, Madison,
Wisconsin). HSV-1 complete genome sequence (Genbank locus HE1CG accession
# X14112) was searched for the core consensus IRF-1 site in interferon
promoters
using the hexamer AAGTGA (Tanaka & Taniguchi, 1992). Sequence matches are
listed in Table 1 by sequence location, and gene name. Ten identical matches
were
found in in HSV-1 strain 17+ sequence (see Figure 7). A strong consensus
identical to
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WO 99/01464 PCT/US98/13733
sequences in IFN promoters was present in the latency associated transcript
(LAT) genome
location which overlaps with the ICPO transcript. Both LAT and ICPO are
important for
reactivation from latency. The applicants found that in vitro translated IRF-1
and IRF-2 can
bind probes derived from this region specifically (Figures 8, 9).Two weaker
matches
mapped to the palindrome in oriL, and one mapped to oriS. These regions
matched to
origin binding protein (OBP or UL9) Binding Site III, which are conserved in
HSV-1 and
HSV-2 strains, as shown in the sequence below [SEQ ID NO: l ].
5' AAAAAAAGTGA,~AA~~CGAAGCGTTCGCACTTTGTCCTAATAATATA-
SITE III
TATATTATTAGGACAAAGTGCGAACGCTT~~T TCACTTTTTTT3'
SITE III
These consensus sites are within regions previously identified as origin
binding
protein (OBP or UL9) Binding Site III. Also, of great interest are previous
observations that, in addition to binding OBP, Binding Site III interacts with
yet
unknown cellular proteins (Dabrowski et al., 1991; Dabrowski et al., 1994).
Taken
together, these observations suggest that IRF-1 may bind to regulatory
elements in
the viral genome such as the' origins of replication, and ICPO/LAT, and
thereby
upregulate viral replication or gene expression. Interestingly, functional IRF
binding
sites recently were identified in the herpesvirus Epstein-Barr virus (EBV)
(Schaffer
et al., 1997; Nonkwelo et al., 1997). IRF-1 and IRF-2 bind directly to
consensus
sequence sites in the EBV Type I latency promoter of EBNA-1 (Qp), and IRF
factors
may play a primary role in transcriptional regulation of EBNA-1 in cell
culture
(Schaffer et al., 1997; Nonkwelo et al., 1997).
Mobility shift assays using probes specific for this region in oriS and oriL
(Dabrowski et al., 1994, Dabrowski & Schaffer, 1991), indicated that certain
cellular
proteins bind to this region. A single base mutation in this consensus
sequence
affected the rate of DNA replication at late times following infection of
cultured
cells. We have been unable to show that in-vitro translated human IRF-1 can
bind
probes from this region. However, it does not rule out the possibility that
IRF-1
bound to other viral or cellular factors can bind at the origin of replication
in its
latent conformation.
IS
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Further sequence analysis of other Herpesviruses genomes revealed other
IRF-1 consensus sequences. These consensus sequences provided by the invention
include, for example, the following sequences in Table 1. Highly conserved
nucleotides surrounding the core are bold and underlined.
Table 1
IRF-1 consensus sites in HSV-1 Strain 17+ by Pattern
Searching using the AAGTGA IRF-1 Core consensus
5,588: GGGAA AAGTGA AAGAC Near the 3' End of LAT
13,654: GACGT AAGTGA CGTCG In UL5 helicase/primase
62,434: AAAAA AAGTGA GAACG Ori L
84,865: CCGCC AAGTGA TCCTG UL38 (VP19C, binds DNA)
131,958: AAAAG AAGTGA GAACG Ori S
TCACTT IRF-1 rev Core consensus
45,524: CACCA TCACTT CCACC Within gH
62,512: CGTTC TCACTT TTTTT Ori L
85,468: GGTAT TCACTT ACCGC UL38 (VP19C)
120,778: GTCTT TCACTT TTCCC ICPO/LAT
146,269: CGTTC TCACTT CTTTT Ori S
Human Cytomegalovirus: Hehcmvcg.Gb_vi
1 AAGTGA
35,131: GCCG_A
AAGTGA
A_AGTA
52,719: GAAAG AAGTGA A_ACCC
53,842: CGCGC AAGTGA A_GCCG
56,326: GTTAA AAGTGA TTTTT
59,418: _ AAGTGA GTCTA
CAATA
81,191: CAAAT AAGTGA TAATG
95,257: AAA_AGAAGTGA TA
CAA Auxilliary
_
replication region
94860-95670
121,335: GGCTA AAGTGA CAGGA
128,147: CTTCG AAGTGA ATATT
155,734: GCAGA AAGTGA TGTGG
158,557: ATCCT AAGTGA GGTGA
158,891: CTACA_AAGTGA A_GAAT
163,229: AGCCT AAGTGA CGGTG
1 /Rev TCACTT
15,215: GTGTG TCACTT GTTGC
15,695: CAACT TCACTT CAAAC
16,798: AAGAT TCACTT AAAGC
50,301: TATAA TCACTT TCCGC
70,132: ACGTA TCACTT TCACG
87,029: GGCCA TCACTT CGGGG
97,452: TCGCA TCACTT AAGAA
130,301: ATCGA TCACTT _TTTTC
139,862: CGACG TCACTT TGAGC
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WO 99/01464 PCT/US98/13733
157,414: AAAG_T TCACTT ATTCT
169,360: GTT_TT TCACTT ACTGA
171,562: AGAAC TCACTT AAGAG
174,668: AAAAA TCACTT _TTGGA
175,038: GTCG_T TCACTT _TGCCG
184,562: GTTAA TCACTT _TAAGT
209,216: GGATC TCACTT ACCGC
224,280: GCCGT TCACTT TTCCG
Varicella Zoster Virus: Vi
Hevzvxx.Gb_
1 AAGTGA
3,179: TCCAAAAGTGA CTTCG
5,514: AAGACAAGTGA A_CCCT
9,053: GCTGA_AAGTGA A_GTGGORF7
15,581: AAGG_AAAGTGA G_ACCGUpsteram of ORF12
25,015: CATACAAGTGA CA_ACC
27,340: ATGCCAAGTGA TCGGT
27,744: GGTTGAAGTGA A_TGCC
36,842: ATTTTAAGTGA TCCAA
38,369: ACGTTAAGTGA ACTTA
42,071: TATCA_AAGTGA TTTTA Upstream of ORF 23
47,587: CTGTTAAGTGA_GCCAA
58,021: TTTATAAGTGA AACAA ORF31
71,172: TTGCAAAGTGA GGTTA
87,744: ACCCAAAGTGA A End of ORF50
CATC
104,738: _ AAGTGA _
CAGCT CATTT
115,777: GTTTTAAGTGA CTATA
121,897: GTGGGAAGTGA A_ACTAORF 71 Downstrem
of
ORI 119547-119810
1 /Rev TCACTT
3S 4,271: AGT_TTTCACTT _TCCCC
43,348: TAATGTCACTT TGCAT
44,942: AGT_TGTCACTT _TTAGC
67,385: AAC_TTTCACTT CTTTT
70,980: CTACATCACTT _TCGAC
73,891: GCCC_TTCACTT _TGTGG
75,664: GAAG_TTCACTT AGCCC
82,708: TTCA_TTCACTT _TGGTCPromoter for ORF46
97,452: AGACATCACTT ACGTG
101,928: TATAATCACTT TTCTT
104,274: TTGTATCACTT ACGAT
112,936: TCGGATCACTT _TATAAUnknown region
Upstream of ORF66
Epstein Barr Virus: Ebv.Gb Vi
1 AAGTGA
6,198: AAGCA AAGTGA A_GGGC Upstream of ORI P the
latency origin (7315-~9312)EBRI promoter (PolIII
transcript associated with SNRPs
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WO 99/01464 PCT/US98/13733
17,640: GACCG AAGTGA AGGCC Bam HI W/W' repeats
near TATAAAG for EBNA LP
(latent protein)
20,712: GACCG AAGTGA A_GGCC
23,784: GACCG AAGTGA A
GGCC
26,856: GACCG AAGTGA _
A_GGCC
29,928: GACCG AAGTGA A_GGCC
33,000: GACCG AAGTGA A_GGCC
36,072: GACCG AAGTGA A_GGCC
39,144: GACCG AAGTGA A_GGCC
42,216: GACCG AAGTGA A_GGCC
45,288: GACCG AAGTGA A_GGCCEnd of Bam HI W/W'
repeat
58,005: TGTAA_AAGTGA A_GCTG924 by downstream
of
BFL2 promoter
TATAAAG
74,427: CTGGA AAGTGA CTCGG
81,082: CAAGA_AAGTGA _AGCAG250 by downstream
of
BMRF2
93,389: GAAGA_ AAGTGA GGACA
149,877: AGCAC AAGTGA TTAGG
1 /Rev TCACTT
179: CCCTC TCACTT CTACT
267: GGTTG TCACTT GTGAG
335: ACAG_T TCACTT CCTCT
5,558: ACCCC TCACTT _TGTAC
9,637: ATAAA TCACTT CCCTA
11,783: CGGGG TCACTT CCCCT
11,845: TGGTG TCACTT CCGCA
53,582: ATGCA TCACTT _TGAGC
97,786: GACCA TCACTT AAGTT
107,114: GATAA TCACTT _TTATC
150,593: TAGGA TCACTT TCATA
In view of Applicants findings provided herein, Applicants believe that
members of
the IRF-1 family are involved in regulation of HSV and other Herpesviruses
replication or
transcription in host cells.
Applicants have also used, for example, DDRT-PCR to identify genes
differentially
expressed following the stress caused by explantation of latently-infected
murine TG. Mice
were infected by the corneal route with HSV-l, and at 4 weeks post infection,
mice were
sacrificed and the TG were explanted. RNA was prepared from TG at various
times
following explantation, followed by DDRT-PCR.
TIS7 is induced by the stress of explantation. Five differentially displayed
bands
were identified as overlapping regions of murine TIS7 (Lim et al., 1987,
Varnum et al.,
1989) (Figure 2) and the results were confirmed by quantitative RPA and RT-PCR
(Figure
3). The TIS (TPA-inducible sequences) family members are early response genes
(Walz et
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WO 99/01464 PCT/US98/13733
al., 1976) which are induced rapidly and transiently in Swiss 3T3 cells by the
tumor
promoter mitogen tetradecanoyl phorbol acetate (TPA) (Lim et al., 1987), or by
serum
(Herschman, 1991). Most of the TIS genes also have been identified in rat PC12
cells,
following induction with NGF, TPA, epidermal growth factor and depolarization
(Kujubu
et al., 1987). Moreover, TIS induction has been detected in primary astrocyte
cultures
following mitogen induction (Arenalder et al., 1989). Thus, a family of TIS
genes appear to
constitute a common pathway or response to many cell stimulatory agents or
physical
stimuli.
The pattern of induction previously observed for TIS7/PC4 genes in these
systems
revealed an increase in the levels of RNA or protein between 2-4 hours post
stimulus
(Varnum et al., 1994), similar to our observation in the mouse explant model
(Figure 2).
Moreover, Applicants have shown that another TIS transcript, TIS28 or c-fos,
was induced
rapidly following explantation (Tal-Singer et al., 1997, Valyi-Nagy et al.,
1991), and again,
the kinetics match those previously observed following mitogen induction
(Arenalder et al.,
1989). These observations suggest that explantation and mitogen stimulation
induce
similar cellular early response pathways which either activate or induce TIS
genes.
Applicants believed that these pathways also may be among the earliest events
involved in
the induction of latent herpesvirus and demonstarted this in the present
invention. It has
been reported that rat PC4 (highly related to marine TIS7) and c-fos are both
induced by
activation of the oncogenenic protein-tyrosine kinase v fps, encoded in the
retrovirus
Fujinami sarcoma virus (Jahner & Hunter, 1991 ). It would be of interest to
determine
whether a similar kinase, either of viral or cellular origin, is activated in
explanted ganglia
or in other HSV reactivation systems.
Interferon ~i is induced by the stress of explantation. TIS7 was originally
identified as a gene induced in marine 3T3 cells following infection with
Newcastle disease
virus (Skup et al., 1982). Nucleotide sequence analysis revealed some
conservation with
human IFN-~i and rat IFN-y (Tirone & Shooter, 1989). The present invention
provides
that, like TIS7, IFN-a and IFN-(3 are induced in neuronal cells within the
first hour
following explantation. These observations indicate that these interferon
related genes,
although different in function, share a common cellular pathway that is
putatively involved
in the early events of HSV reactivation.
IFN-(3 expression is modulated at the transcriptional level by multiple
regulatory
factors that bind upstream of the initiation site, such as the activators IRF-
1 and NFkB, and
the repressor IRF-2 (reviewed in Tanaka & Taniguchi, 1992). Applicants showed
that IRF-
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WO 99/01464 PCT/US98/13733
1 was induced by the stress of explantation. In contrast, neither IRF-2 nor
the IFN receptor
were induced (Figure 4). Since the induction of IRF-1 followed the same
temporal pattern
as that of IFN, IFN- alpha and beta are believed to be induced via an IRF-1
dependent
pathway in explanted TG cells (Kawakami et al., 1995). Induction of IFN
expression has
previously been observed following HSV infection (Gobi et al., 1988b, Green et
al., 1991,
Stanwick et al., 1982a). However, Applicants detected induction of IFN at 1-2
hours, prior
to viral gene induction, which was detected at 2-4 h p.e. (Tal-Singer et al.,
1997).
Furthermore, IFN induction was detected in neurons of both latently-infected
and
uninfected TG. IFN-b and IRF-1 were induced in the TG in the absence of serum
in the
explantation media. Taken together, these data indicate that IFN induction is
a consequence
of the stress of explantation, and did not result from viral gene expression,
or from
incubation in the presence of serum factors.
It has been suggested that, during HSV infection, transcription of IFN-b is
induced
by complex formation between cellular Oct-1 and viral VP16, and the
interaction of this
complex with Oct-1 binding sites in the IFN promoter (Leblanc & Hiscott,
1992).
Consistent with this, Applicants have shown previously that Oct-1 is induced
in neuronal
cells within 1 hour p.e. (Tal-Singer et al., 1997, Valyi-Nagy et al., 1991 ).
However, IFN
induction occurred even in the absence of latent virus. Thus, Applicants
believe that
cellular factors that are functionally analogous to the viral late gene and
virion tegument
component VP16 are induced in TG explants. A precedent for this is the
identification of a
cellular Oct-I-binding protein in lymphocytes (called either Bob-1, OCAB, or
OCBP), that
appears to be required for the tissue-specific induction of immunoglobulin
gene promoters
(Gstaiger et al., 1995, Luo & Roeder, 1995, Strubin et al., 1995). Thus,
Applicnats believe
that neuronal-specific Oct-1 binding proteins are induced in explanted
ganglia. This gene-
activation pathway could be shared by IFN and viral promoters since both
contain Oct-I
binding consensus sequences (MacDonald et al., 1990, O~iare & Goding, 1988).
Relationship between reactivating HSV and IFNs. IFN is known to possess
antiviral properties, and indeed is induced by HSV infection (Gobi et al.,
1988a, Green et
al., 1981, Stanwick et al., 1982b). Several mechanisms have been elucidated
for specific
effects of IFN on HSV. For example, IFN is an inhibitor of activation of HSV
immediate
early (IE) genes in vitro by VP16 (LaMarco & McKnight, 1989). Applicants
recently found
induction of both Early and IE HSV genes during the first hours of viral
reactivation (Tal-
Singer et al., I997). The absence of viral IE gene expression prior to Early
gene expression
CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
during the first hours following explantation of TG may be a result of the
inhibitory effect
of IFN on a neuron-specific VP16 homologue.
Furthermore, IFN blocks both HSV morphogenesis and release of viral particles
from infected cells (Chatterjee et al., 1985). Therefore, an interesting
relationship between
IFN and reactivating HSV may involve one round of viral replication, allowing
the virus to
travel from TG neurons to the site of recrudescence in corneal epithelium.
Immediate
induction of IFN may prevent the spread of reactivating virus within the
nervous system by
inhibiting release of viral particles and activating host defense mechanisms
such as natural
killer cells (Habu et al., 1984). Moreover, in another virus system,
neuroblastoma cells
expressing high levels of IFN-b support persistent rabies virus infections
(Honda et al.,
1985). This suggests that the IFN response may be involved in ensuring the
viability of
infected host cells.
In a different scenario, IFN may inhibit viral reactivation in neurons and, in
a few
cells, other cellular factors override its effects by inducing high levels of
viral gene
expression. For example, Walev et al. (Walev et al., 1995) have shown that
treatment of TG
explants with soluble IFN inhibits reactivation, detected by reduction of
infectious virus in
the presence of IFN. In contrast, TNF-a treatment induced the efficiency of
reactivation in
that study. These data support the hypothesis that induction of IFN inhibits
multiple rounds
of viral replication in neuronal cells of the TG. Consistent with this
scenario, we detected
induction of TNF-a transcription in TG explants (Figure b) under conditions
leading to
viral reactivation. Thus, it is possible that the levels of TNF-a in few
neurons (1% of latent
neurons) are higher than the levels of IFN, allowing the latent viral genome
in those
neurons to reactivate.
The relationship of TIS7, IFN, and viral responses. TIS7 and interferons are
induced by viral infection (Skup et al., 1982, Tanaka & Taniguchi, 1992) and,
as shown in
this study, by the stress of explantation. The precise function of TIS7 in the
cell is yet to be
elucidated, it is however clear that it plays a role in cellular growth and
differentiation
(Guardavaccaro et al., 1995, Herschman et al., 1994). Furthermore, it is known
that TIS7
has no antiviral activity (Tirone & Shooter, 1989) which is a property of
interferons
{Tanaka & Taniguchi, 1992). Applicants performed studies to determine whether
these
cellular components share a common induction pathway with HSV. Applicnts
believe that
that TIS, interferon family members, and HSV share a common regulatory element
such as
IRF-1, and that HSV-1 has evolved reactivation strategies which take advantage
of these
cellular stress-induced activation pathways.
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WO 99/01464 PCT/US98/13733
Using DDRT-PCR several genes involved in the cellular response to the stress
of
explantation were identified. These results also yielded insights into the
cellular
environment which is present during HSV reactivation. Therefore, PCR
differential-
display represents an excellent method to screen for species of RNA which are
transcriptionally regulated in TG following explantation. Genes identified
using this
IO method can be studied in other HSV reactivation systems, resulting in a
database of specific
genes which may be involved in the reactivation process.
Also described by the invention are differential display results showing that
TIS7
was found in 5 separate bands, while only one other band was found more than
one time,
thus prompting the focus on TIS7.
IS TIS7 induction was shown in neurons at the protein level, using
immunohistochemistry of TG sections.
Provided herein, to better describe the invention, are putative IRF-1 binding
in the
viral genome, Figure 7 (there were significant matches to the IRF-1 consensus
binding
site). The disclosure of these sites in no way limits the manner in which
cellular factors
20 interact with virus sequences during reactivation. Applicants have also
demonstrated that
IRF-1 binding sites occur in potentially critical regions of the viral genome,
and thus are
believed to be crucial in the reactivation process.
The mechanisms provided herein by which the Factors of the invention function
in
viral infection and latency in no way limit the scope of the invention. These
mechanisms
25 are provided to clarify the invention and certain bases for the invention.
Identified herein are cellular genes induced in response to stimuli that
reactivate
virus and methods for screening compounds to treat disease based on this
observation.
Therapies based on this observation are also provided. The invention provides
that these
genes are potential causative agents in reactivation, but this model in no way
limit the
30 scope of the invention.
Certain of the polynucleotides of the invention, such as IRF-I, TIS7,
interfereon
alpha, and interfereon beta, are well known in the art, and references
disclosing their
sequences are provided herein. Each of the references provided herein are
incorporated by
reference herein in their entirety.
35 Any polynucleotide of the present invention may be in the form of RNA or in
the
form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA
may be double-stranded or single-stranded, and if single stranded may be the
coding strand
or non-coding (anti-sense) strand. The coding sequence which encodes the
polypeptide
may be identical to the coding sequence shown or may be a different coding
sequence
22
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which coding sequence, as a result of the redundancy or degeneracy of the
genetic code,
encoding the same polypeptide.
The present invention includes variants of the hereinabove described
polynucleotides which encode fragments, analogues and derivatives of the
polypeptide
characterized by the deduced amino acid sequence given herein. The variant of
the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a non-
naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same
polypeptide characterized by the deduced amino acid sequence given herein as
well as
variants of such polynucleotides which variants encode for a fragment,
derivative or
analogue of the polypeptide. Such nucleotide variants include deletion
variants,
substitution variants and addition or insertion variants.
The polynucleotide may have a coding sequence which is a naturally occurring
allelic variant of the coding sequence characterized by the DNA sequence
disclosed herein.
As known in the art, an allelic variant is an alternate form of a
polynucleotide sequence
which may have a substitution, deletion or addition of one or more
nucleotides, which does
not substantially alter the function of the encoded polypeptide.
The polynucleotide which encodes for the mature polypeptide, may include only
the coding sequence for the mature polypeptide or the coding sequence for the
mature
polypeptide and additional coding sequence such as a leader or secretory
sequence or a
proprotein sequence.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequence for the polypeptide as well
as a
polynucleotide which includes additional coding and/or non-coding sequence.
The present invention therefore includes polynucleotides, wherein the coding
sequence for the mature polypeptide may be fused in the same reading frame to
a
polynucleotide sequence which aids in expression and secretion of a
polypeptide from a
host cell, for example, a leader sequence which functions as a secretory
sequence for
controlling transport of a polypeptide from the cell. The polypeptide having a
leader
sequence is a preprotein and may have the leader sequence cleaved by the host
cell to form
the mature form of the polypeptide. The polynucleotides may also encode for a
proprotein
which is the mature protein plus additional 5' amino acid residues. A mature
protein having
a prosequence is a proprotein and may be an inactive form of the protein. Once
the
prosequence is cleaved an active mature protein remains.
23
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WO 99/01464 PCT/US98/13733
Thus, for example, the polynucleotide of the present invention may code for a
mature protein, or for a protein having a prosequence or for a protein having
both a
prosequence and a presequence (leader sequence). Further, the amino acid
sequences
provided herein show a methionine residue at the NHz terminus. It is
appreciated, however,
that during post-translational modification of the peptide, this residue may
be deleted.
Accordingly, this invention contemplates the use of both the methionine-
containing and the
methionineless amino terminal variants of each protein disclosed herein.
The polynucleotides of the present invention may also have the coding sequence
fused in frame to a marker sequence at either the 5' or 3' terminus of the
gene which allows
for purification of the polypeptide of the present invention. The marker
sequence may be a
hexa-histidine tag supplied by the pQE series of vectors (supplied
commercially by
Quiagen Inc.) to provide for purification of the polypeptide fused to the
marker in the case
of a bacterial host.
The present invention further relates to polynucleotides which hybridize to
the
hereinabove-described sequences if there is at least 50% and preferably at
least 70%
identity between the sequences. The present invention particularly relates to
Streptococcal
polynucleotides which hybridize under stringent conditions to the hereinabove-
described
polynucleotides . As herein used, the term "stringent conditions" means
hybridization will
occur only if there is at least 95% and preferably at least 97% identity
between the
sequences. The polynucleotides which hybridize to the hereinabove described
polynucleotides in a preferred embodiment encode polypeptides which retain
substantially
the same biological function or activity as the polypeptide characterised by
the deduced
amino acid sequence given herein.
The terms "fragment," "derivative" and "analogue" when referring to the
polypeptide characterized by the deduced amino acid sequence herein, means a
polypeptide
which retains essentially the same biological function or activity as such
polypeptide.
Thus, an analogue includes a proprotein which can be activated by cleavage of
the
proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a
natural polypeptide or a synthetic polypeptide, preferably a recombinant
polypeptide.
The fragment, derivative or analogue of the polypeptide characterized by the
deduced amino acid sequence herein may be (i) one in which one or more of the
amino acid
residues are substituted with a conserved or non-conserved amino acid residue
(preferably a
conserved amino acid residue) and such substituted amino acid residue may or
may not be
one encoded by the genetic code, or (ii) one in which one or more of the amino
acid
24
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WO 99/01464 PCT/US98/13733
residues includes a substituent group, or (iii) one in which the polypeptide
is fused with
another compound, such as a compound to increase the half life of the
polypeptide (for
example, polyethylene glycol), or (iv) one in which the additional amino acids
are fused to
the polypeptide, such as a leader or secretory sequence or a sequence which is
employed for
purification of the polypeptide or a proprotein sequence. Such fragments,
derivatives and
analogues are deemed to be within the scope of those skilled in the art from
the teachings
herein.
The polypeptides and polynucleotides of the present invention are preferably
provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original
IS environment (e.g., the natural environment if it is naturally occurring).
For example, a
naturally-occurring polynucleotide or polypeptide present in a living animal
is not isolated,
but the same polynucleotide or polypeptide, separated from some or all of the
coexisting
materials in the natural system, is isolated. Such polynucleotides could be
part of a vector
and/or such polynucleotides or polypeptides could be part of a composition,
and still be
isolated in that such vector or composition is not part of its natural
environment.
The present invention also relates to vectors which include polynucleotides of
the
present invention, host cells which are genetically engineered with vectors of
the invention
and the production of polypeptides of the invention by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is
therefore
provided a process for producing the polypeptide of the invention by
recombinant
techniques by expressing a polynucleotide encoding said polypeptide in a host
and
recovering the expressed product. Alternatively, the polypeptides of the
invention can be
synthetically produced by conventional peptide synthesizers.
Host cells are genetically engineered (transduced or transformed or
transfected)
with the vectors of this invention which may be, for example, a cloning vector
or an
expression vector. The vector may be, for example, in the form of a plasmid, a
cosmid, a
phage, etc. The engineered host cells can be cultured in conventional nutrient
media
modified as appropriate for activating promoters, selecting transformants or
amplifying the
genes. The culture conditions, such as temperature, pH and the like, are those
previously
used with the host cell selected for expression, and will be apparent to the
ordinarily skilled
artisan.
Suitable expression vectors include chromosomal, nonchromosomal and synthetic
DNA sequences, e.g., bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors
CA 02295933 1999-12-23
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derived from combinations of plasmids and phage DNA. However, any other vector
may
be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of
procedures. In general, the DNA sequence is inserted into an appropriate
restriction
endonuclease sites) by procedures known in the art.
The DNA sequence in the expression vector is operatively linked to an
appropriate
expression control sequences) (promoter) to direct mRNA synthesis. As
representative
examples of such promoters, there may be mentioned: LTR or SV40 promoter, the
E. coli.
lac or trp, the phage lambda PL promoter and other promoters known to control
expression
of genes in eukaryotic or prokaryotic cells or their viruses. The expression
vector also
contains a ribosome binding site for translation initiation and a
transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable
marker genes to provide a phenotypic trait for selection of transformed host
cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or
such as
tetracycline or ampicillin resistance in E. coli.
The gene can be placed under the control of a promoter, ribosome binding site
(for
bacterial expression) and, optionally, an operator (collectively referred to
herein as
"control" elements), so that the DNA sequence encoding the desired protein is
transcribed
into RNA in the host cell transformed by a vector containing this expression
construction.
The coding sequence may or may not contain a signal peptide or leader
sequence. The
polypeptides of the present invention can be expressed using, for example, the
E. coli tac
promoter or the protein A gene (spa) promoter and signal sequence. Leader
sequences can
be removed by the bacterial host in post-translational processing. See, e.g.,
U.S. Patent
Nos. 4,431,739; 4,425,437; 4,338,397. Promoter regions can be selected from
any desired
gene using CAT (chloramphenicol transferase) vectors or other vectors with
selectable
markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named
bacterial
promoters include lacI, lack T3, T7, gpt, lambda PR, PL and trp. Eukaryotic
promoters
include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs
from
retrovirus, and mouse metallothionein-I. Selection of the appropriate vector
and promoter is
well within the level of ordinary skill in the art.
In addition to control sequences, it may be desirable to add regulatory
sequences
which allow for regulation of the expression of the protein sequences relative
to the growth
of the host cell. Regulatory sequences are known to those of skill in the art,
and examples
include those which cause the expression of a gene to be turned on or off in
response to a
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WO 99/01464 PCTNS98113733
chemical or physical stimulus, including the presence of a regulatory
compound. Other
types of regulatory elements may also be present in the vector, for example,
enhancer
sequences.
An expression vector is constructed so that the particular coding sequence is
located in the vector with the appropriate regulatory sequences, the
positioning and
orientation of the coding sequence with respect to the control sequences being
such that the
coding sequence is transcribed under the "control" of the control sequences
(i.e., RNA
polymerise which binds to the DNA molecule at the control sequences
transcribes the
coding sequence). Modification of the coding sequences may be desirable to
achieve this
end. For example, in some cases it may be necessary to modify the sequence so
that it may
be attached to the control sequences with the appropriate orientation; i.e.,
to maintain the
reading frame. The control sequences and other regulatory sequences may be
ligated to the
coding sequence prior to insertion into a vector, such as the cloning vectors
described
above. Alternatively, the coding sequence can be cloned directly into an
expression vector
which already contains the control sequences and an appropriate restriction
site.
Generally, recombinant expression vectors will include origins of replication
and
selectable markers permitting transformation of the host cell, e.g., the
ampicillin resistance
gene of E. coli and S. cerevisiae TRPI gene, and a promoter derived from a
highly-
expressed gene to direct transcription of a downstream structural sequence.
The
heterologous structural sequence is assembled in appropriate phase with
translation
initiation and termination sequences, and preferably, a leader sequence
capable of directing
secretion of translated protein into the periplasmic space or extracellular
medium.
Optionally, the heterologous sequence can encode a fusion protein including an
N-terminal
identification peptide imparting desired characteristics, e.g., stabilization
or simplified
purification of expressed recombinant product.
The vector containing the appropriate DNA sequence as hereinabove described,
as
well as an appropriate promoter or control sequence, may be employed to
transform an
appropriate host to permit the host to express the protein.
More particularly, the present invention also includes recombinant constructs
comprising one or more of the sequences as broadly described above. The
constructs
comprise a vector, such as a plasmid or viral vector, into which a sequence of
the invention
has been inserted, in a forward or reverse orientation. In a preferred aspect
of this
embodiment, the construct further comprises regulatory sequences, including,
for example,
a promoter, operably linked to the sequence. Large numbers of suitable vectors
and
promoters are known to those of skill in the art, and are commercially
available. The
27
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WO 99/01464 PCT/US98/13733
following vectors are provided by way of example. Bacterial: pET-3 vectors
(Stratagene},
pQE70, pQE60, pQE-9 (Qiagen), pbs, pDlO, phagescript, psiX174, pbluescript SK,
pbsks,
pNHBA, pNHl6a, pNHlBA, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,
pDR540, pRITS (Pharmacia). Eukaryotic: pBlueBacIII (Invitrogen), pWLNEO,
pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
However, any other plasmid or vector may be used as long as they are
replicable and viable
in the host.
Examples of recombinant DNA vectors for cloning and host cells which they can
transform include the bacteriophage 1 (E. coli), pBR322 (E. coli), pACYCl77
(E. coli),
pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl
(gram-
IS negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHVl4 (E.
toll and
Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6
(Streptomyces), YIpS
(Saccharomyces), a baculovirus insect cell system, YCpl9 (Saccharomyces). See,
generally, "DNA Cloning": Vols. I & II, Glover et al. ed. IRL Press Oxford
(1985) (1987)
and; T. Maniatis et al. ("Molecular Cloning" Cold Spring Harbor Laboratory
(1982).
In some cases, it may be desirable to add sequences which cause the secretion
of
the polypeptide from the host organism, with subsequent cleavage of the
secretory signal.
Polypeptides can be expressed in host cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to produce such
proteins
using RNAs derived from the DNA constructs of the present invention.
Appropriate
cloning and expression vectors for use with prokaryotic and eukaryotic hosts
are described
by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold
Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by
reference.
Following transformation of a suitable host strain and growth of the host
strain to
an appropriate cell density, the selected promoter is induced by appropriate
means (e.g.,
temperature shift or chemical induction) and cells are cultured for an
additional period.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical
means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption, or
use of cell lysing agents, such methods are well known to those skilled in the
art.
Depending on the expression system and host selected, the polypeptide of the
present invention may be produced by growing host cells transformed by an
expression
vector described above under conditions whereby the polypeptide of interest is
expressed.
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WO 99/01464 PCT/US98/13733
The polypeptide is then isolated from the host cells and purified. If the
expression system
secretes the polypeptide into growth media, the polypeptide can be purified
directly from
the media. If the polypeptide is not secreted, it is isolated from cell
lysates or recovered
from the cell membrane fraction. Where the polypeptide is localized to the
cell surface,
whole cells or isolated membranes can be used as an assayable source of the
desired gene
product. Polypeptide expressed in bacterial hosts such as E. coli may require
isolation from
inclusion bodies and refolding. Where the mature protein has a very
hydrophobic region
which leads to an insoluble product of overexpression, it may be desirable to
express a
truncated protein in which the hydrophobic region has been deleted. The
selection of the
appropriate growth conditions and recovery methods are within the skill of the
art.
The polypeptide can be recovered and purified from recombinant cell cultures
by
methods including ammonium sulphate or ethanol precipitation, acid extraction,
anion or
cation exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography and
lectin chromatography. Protein refolding steps can be used, as .necessary, in
completing
configuration of the mature protein. Finally, high performance liquid
chromatography
(HPLC) can be employed for final purification steps.
Depending upon the host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be non-
glycosylated.
Polypeptides of the invention may also include an initial methionine amino
acid residue.
The polypeptide may be used as an antigen for vaccination of a host to produce
specific antibodies which have anti-bacterial action. This invention also
contemplates the
use of the DNA encoding the antigen as a component in a DNA vaccine as
discussed more
fully below.
The polypeptides or cells expressing them can be used as an immunogen to
produce
antibodies thereto. These antibodies can be, for example, polyclonal or
monoclonal
antibodies. The term antibodies also includes chimeric, single chain, and
humanized
antibodies, as well as Fab fragments, or the product of an Fab expression
library. Various
procedures known in the art may be used for the production of such antibodies
and
fragments.
Antibodies generated against the polypeptides of the present invention can be
obtained by direct injection of the polypeptides into an animal or by
administering the
polypeptides to an animal, preferably a nonhuman. The antibody so obtained
will then bind
the polypeptides itself. In this manner, even a sequence encoding only a
fragment of the
poiypeptides can be used to generate antibodies binding the whole native
polypeptides.
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WO 99/01464 PCT/US98/13733
Such antibodies can then be used to isolate the polypeptide from tissue
expressing that
polypeptide.
Polypeptide derivatives include antigenically or immunologically equivalent
derivatives which form a particular aspect of this invention.
The term 'antigenically equivalent derivative' as used herein encompasses a
IO polypeptide or its equivalent which will be specifically recognised by
certain antibodies
which, when raised to the protein or polypeptide according to the present
invention,
interfere with the interaction between pathogen and mammalian host.
The term 'immunoiogically equivalent derivative' as used herein encompasses a
peptide or its equivalent which when used in a suitable formulation to raise
antibodies in a
IS vertebrate, the antibodies act to interfere with the interaction between
pathogen and
mammalian host.
In particular derivatives which are slightly longer or slightly shorter than
the native
protein or polypeptide fragment of the present invention may be used. In
addition,
polypeptides in which one or more of the amino acid residues are modified may
be used.
20 Such peptides may, for example, be prepared by substitution, addition, or
rearrangement of
amino acids or by chemical modification thereof. All such substitutions and
modifications
are generally well known to those skilled in the art of peptide chemistry.
The polypeptide, such as an antigenically or immunologically equivalent
derivative
or a fusion protein thereof is used as an antigen to immunize a mouse or other
animal such
25 as a rat or chicken. The fusion protein may provide stability to the
polypeptide. The
antigen may be associated, for example by conjugation , with an immunogenic
carrier
protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin
(KLH).
Alternatively a multiple antigenic peptide comprising multiple copies of the
protein or
polypeptide, or an antigenically or immunologically equivalent polypeptide
thereof may be
30 sufficiently antigenic to improve immunogenicity so as to obviate the use
of a carrier.
For preparation of monoclonal antibodies, any technique which provides
antibodies
produced by continuous cell line cultures can be used. Examples include the
hybridoma
technique (Kohler and Milstein, Nature, 256:495-497(1975)), the trioma
technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology TodaX 4:72(
1983)), and the
35 EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et
al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S.
Patent
4,946,778) can be adapted to produce single chain antibodies to immunogenic
polypeptide
products of this invention.
CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
Using the procedure of Kohler and Milstein (supra, (1975)), antibody-
containing
cells from the immunised mammal are fused with myeloma cells to create
hybridoma cells
secreting monoclonal antibodies.
The hybridomas are screened to select a cell line with high binding affinity
and
favorable cross reaction with other Streptococcal species using one or more of
the original
IO polypeptide and/or the fusion protein. The selected cell line is cultured
to obtain the
desired Mab.
Hybridoma cell lines secreting the monoclonal antibody are another aspect of
this
invention.
Alternatively phage display technology could be utilised to select antibody
genes
with binding activities towards the polypeptide either from repertoires of PCR
amplified v-
genes of lymphocytes from humans screened for possessing anti-Fbp or from
naive libraries
(McCafferty, J. et al., Nature 348:552-554( 1990), and Marks, J. et al.,
Biotechnolo~y
10:779-783(1992)). The affinity of these antibodies can also be improved by
chain
shuffling (Clackson, T. et al., Nature 352:624-628(1991)).
The antibody should be screened again for high affinity to the polypeptide
and/or
fusion protein.
As mentioned above, a fragment of the final antibody may be prepared.
The antibody may be either intact antibody of Mr approx 150,000 or a
derivative of
it, for example a Fab fragment or a Fv fragment as described in Skerra, A and
Pluckthun,
A., Science 240:1038-1040 ( 1988). If two antigen binding domains are present
each
domain may be directed against a different epitope - termed 'bispecific'
antibodies.
The antibody of the invention may be prepared by conventional means for
example
by established monoclonal antibody technology (Kohler, G. and Milstein, C.
(supra, (1975)
or using recombinant means e.g. combinatorial libraries, for example as
described in Huse,
W.D. et al., Science 246:1275-1281 (1989).
Preferably the antibody is prepared by expression of a DNA polymer encoding
said
antibody in an appropriate expression system such as described above for the
expression of
polypeptides of the invention. The choice of vector for the expression system
will be
determined in part by the host, which may be a prokaryotic cell, such as E.
coli (preferably
strain B) or Streptomyces sp. or a eukaryotic cell, such as a mouse C127,
mouse myeloma,
human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect
cell. The
host may also be a transgenic animal or a transgenic plant (for example, as
described in
Hiatt, A. et al., Nature 340:76-78(1989). Suitable vectors include plasmids,
31
CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
bacteriophages, cosmids and recombinant viruses, derived from, for example,
baculoviruses
and vaccinia.
The Fab fragment may also be prepared from its parent monoclonal antibody by
enzyme treatment, for example using papain to cleave the Fab portion from the
Fc portion.
Preferably the antibody or derivative thereof is modified to make it less
immunogenic in the patient. For example, if the patient is human the antibody
may most
preferably be fiumanised'; where the complimentarity determining regions) of
the
hybridoma-derived antibody has been transplanted into a human monoclonal
antibody , for
example as described in Jones, P. et al., Nature 321:522-525 ( 1986), or
Tempest et al.,
Biotechnoloev 9:266-273 (1991).
The modification need not be restricted to one of fiumanisation' ; other
primate
sequences (for example Newman, R. et al., Biotechnolo~y 10:1455-1460 (1992))
may also
be used.
The humanised monoclonal antibody, or its fragment having binding activity,
form
a particular aspect of this invention.
This invention provides a method of screening drugs to identify those which
interfere with the proteins selected as targets herein, which method comprises
measuring
the interference of the activity of the protein by a test drug. For example if
the protein
selected has a catalytic activity, after suitable purification and formulation
the activity of
the enzyme can be followed by its ability to convert its natural substrates.
By incorporating
different chemically synthesised test compounds or natural products into such
an assay of
enzymatic activity one is able to detect those additives which compete with
the natural
substrate or otherwise inhibit enzymatic activity.
The invention also relates to inhibitors identified thereby.
The use of a polynucleotide of the invention in genetic immunisation will
preferably employ a suitable delivery method such as direct injection of
plasmid DNA into
muscles (Wolff et al., Hum. Mol. Genet. 1:363 (1992); Manthorpe et al., Hum.
Gene Ther.
4:419 (1963)), delivery of DNA complexed with specific protein carriers ( Wu
et al., J.
Biol. Chem. 264:16985 (1989)), coprecipitation of DNA with calcium phosphate
(Benvenisty & Reshef, Proc. Nat'1 Acad. Sci. USA, 83:9551 (1986)),
encapsulation of DNA
in various forms of liposomes (Kaneda et al., Science 243:375 (1989)),
particle
bombardment (Tang et al., Nature 356:152 (1992)); Eisenbraun et al., DNA Cell
Biol.
12:791 (1993)) and in vivo infection using cloned retroviral vectors (Seeger
et al., Proc.
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WO 99/01464 PCTNS98/13733
Nat'l. Acad. Sci. USA 81:5849 (1984)). Suitable promoters for muscle
transfection include
CMV, RSV, SRa, actin, MCK, alpha globin, adenovirus and dihydrofolate
reductase.
In therapy or as a prophylactic, the active agent i.e., the polypeptide,
polynucleotide
or inhibitor of the invention, may be administered to a patient as an
injectable composition,
for example as a sterile aqueous dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical application
for example in the form of ointments, creams, lotions, eye ointments, eye
drops, ear drops,
mouthwash, impregnated dressings and sutures and aerosols, and may contain
appropriate
conventional additives, including, for example, preservatives, solvents to
assist drug
penetration, and emollients in ointments and creams. Such topical formulations
may also
IS contain compatible conventional carriers, for example cream or ointment
bases, and ethanol
or oleyl alcohol for lotions. Such carriers may constitute from about 1 % to
about 98% by
weight of the formulation; more usually they will constitute up to about 80%
by weight of
the formulation.
For administration to human patients, it is expected that the daily dosage
level of
the active agent will be from 0.01 to 10 mg/kg, typically around 1 mg/kg. The
physician in
any event will determine the actual dosage which will be most suitable for an
individual
patient and will vary with the age, weight and response of the particular
patient. The above
dosages are exemplary of the average case. There can, of course, be individual
instances
where higher or lower dosage ranges are merited, and such are within the scope
of this
invention.
A vaccine composition is conveniently in injectable form. Conventional
adjuvants
may be employed to enhance the immune response.
A suitable unit dose for vaccination is 0.5-Sug/kg of antigen, and such dose
is
preferably administered 1-3 times and with an interval of I-3 weeks.
Within the indicated dosage range, no adverse toxicologicals effects are
expected
with the compounds of the invention which would preclude their administration
to suitable
patients.
Screening Assays
A Factor of the invention may be employed in a screening process for compounds
which bind the receptor and which activate (agonists) or inhibit activation of
(antagonists) the
receptor polypeptide of the present invention. Thus, polypeptides of the
invention may also
be used to assess the binding of small molecule substrates and ligands in, for
example, cells,
cell-free preparations, chemical libraries, and natural product mixtures.
These substrates and
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WO 99/01464 PCT/US98/13733
S ligands may be natural substrates and ligands or may be structural or
functional mimetics.
See Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).
Factors of the invention are responsible for many biological functions,
including
many pathologies. Accordingly, it is desirous to fmd compounds and drugs which
stimulate a
Factor of the invention on the one hand and which can inhibit the function of
a Factor of the
invention on the other hand. In general, agonists are employed for therapeutic
and
prophylactic purposes for such conditions as Herpesvirus infections, such as
HSV, VZV,
HCMV and EBV infections. Antagonists may be employed for a variety of
therapeutic and
prophylactic purposes for such conditions as Herpesvirus infection, such as
HSV, VZV,
HCMV and EBV infections.
In general, such screening procedures involve producing appropriate cells
which
express the receptor polypeptide of the present invention on the surface
thereof. Such cells
include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the
receptor (or
cell membrane containing the expressed receptor) are then contacted with a
test compound to
observe binding, or stimulation or inhibition of a functional response.
The assays may simply test binding of a candidate compound wherein adherence
to
the cells bearing the receptor is detected by means of a label directly or
indirectly
associated with the candidate compound or in an assay involving competition
with a
labeled competitor. Further, these assays may test whether the candidate
compound results
in a signal generated by activation of the receptor, using detection systems
appropriate to
the cells bearing the receptor at their surfaces. Inhibitors of activation are
generally
assayed in the presence of a known agonist and the effect on activation by the
agonist by
the presence of the candidate compound is observed.
cDNA, protein and antibodies to a Factor of the invention may also be used to
configure assays for detecting the effect of added compounds on the production
of mRNA
and protein from a Factor of the invention in cells. For example, an ELISA may
be
constructed for measuring secreted or cell associated levels of protein feom a
Factor of the
invention using monoclonal and polyclonal antibodies by standard methods known
in the
art, and this can be used to discover agents which may inhibit or enhance the
production of
a Factor of the invention (also called antagonist or agonist, respectively}
from suitably
manipulated cells or tissues. Standard methods for conducting screening assays
are well
understood in the art.
Examples of potential antagonists of a Factor of the invention include
antibodies or,
in some cases, oligonucleotides or proteins which are closely related to the
ligand of a Factor
34
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WO 99/01464 PCT/US98/13733
of the invention, e.g., a fragment of the ligand, or small molecules which
bind to the receptor
but do not elicit a response, so that the activity of the receptor is
prevented.
Prophylactic and Therapeutic Methods
This invention provides methods of treating abnormal conditions related to
both an
excess of and insufficient amounts of the biological activity of a Factor of
the invention.
If the activity of a Factor of the invention is in excess, several approaches
are
available. One approach comprises administering to a subject an inhibitor
compound
(antagonist) as hereinabove described along with a pharmaceutically acceptable
carrier in an
amount effective to inhibit activation by blocking binding of ligands to a
Factor of the
invention, or by inhibiting a second signal, and thereby alleviating the
abnormal condition.
In another approach, soluble forms of a Factor of the invention polypeptides
still
capable of binding the ligand in competition with endogenous Factor of the
invention may
be administered. Typical embodiments of such competitors comprise fragments of
the
polypeptide from a Factor of the invention.
In still another approach, expression of the gene encoding endogenous Factor
of the
invention can be inhibited using expression blocking techniques. Known such
techniques
involve the use of antisense sequences, either internally generated or
separately
administered. See, for example, O'Connor, J Neurochem ( 1991 ) 56:560 in
Oli~odeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press,
Boca
Raton, FL (1988). Alternatively, oligonucleotides which form triple helices
with the gene
can be supplied. See, for example, Lee et al., Nucleic Acids Res ( 1979)
6:3073; Cooney et
al., Science ( I 988) 241:456; Dervan et al., Science ( 1991 ) 251:1360. These
oligomers can
be administered per se or the relevant oligomers can be expressed in vivo.
For treating abnormal conditions related to an under-expression of Factor of
the
invention and its activity, several approaches are also available. One
approach comprises
administering to a subject a therapeutically effective amount of a compound
which activates
Factor of the invention, i.e., an agonist as described above, in combination
with a
pharmaceuticaliy acceptable carrier, to thereby alleviate the abnormal
condition.
Alternatively, gene therapy may be employed to effect the endogenous
production of a Factor
of the invention by the relevant cells in the subject. For example, a
polynucleotide of the
invention may be engineered for expression in a replication defective
retroviral vector, as
discussed above. The retroviral expression construct may then be isolated and
introduced into
a packaging cell transduced with a retroviral plasmid vector containing RNA
encoding a
polypeptide of the present invention such that the packaging cell now produces
infectious
CA 02295933 1999-12-23
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viral particles containing the gene of interest. These producer cells may be
administered to a
subject for engineering cells in vivo and expression of the polypeptide in
vivo. For overview
of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-
based
Therapeutic Approaches, (and references cited therein) in Human Molecular
Genetics, T
Strachan and A P Read, BIOS Scientific Publishers Ltd ( 1996).
Diagnostic Assays
This invention also relates to the use of a Factor of the invention
polynucleotides for
use as diagnostic reagents. Detection of a differentially expressed Factor
gene associated with
a viral infection and/or reactivation, as compared to a normal individual,
will provide a
diagnostic tool that can add to or define a diagnosis of a disease or
susceptibility to a disease
which results from under-expression, over-expression or altered expression of
a Factor gene.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as
from
blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may
be used
directly for detection or may be amplified enzymatically by using PCR or other
amplification
techniques prior to analysis. RNA or cDNA may also be used in similar fashion.
Deletions
and insertions can be detected by a change in size of the amplified product in
comparison to
the normal genotype. Point mutations can be identified by hybridizing
amplified DNA to
labeled Factor gene-derived nucleotide sequences. Perfectly matched sequences
can be
distinguished from mismatched duplexes by RNase digestion or by differences in
melting
temperatures. DNA sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without denaturing
agents, or by
direct DNA sequencing. See, e.g., Myers et al., Science ( 1985) 230:1242.
Sequence changes
at specific locations may also be revealed by nuclease protection assays, such
as RNase and
S 1 protection or the chemical cleavage method. See Cotton et al., Proc Natl
Acad Sci USA
(1985) 85: 4397-4401. In another embodiment, an array of oligonucleotides
probes
comprising Factor gene nucleotide sequence or fragments thereof can be
constructed to
conduct efficient screening of e.g., genetic mutations. Array technology
methods are well
known and have general applicability and can be used to address a variety of
questions in
molecular genetics including gene expression, genetic linkage, and genetic
variability. (See
for example: M.Chee et al., Science, Vol 274, pp 610-613 ( 1996)).
The diagnostic assays offer a process for diagnosing or determining a
susceptibility to
Herpesvirus infection, such as HSV, VZV, HCMV and EBV infection, through
detection of
mutation or difference in expression in the Factor of gene as compared to
normal individuals
by the methods described.
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In addition, Herpesvirus infection, such as HSV, VZV, HCMV and EBV infection,
can be diagnosed by methods comprising determining from a sample derived from
a subject
an abnormally decreased or increased level of a polypeptide or mRNA or a
Factor of the
invention. Decreased or increased expression can be measured at the RNA level
using any
of the methods well known in the art for the quantitation of polynucleotides,
such as, for
example, PCR, RT-PCR, RNase protection, Northern blotting and other
hybridization
methods. Assay techniques that can be used to determine levels of a protein,
such as an a
Factor of the invention, in a sample derived from a host are well-known to
those of skill in the
art. Such assay methods include radioimrnunoassays, competitive-binding
assays, Western
Blot analysis and ELISA assays.
The examples below are carried out using standard techniques, which are well
known
and routine to those of skill in the art, except where otherwise described in
detail. The
examples illustrate, but do not limit the invention.
EXAMPLES
Example 1: Infection of mice and reactivation experiments.
4 to 6 week old female BALB/c BYJ mice were obtained from Jackson
Laboratories. Mice were anesthetized with intraperitoneal injection of
ketamine
(87mg/kg)/xylazine (13 mg/kg), then, after corneal scarification, were
inoculated in the eye
with 10° PFU of HSV-1 17~ (Brown et al., 1973). At a minimum of 28 days
post infection,
mice were sacrificed by cervical dislocation and TG were isolated. Groups of 6-
10
explanted TG were incubated in Dulbecco's Modified Eagle medium supplemented
with
5% fetal bovine serum at 37"C for 0, I, 2, 4 or 24 hours post-explant (p.e.).
In a single
experiment, TG were explanted in the absence of serum. For hyperthermia
experiments,
mice were infected following light scarification of both ear pinnae with 105
PFU of HSV-1
SC-16 (Harbour et al. 1981). At a minimum of 28 days post infection, transient
hyperthermia was induced as described previously (Sawtell et al. 1992).
Briefly, mice were
placed in a 43"C water bath for 10-12 minutes, then dried and placed in a warm
incubator
(34"C) for approximately 30 min to prevent hypothermia. At varying times post
treatment,
mice were sacrificed by cervical dislocation and TG were removed.
Example 2: Extraction of RNA.
Ganglia used for RNA preparation were snap frozen in liquid nitrogen. RNA was
isolated from TG and brain stems using the TRIzoI reagent, as described by
manufacturer
(Gibco BRL), followed by extensive digestion with RNase-free DNase I (BMB) and
37
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ethanol precipitation. RNA concentrations were determined by spectrophotometer
and
agarose gel electrophoresis (Maniatis et al., 1982).
Example 3: Differential Display RT-PCR.
Complementary DNA (cDNA) was prepared from 300 ng RNA from latently-
infected TG at 0, 1, 2, and 4 hours p.e. using the Differential Display Kit
(TM) (Display
Systems Biotech, Inc., Los Angeles, CA), as described by manufacturer. Primers
presented
in this study are listed in Table 2. Briefly, RNA from each sample was
incubated, with one
of nine downstream primers containing 11T residues and 2 nucleotide anchors
(AA, AC,
AG, CA, CC, CG, GA, GC, GG), for one hour at 40"C, followed by 5 min at 95"C
to
inactivate the M-MuLV enzyme. cDNA was stored at -70"C. Each cDNA was
subjected to
PCR amplification with DispIayTaq (TM) (Display Systems Biotech) using the
original
downstream primer, one of 24 10-mer 5' primers, and a-"P-dATP (Warthoe, 1995).
PCR
conditions were 35 cycles of 30 sec denaturation at 94"C, 60 sec primer
annealing at 40"C,
and 60 sec extension at 72"C, employing a Perkin Elmer Cetus Gene Amp PCR
System
thermocycler. A final extension reaction was then performed for S min 72"C .
Radiolabeled
reaction products were subjected to high resolution polyacrylamidelurea gel
electrophoresis
as described (Liang & Pardee, 1992). Gels were dried on Whatman filters and
analyzed by
autoradiography. Differentially-displayed PCR bands were cut out from the
filter paper and
dissolved in DEPC-treated water (Ambion) for 30 min at room temperature
followed by 10
min at 100"C.
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Table 2. Differential Display primers presented in this study*.
Name Sequence
5' # GATCATAGCC
1
5' # CTGCTTGATG
2
5' # GATCCAGTAC
3
5'#4 GATCGCATTG
5'#5 AAACTCCGTC
5' # TGGTAAAGGG
6
5'#7 GATCATGGTC
5'#9 GTTTTCGCAG
5'#10 TACCTAAGCG
5'#11 GATCTGACAC
5'#12 GATCTAACCG
5'# 13 TGGATTGGTC
5'#14 GGAACCAATC
S'#15 GATCAATCGC
5'#20 GATCAAGTCC
5'#21 GATCTCAGAC
5'#22 GGTACTAAGG
3' # TTTTTTTTTTTAC
2
3' #4 TTTTTTTTTTTCA
3' #5 TTTTTTTTTTTCC
3' #6 TTTTTTTTTTTCG
3' #8 TTTTTTTTTTTGC
* Primers obtained from Display Systems Biotech, lnc., Los Angeles, CA
Example 4: Reamplification PCR.
To assure that each band analyzed contained a single cDNA species, each
differentially displayed band was reampiified in 4 individual PCR reactions.
Four T7-
T11VVN 3' primers were used, where VV was the original DDRT nucleotide anchor,
T7
was a 23 nucleotide portion of the T7 promoter (TAATACGACTC ACTATAGGGCCC),
and N was A,G,T, or C. The Reamplification reactions included the- original
upstream
primers, 2mM dNTP and the Stoffel fragment of Taq PoIymerase (Perkin Elmer
Cetus).
Reactions were performed under the original DDRT-PCR conditions. PCR products
were
separated by agarose electrophoresis and the most prominent product among the
four
parallel reactions was isolated for automated sequencing and further
confirmation.
Sequence analysis using these primers showed differntially displayed bands as
outlined in Table 2 below.
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Table 2
Band Expression GenBank
Number3' S' PatterncDNA Name AccessionP Value
PrimerPrimer Number
64' 2 3 Ind Mouse TIS7 V00756 ! a
-210
56' 2 7 lnd Mouse TIS7 J00424 1 a
-75
116' 6 2 Ind MouseTIS7 X17400 1 e-l40
125' 6 7 lnd Mouse TIS7 X17400 1 a
-37
201 8 2 lnd Mouse TIS7 X 17400I a
' -47
I 14" 6 1 Re Unknown mouse W97484 I a
-66
I 17 6 2 Ind Human DNA Bindin ProteinD28468 1 a
-21
200 6 1 Re Unknown human W38244 1 a
-70
229 8 12 Re Mouse Sema horin X85990 I a
-189
1 I 6 I Ind Unknown human 861599 1 a
S" -36
124" 6 7 Re Unknown human 861599 1 a
-29
39 2 I lnd Unknown human D 197921 a
-18
42 2 2 Ind Unknown human D62695 I a
-66
44 2 2 Ind Unknown human T34888 1 a
-45
65 2 4 Re Mouse kallikrein tumorM 186201 a
anti en -13
97 5 I lnd Mouse T-cell Anti 002567 1 a
S en 4-1 BB -52
27 4 9 Ind Rat ATPase H39388 1 a
-22
123 6 5 ind LASS mouse rowth arrestX67267 1 a
ene -45
20 4 20 Rep Human NADH ubiquinoneT58895 1 a
oxyreductase -185
subunit B14
129 6 11 Ind Human nuclear encodedM25484 I a
mitochondria) -168
NADH ubiquinone oxyreductase
24KD
subunit
232 8 12 lnd Mouse faminin T54408 1 a
-I
16
41 2 2 Re Mouse al ha-tubulin H34265 1 a
-73
54 2 6 Ind Mouse retrorans oson-likeM21123 I a
element -66
138 6 l5 Ind Mouse beta-tubulin X04663 1 a
-38
98 5 IS Re Mouse beta-tubulin X04663 1 a
-32
21 4 21 Re Human ribosomal roteinX77770 0
S26
218 8 6 Re Mouse lecithin cholesterolacX54095 1 a
I transferase -IS
53 2 6 Ind Human retrovirus-relatedK02590 1 a
reverse -153
transcri tale
Key to Table 2:
a Ind= induced, Rep= repressed
b Represents Poisson distribution value. The cutoff number representing
significant hits
was I a -9
c Bands 56, 64, 116, 125, and 201 share sequences (see Figure 2)
d Bands pairs 114/117 and 115/124 share overlapping sequences and database
hits.
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WO 99/01464 PCT/US98/13733
Example S: Confirmation of differentially regulated expression.
Isolated reamplification bands were used as templates for synthesis of 'z-P
labeled
riboprobes with the MAXIscript'~'~' kit (Ambion., Austin, TX) as described by
manufacturer.
RNase protection assays (RPA) were performed using Hybspeed~'"' RPA kit
(Ambion), and
0.5-1 mg total RNA from a second, new set of latently-infected and uninfected
TG
explants. Mouse ~i-actin riboprobes provided with the MAXIscript""' kit were
used as
controls. Probes and protected fragments were analyzed by denaturing
polyacrylamide gel
electrophoresis (PAGE) and phosphorimaging.
Example 6: PCR amplification of cDNA.
The second confirmatory PCR using specific primer sets was performed as
follows.
cDNA was generated from 2 mg of total RNA using Superscript Preamplification
kit
priming with oligo (dT) and random hexamers (Gibco BRL). Reactions were
performed in
25-ml volumes containing 4% of cDNA, 200 mM each deoxynucleoside triphosphate
(Pharmacia), 1 mM of each primer , 1.25 U of AmpliTaq Gold'"'' (Perkin Elmer)
with PCR
Buffer A (Fisher). Primer pairs used are described in Table 3
Table Primer Pairs_Used in This
3. PCR Study
1
Name Sequence P ~ Reference
duct
TIS7SA CTCTTATCTCGGCATTTG
GGACAAGAGAAAGCAGCG 342
TIS7B CGATGCCGAAGAACAAGA
CTGCCTGTCTTGTCTTCG 300
IFN-(3 GAAAAGCAAGAGGAAAGATT
AAGTCTTCGAATGATGAGAA 165
(Nickolaus&
Zawatzky, 1994)
IFN-a AATGACCTCCACCAGCAGCT
TCTCAGGTACACAGTGATCC 201
(Nickolaus&
Zawatzky, 1994)
IFN-a/~iRACATGAGCCCCCCAGAAGTACG
ATGACCGGAGGAGGAGGGAGAA 613 (Kita et al.,
1994)
IRF-1 CAGAGGAAAGAGAGAAGTCC
CACACGGTGACAGTGCTGG 201 (Barber et al.,
1995)
IRF-2 CCTGAGTATGCGGTCCTGACTT
CCGGGTCTCCCGGTCTGGCCGA 528
(Kita et al.,
1994)
TNF-a GAAAGCATGATCCGCGACGTGG
GTAGACCTGCCCGGACTCCGCAA 678 (Kita et al.,
1994)
p-Actin ATAGCACAGCTTCCCTTTGAT
AACATGCATTGTTACCAACT 452 (Tal-Singer et
al., 1997)
CyclophilinATTCGAGTTGTCCACAGTCAGCAATGG
ATGGTCAACCCCCACCGTGTTCTTCGAC469 (Ber sma et al.,
1991 )
41
SUBSTITUTE SHEET (RULE 26)
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WO 99/01464 PCTlUS98/13733
Primers specific for TIS7 were designed based on the published sequence
(Varnum
et al., 1989). Cycling reactions were performed with a Perkin Elmer Cetus Gene
Amp PCR
System thermocycler. After one cycle of 9 min denaturation at 94°C,
cycles were as
follows: (i) 1 min denaturation at 94"C, (ii) annealing at 60"C for 1 min,
(iii) extension for 2
min at 72°C. The final cycle was terminated with a 7 min extension at
72"C. Amplification
was carried out for 25-35 cycles. RT reactions were included in each set of
experiments as
negative controls, and 10 ng of mouse DNA was used as a positive control. In
every case,
the size of PCR product bands corresponded to the predicted MW.
Example 7: Detection of PCR products.
Aliquots of 40% of the amplification products were fractionated on 2.5%
NuSieve
Agarose {FMC). Gels were stained with ethidium bromide (Sigma) and the amount
of
products were quantitated by fluorimetry. The relative amount of PCR product
was
determined in arbitrary numbers as the ratio between the PCR product band
intensity to that
of cellular housekeeping genes cyclophilin or (3-actin (Devi-Rao et al., 1994,
Tal-Singer et
al., 1997). Statistical analysis was performed using Microsoft Excel (Redmond,
WA).
Example 8: Immunohistochemical procedures.
Ganglia used for immunohistochemistry were fixed for 24 h with 4%
paraformaldehyde in PBS then immersed in 70% ethanol/ 150 mM NaCI for 24 h and
embedded in paraffin wax, and 6 mm serial sections were cut and processed as
described
elsewhere (Randazzo et al., 1995}. Rabbit polyclonal antisera to HSV-1 (Dako
Corporation, Carpinteria, CA) was used for detection of replicating virus as
described
(Adams et al., 1984, Kesari et al., 1995). Rabbit polyclonal anti-mouse
interferon-a/b (Lee
Biomolecular Research, San Diego, CA), and rabbit polyclonal anti-mouse TNF-a
(Genzyme Diagnostics, Cambridge, MA) were used to probe for cytokines. Rabbit
polyclonal anti-TIS7 was a generous gift from B. Varnum, Amgen, CA. Antigen-
expressing
cells were detected by an indirect avidin-biotin immunoperoxidase method
{Vectastain
ABC kit, Vector Labs, Burlingham, CA), with 3,3'-diaminobenzadine (DAB) as the
chromagen (Trojanowski et al., 1993).
We have recently shown that cellular Immediate Early factors, such as c-jun, c
myc, and Oct-1, are induced in neuronal cells at early times following
explantation of latent
HSV-1 -infected murine ganglia (Tal-Singer et al., 1997, Valyi-Nagy er al.,
1991). DDRT
PCR was used in the present study as an approach to identify previously
unknown cellular
genes which are induced or repressed by explantation of latently-infected TG.
This method
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allows the visualization and subsequent isolation of cDNAs corresponding to
mRNAs that
are differentially expressed in various cell populations (Liang & Pardee,
1992).
Example 9: Explantation of TG induces differential expression of multiple
mRNAs.
RNA was prepared from latently-infected TG at different time points (0, 1, 2,
and 4
hours) following explantation into culture media. Complementary DNA was
amplified
using a set of arbitrary PCR primers. The PCR products were resolved by PAGE
and
visualized by autoradiography. Every pair of primers (216 primer combinations)
identified
a limited number of target sequences within the pool of cDNAs. Thus, a typical
reaction
generated 50-200 distinct radiolabeled PCR products between SO and 600 by in
length. As
expected from previous studies (Liang et al., 1995), the majority of PCR
products were
present at identical levels in samples derived from different time points
(Figure 1 ).
However, over 100 differentially-displayed PCR products were detected and
isolated. All
differentially-displayed products were isolated for further characterization.
Four
overlapping products (#56, #64, #116, and #125 as shown in Figure 2B) are
described in
this report. The intensity of the four PCR products was clearly increased in
samples
prepared 1 and 2 hours after explantation (#56, and #64 shown in Figure 1 ).
Reamplified PCR products were subjected to sequencing followed by BLAST
(Altschul et al., 1990) sequence analysis. The sequences of bands #56, #64,
#116, and #125
were identical to sequences in the coding region of mouse TIS7 mRNA (Varnum et
al.,
1989) ( Figure 2), and were conserved with rat PC4 mRNA (Tirone & Shooter,
1989). TIS7
and rat PC4 were previously shown to be highly related in protein sequence and
are thought
to be functional homologs (Tirone & Shooter, 1989, Varnum et al., 1989).
Example 10: Confirmation of differential display.
We next determined whether RNA corresponding to the isolated bands was
differentially expressed in either uninfected or latently-infected TG
explants. Reamplified
PCR products were used as probes in quantitative RNase protection assays
(RPA). RNA
corresponding to band #56 was induced by 2 hours following explantation of
uninfected
(Figure 3A) and infected (results not shown) explants. Phosphorimager
quantitation
(Figure 3B) indicated that the levels of RNA were induced nearly 7-fold by
four hours.
Similar results were obtained using probes generated from band #64 (results
not shown).
Thus, RNA transcripts corresponding to differentially-displayed bands #56, and
#64 were
clearly induced in TG by explantation.
To further confirm the DDRT results, cDNA prepared from a different set of
latently infected TG explants was subjected to PCR using primers specific for
TIS7 (Table
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CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
3. Each PCR reaction yielded single product bands whose expected sizes
corresponded to
TIS7 cDNA. Importantly, TIS7 detected with these primers was rapidly induced
in both
infected (Figure 3C) and uninfected explants (not shown), confirming the RPA
results
(Figure 3A). Furthermore, by 24 hours p.e., TIS7 expression returned to basal
levels
(Figure 3C). We also confirmed TIS7 induction in neuronal cells by
immunostaining using
affinity purified polyclonal antisera directed against TIS7 (not shown). Thus,
induction of
TIS7 following explantation of ganglia was detected by three independent
methods: DDRT-
PCR, RPA, and immunostaining.
Example 11: IFN-~3 is induced by explantation of TG.
Several reports have indicated that TIS7/PC4 are related in sequence to IFN-(3
(Skup et al., 1982, Tirone & Shooter, 1989). To determine whether interferons
are also
induced by explantation, we again used qualitative RT-PCR. cDNA derived from
infected
and uninfected TG explants was analyzed by PCR using primers specific for IFN-
a or IFN-
~3 (Table 3. Again, single specific PCR products were obtained from each
reaction. IFN-(3
was induced 2-3.5-fold during the four hours following explantation, as
compared to the
amount of cellular housekeeping gene cyclophilin (Figure 4A). Similarly, IFN-a
levels
were 1.2-1.4-fold higher (not shown). Similar results were obtained for
uninfected samples
(results not shown).
Example 12: IFN expression is induced in neuronal cells.
HSV latency occurs primarily in neuronal cells which innervate the cornea
(Cook et
al., 1974, Ramakrishnan et al., 1994, Ramakrishnan et al., 1996). These
neurons represent
approximately 2% of the neuronal population in the TG (Arvidson, 1977, Marfurt
et al.,
1989). To determine whether IFN expression co-localizes with reactivating
virus in
neuronal cells, TG sections from latently-infected and uninfected explants
were analyzed
by immunohistochemistry. IFN al[3 protein expression was not detected at 0 h
p.e., and was
induced at 4, 8, and 24 h p.e. in both infected and uninfected explants
(Figure 5).
Furthermore, as judged by this technique, all of the neuronal cells expressed
IFN. Since
virus reactivates in approximately 1% of neuronal cells (Valyi-Nagy et al.,
1991), we
conclude that viral gene expression occurs in cells expressing IFN.
Example 13: Induction of Interferon Regulatory Factor -1 (IRF-1).
The IFN regulatory factors (IRF) bind to interferon consensus sequences (ICSs)
found in many promoters of IFN gene family members (Herschman, 1991, Tanaka &
Taniguchi, 1992, Taniguchi et al., 1995). IRF-1 is an activator of IFN-b,
whereas IRF-2
functions as a repressor (Watanabe et al., 1991). As shown in Figure 4B, IRF-1
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WO 99/01464 PCTNS98/13733
transcription was induced within the first hour p.e., and its profile of
induction was
strikingly similar to IFN-(3 (Figure 4A). In contrast, no significant change
was observed in
the levels of the IFN-a and ~ receptor (IFNa~iR or IRF-2 (Figure 4 C, 4D).
Induction of
IFN and IRF-1 also occured in the absence of serum in the explantation media
(not shown),
indicating that serum factors are not the cause for our observations. These
results were
reproducible in both infected and uninfected preparations (not shown), as also
found for
TIS7 (Figure 3). Our results suggest that IFN induction after explantation of
TG involves an
IRF-1-dependent pathway.
Example 14: TNF-a is induced by TG explantation.
Soluble tumor necrosis factor (TNF)-a enhances the reactivation frequency and
replication of HSV-1 during explant reactivation (Walev et al., 1995). To
determine
whether endogenous TNF-a is induced during explantation, RT-PCR was performed
using
primers specific for TNF-a. TNF-a transcripts were induced rapidly following
explant
(Figure 6). However, we were unable to detect TNF-a, a secreted factor, in TG
sections by
immunostaining.
Example 15: Oligonucleotides, probes, and electromobility shift assays.
To generate probes for gel mobility shift assays and competition experiments,
single-stranded oligo nucleotides were synthesized (Gibco BRL Life
Technologies)
annealed with complementary oligonucleotides and labeled as described
previously
(Scahffer et al., 1997, Frazier et al., 1996). The sequence of each probe and
its mutant
derivatives is descibed below:
ISRE 5' GATCCTAGAAGGGAAACCGAAACTGAGGATC
LAT 5' GATCGAGGGGAAAAGTGAAAGACACGGGCA
LATmAT 5' GATCGAGGG~GAAAAGATAAAGACACGGGCA
LATmm 5' GATCGAGGGGARAAGTGGCTGACACGGGCA
OriS 5' GATCATTATAAAAAAAGTGC~AACGCGAG
CA 02295933 1999-12-23
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DNA binding assays were carried out as described previously (Frazier et al.,
199b).
The reaction was in 20 ul total volume containing 2 ul (20 ug) BSA (NEB) 0.2
ul (2
ug) sheared salmon sperm DNA9 ul buffer D (20 mM herpes, pH7.9, 100 mM KCI,
20% glycerol, 0.2 mM EDTA, 1 mM DTT, protein (usually 1-2 ul) and water. In
samples where antibody incunbation was added, the reaction was incubated with
unlabeled probe (100 fold) at room T for 20 minutes before the probe (<1 ng,
30K
cpm) being added. Reaction was done at RT for 20 minutes. For supershift,
antiserum (Santa Cruz) was added and incubated for one hour at RT. The gels
were
run at 4C at 170 V for about 2-3 hours. Gels were transfered to 3M paper,
dried and
exposed to X-ray film.
Example 16: In vitro translated IRF-1 binds to LAT ISRE.
1RF-1 protein sample was generated by in vitro transcription and translation
of a human IRF-1 DNA template (generous gift from Richard Pine, New York
University) by using the rabbit reticulate lysate system (Promega) and
incubated
with LAT probe. As shown in figure 8, IRF-I formed a complex LAT ISRE DNA
sequence. This complex was supershifted with IRF-1 specific antisera but not
with
IRF-2 antisera, indicating the specificity of the interaction. We have been
unable to
show in gel mobility shift assays competition experiments with oriL site III
oligo,
and oriS site I oligo possess IRF binding sites (not shown). However, the LAT
region ISRE was shown to be able to associate with IRF-2 in phorbol ester
stimulated Jurkat cells extracts (Santa Cruz) in gel mobility shift assays
(not shown).
Moreover, the IRF-2/ISRE complex {Complex B } formed when the LAT probe was
incubated with Jurkat cell extracts was competed by wild-type LAT and ISRE
probes (Santa Cruz) but not by mutant ISRE mQpinr (Santa Cruz), mutated LAT
probes LATmAT and LATmm that contain two and three nucleotide changes,
respectively, or and oriS Site I oligo {figure 9). These experiments indicate
that the
LAT site is truly an IRF binding site capable of binding both IRF- l and IRF-
2.
Example 17: Induction of TIS7 and IRF-1 is induced by hyperthermia.
Transient hyperthermia is known to cause reactivation of HS V-1 from
latency (Sawtell et al., 1992). To determine whether IFN-related genes such as
TIS7
and IRF-1 are induced by another reactivation stimulus, samples from
individual
hyperthermia-treated mice were prepared. As shown in Figure 10A, cellular
46
CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
housekeeping genes were realtively similar in all the animals, whereas HSP70,
TIS7,
and IRF-1 transcription was induced within the first 1-2 hours following
hyperthermia (Figure 10 B, C, D). Similar results were obtained in two
experiments
using samples obtained from different mice (not shown). Our results suggest
that the
pathway leading to induction of TIS7 and IRF-1 is common to different
reactivation
stimuli.
47
CA 02295933 1999-12-23
WO 99/01464 PCT/US98/13733
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(B) TELEFAX: (610) 270-5090
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
AAAAAAAGTG AGAACGCGAA GCGTTCGCAC TTTGTCCTAA TAATATATAT ATTATTAGGA 60
CAAAGTGCGA ACGCTTCGCG TTCTCACTTT TTTT
