Note: Descriptions are shown in the official language in which they were submitted.
~ w 095/060SS 2 1 6 9 7 4 8 PcTrusg4/093n3
HSV-2 UL26 GENE, CAPSID PROTEINS, IMMUNOASSAYS AND
PROTEASE INHIBITORS
CROSS-REFERENCE
5 TO RELATED APPLICATIONS
This appli~tion is a continll~tiQn-in-part of copending U. S. patent
application serial number 08/110,522, filed August 20, 1993, the entire contents of
which are incol~ol,l~ed herein by ~ferellce.
l~;LD OF THE INVENTION
The present invention relates to HSV-2 UL26 and HSV-2 UL26.5 genes; to
essenti~lly pure HSV-2 UL26 and HSV-2 UL26.5 gene products; to compositions
and m~.thotlc of producing and using HSV-2 UL26 and HSV-2 UL26.5 DNA
15 sequences and gene products.
BACKGROUND OF THE INVENTION
The herpes viruses consist of large icosahedral enveloped virions cont~ining
a linear double stranded genome. Currently, six human herpes viruses have been
20 isolated and are known to be responsible for a variety of disease states fromsub-clinical infections to fatal disease states in the i~ UIlOcc Illplolllised. One
human herpes virus, herpes simplex virus type 2, rl~sign~t~l HSV-2, is usually
ac~luil._d through sexual contact and gives rise to genital herpes. The frequency of
l~cul~ ce of secondary genital herpes ranges between one and six times per year.25 It is estim~te~1 that genital HSV-2 infections occur in ten to sixty million individuals
in the USA. Currently, there are no vaccines available to protect against HSV-2
infection.
Little is known regarding the genome composition of HSV-2. Nevertheless,
HSV-2 presents a major public health problem. Individuals continue to become
30 infected by the virus and no completely satisfactory anti-viral agents or vaccines are
available. There is a need for a method of identifying anti-HSV-2 agents. There is
a need for reagents useful in such methods. There is a need for a method of
identifying compounds which mc~ te~ the activity of HSV-2 proteins and affect
the ability of the virus to replicate and produce multiple infectious virions in an
35 infected cell. There is a need for methods of and kits for distinguishing HSV-2
infections from other herpesvirus infections.
-1-
woss/060~s 2~691 t PCT/USg~/09303
SUMl\IARY OF THE INVENTION
The present invendon relates to e.ssenti~lly pure HSV-2UL26 gene products
and fr~gm~nt~ thereof including HSV-2 protease precursor protein, mature HSV-2
protease and active fr~gmçntc thereof, HSV capsid precursor protein and mature
HSV-2 capsid protein.
The present invention relates to essenti~lly pure HSV-2UL26.5 gene
products and fr~ ment~ thereof inc!utling HSV-2 capsid ~l~ul~.or protein and
mature HSV-2 capsid protein.
The present invendon relates to isolated nucleic acid molecules compri~ing
10 the HSV-2~L26 gene or pordons thereof including isolated nucleic acid molecules
that encode mature HSV-2 protease and acdve fr~gm~nt~ thereof and nucleic acid
molesllles that encode ~ or or mature HSV-2 capsid protein, regulatory, e.g.,
~rulllotel regions, or funcdonal fragments thereof.
The present invendon relates to expression vectors comprising the HSV-2
UL26 gene or porhons thereof including nucleotide sequences that encode mature
HSV-2 protease and active fr~gmentc thereof and nucleotide sequences that encodeprecursor or mature HSV-2 capsid protein or funcdonal fr~ m~nt~ thereof.
The present invendon relates to host cells that contain expression vectors
compri~ing the HSV-2UL26 gene or pordons thereof including nucleotide
sequences that encode mature HSV-2 protease and acdve fr~gmPnt~ thereof and
nucleotide sequences that encode precursor or mature HSV-2 capsid protein or
functional f~grnPnt~ thereof.
The present invendon relates to isolated nucleic acid molecules compri~ing
the HSV-2VL26.5 gene or pordons thereof inclll~ling isolated nucleic acid
molecules tnat encode mature HSV-2 capsid protein, regulatory, e.g., Promoter
regions or fragm~nt~ thereof and nucleotide sequences that encode precursor or
mature HSV-2 capsid protein or funcdonal fr~gm~nt~ thereof.
The present invention relates to expression vectors comprising the HSV-2
UL26.5 gene or portions thereof including nucleotide sequences that encode mature
HSV-2 capsid protein or fr~ m~nt~ thereof and nucleodde sequences that encode
precursor or mature HSV-2 capsid protein or functional fr~gme~t~ thereof.
The present invention relates to host cells that contain expression vectors
comprising the HSV-2UL26.5 gene or portions thereof including nucleodde
sequences that encode mature HSV-2 capsid protein or fr~T ent~ thereof and
35 nucleodde sequences that encode precursor or mature HSV-2 capsid protein or
functional fr~ nt~ thereof.
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WO 95/06055 ` ' PCT/US94/09303
The present invention relates to methods of identifying compounds that
inhibit HSV-2 protease activity comprising contacting HSV-2 protease or active
fr~gmPntc thereof with an HSV-2 protease substrate in the presence of a test
co,l,l~ound, detecting the level of proteolytic cleavage of the substrate and
5 collll)hling that level to the level that occurs in the absence of dhe test col"poulld.
The present invention relates to methods of identifying compounds that
inhibit HSV-2 virion assembly by contacting HSV-2 capsid proteins in the presence
of a test co,ll~oulld, detec*ng the level of capsid-capsid association and cOI~p~ ;ng
that level to dhe level that occurs in the absence of dle test compound.
The present invention relates to HSV-2 protease substrates produced by
means of ch~mic~l synthesis or recombinandy produced and preflic~tYl on
f~, mentC or all of the UL26 gene product.
The present invention relates to antibodies that selectively bind to HSV-2
protease procesced substrates but not unprocessed substrates or that selectively bind
15 to unprocessed substrates but not to processed substrates.
The present invention relates to m~thocls of distinguishing between HSV-l
DNA and HSV-2 DNA compricing PCR amplifi~tion of DNA using ~ which
will amplify HSV-l DNA but not HSV-2 DNA and/or PCR amplific~tion of DNA
using plilllCl~ which will amplify HSV-2 DNA but not HSV-l DNA.
The present invention relates to PCR primers which will amplify HSV-l
DNA but not HSV-2 DNA and PCR ~ which will amplify HSV-2 DNA but
not HSV-l DNA.
The present invention relates to kits for distinguishing between HSV-l DNA
- and HSV-2 DNA compricing a container comprising PCR ~lime.~ which will
25 amplify HSV-l DNA but not HSV-2 DNA and a positive control and size marker todet~ e if HSV-l DNA has been amplified by the primers and/or a container
comprising PCR ~limcl~ which will amplify HSV-2 DNA but not HSV-l DNA and
a positive control and size marker to determine if HSV-2 DNA has been amplified
by the ~liLIlel~.
The present invention relates to methods of distinguishing between HSV-l
protein and HSV-2 protein comprising an immllno~cs~y using antibodies that
selectively bind to HSV-l protein but not HSV-2 protein and/or an immunoassay
using antibodies that selectively bind to HSV-2 protein but not HSV-l protein.
The present invention relates to antibodies which selectively bind to HSV-l
35 protein but not HSV-2 protein or antibodies which selectively bind to HSV-2
protein but not HSV-l protein.
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wo ss/060ss 2~69~ PcT/usg1/093n3 --
The present invention relates to kits for distinguishing between HSV-1
protein and HSV-2 protein. Said kit comprising a carrier being cc ~ ,cnted to
receive a series of containers in close confineme~t which comprises a first container
comrricing antibodies which selectively bind to HSV-1 protein but not HSV-2
protein and a means to detect whether the antibodies are bound to HSV-1 protein
and/or a second cont~in~or comprising antibodies which selectively bind to HSV-2protein but not HSV-1 protein and a means to detect whether the antibodies are
bound to HSV-2 protein.
The present inventional relateds to the HSV-2 protease plUlllOt~l and/or
enh~n~er elementc and their uses.
The present invention relates to the HSV-2 capsid protein pl~ olel and/or
Pnh~ncer elemelltc and their uses.
BRIEF DESCRIPI ION OF THE FIGURES
Figure 1 illu~llates the HSV-2 UL26 gene. The symbol < > denotes the limits of the
HSV-2 UL26 gene product. A putative termination codon is underlined.
The symbol [[ ]] denotes the limits of the HSV-2 UL26.5 gene product.
The symbol [ ] denotes the limits of two major proteolytic sites. The cissile bond is
inrli~ted by the *.
The symbol ¦ ¦ denotes the promoter region of the HSV-2 UL26.5 gene, a putative
"TATA box" is ~-nrl-~.rlin~cl
Figure 2 illustrates the ~ ;s~ion of chloramphenicol acetyltransferase (CAT) when
regl-l~ted in the HSV-2 UL26.5 promoter.
DETAILED DESCRIPI ION OF THE INVENTION
As used herein, the term UL26 gene refers to a DNA molecule compricing a
nucleotide sequence that encodes the HSV-2 protease and a form of the HSV-2
capsid protein. The UL26 gene is disclosed in SEQ ID NO: 1. The coding region ofthe UL26 gene concictc of nucleotides 534-2447 of SEQ ID NO: 1. When e~ ssed,
the UL26 gene enco~les a 638 amino acid active protease precursor disclosed in SEQ
ID NO:1 and SEQ ID NO:2.
As used herein, the term "active protease precursor" refers to the
unprocessed UL26 tr~ncl~tion product. The active protease precursor is an activeHSV-2 protease. When produced, the active protease precursor autocleaves at an
internal protease cleavage site between amino acid residues 247 and 248. The
amino terminal 247 amino acid portion retains protease activity.
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wo 95/06055 9 ~ PCTIUS94/09303
As used herein, the term "mature protease" refers to the amino terminal 247
amino acid protein that is produced by autocleavage of the active protease precursor.
The amino acid sequence of the mature protease is disclosed as amino acids 1-247of SEQ ID NO: 1 and SEQ ID NO:2.
As used herein, the term "HSV-2 protease" is meant to refer to,
in~ ngç~bly, active protease precursor, mature protease or active fragment~
thereof.
As used herein, the term "UL26.5" gene refers to a DNA molecule
comprising a nucleotide sequence that encodes the HSV-2 capsid protein. The
UL26.5 gene is an internal sequence within the UL26 gene which is separately
transcribed. The UL26.5 gene is disclosed in SEQ ID NO: 1 and includes the coding
region from nucleotide 1461-2447. When expressed, the UL26.5 gene encodes a
329 amino acid capsid precursor disclosed in SEQ ID NO: 1 and SEQ lD NO:2 as
amino acids 310-638.
As used herein, the term "capsid precursor" refers to the unprocessed UL26.5
tr~n~l~tion product. While not wishing to be bound by any particular mech~nistictheory regarding the function of the gene products of this invention, but based in
part on the literature concerning HSV-1, it is believed that after it is produced, the
capsidl precursor is cleaved by the HSV-2 protease at an internal protease cleavage
site be~ amino acid residues 613 and 614 of SEQ ID NO:1 and SEQ ID NO:2.
The 304 amino acid portion is the capsid protein used in viral assembly and viral
DNA p~ ging It is the C-terminal processing of UL26.5 that enables packaging
of viral DNA into mature capsids. Inhibition of this processing event results in the
inability to package DNA into mature capsids.
As used herein, the term "mature capsid protein" refers to the 304 amino acid
protein that is produced by cleavage of the capsid precursor by the HSV-2 protease.
The amino acid sequence of the mature capsid protein is disclosed as amino acids310-613 of SEQ ID NO:l and SEQ ID NO:2.
As used herein, the term "HSV-2 capsid protein" is meant to refer to,
interchangeably, capsid precursor and mature capsid protein.
As used herein the term "functional fragments" when used to modify a
specific gene or gene product means a less than full length portion of the gene or
gene product which retains substantially all of the biological function ~soci~terl
with the full length gene or gene product to which it relates. To determine whether
a fragment of a particular gene or gene product is a functional fragment one merely
_5
~97 4~
WO 95/06055 PCT/US94/093(~3 --
generates the fr~gm~nt~ by well-known nucleolytic or proteolytic techniques and
tests the thus generated fragments for the described biological function.
The present invention relates to es~enti~lly pure HSV-2 protease, to
co,l,~osi~ions and methotl~ for producing and using HSV-2 protease, to nucleic acid
S molecllles that encode HSV-2 protease and to methods for producing and using
nucleic acid moleclllss that encode HSV-2 protease. The present invention relates
to es~çnti~lly pure HSV-2 capsid protein, to co"l~osiLions and methods for
pro lllring and using HSV-2 capsid protein, to nucleic acid molecules that encode
HSV-2 capsid protein, to m~tho lc for producing and using nucleic acid molecules10 that encode HSV-2 capsid protein. The present invention relates to substrates which
are cleaved by HSV-2 protease, to m.otho~l~ of identifying compounds that inhibit
HSV-2 protease activity, to methods of identifying compounds which inhibit HSV-2capsid ~csçmh1y, to methods of distinguishing between samples cont~ining HSV-l
DNA and samples co~Laini-lg HSV-2 DNA, to methods of distinguishing between
15 samples col~ g HSV-1 protein and samples cont~ining HSV-2 protein, and to
re~g~nt~, including oligonucleotides and antibodies, for perforrning such methods.
Some embo~ -tc of the present invention provide m.otho 1~ for identifying
compounds which inhibit or otherwise mo~llll~te the activity of HSV-2 protease.
Thus, the present invention provides methods for identifying compounds useful as20 anti-HSV-2 agents since the activity of the HSV-2 protease is es~enti~l for the viral
life cycle. According to the present invention, HSV-2 protease is contacted with an
HSV-2 protease substrate (substrate) in the presence of a test compound to
dete.~ e whether or not the test compound affects proteolytic activity. The effect
of the test compound on the HSV-2 protease may be determined by cc .il~?~ing the25 proteolytic activity in the presence of the test compound to the proteolytic activity
that would be observed in the absence of the compound.
Prt~teolytic activity refers to the ability of the HSV-2 protease to
e.nzy-nzit;~lly process the substrate into products, i.e. cleave a single substrate
peptide molecule into two or more peptide molecules (proteolytic products). In the
30 viral life cycle, protease precursor is processed into mature protease and capsid-
precursor is processed into mature capsid by such proteolytic cleavage. This
conversion is necess~ry for virion assembly and viral DNA packaging. The level of
proteolytic activity may be determined by a variety of means well known by thosehaving ordinary skill in the art. F.~senti~lly, a means is provided to ~ tingui~h
35 unprocessed substrate from proteolytic product. Thus, after the substrate, HSV-2
protease and test compound are contacted, the level of proteolytic activity can be
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~ = ~
wo 95/06055 21 69 7 i ~ PCT/US94/093n3
observed by detecting the amount of unprocessed substrate rem~ining, the amount of
unprocessed substrate depleted, or the amount of proteolytic product generated.
The present invention provides essentially pure HSV-2 protease which is
useful in an assay to identify compounds which mofl~ teHSV-2 protease activity.
S The present invention provides mçthotl~ of producing essentially pure HSV-2
protease. The amino acid sequence of HSV-2 protease is disclosed in SEQ ID NO:l
and SEQ ID NO:2. As described above, the 638 amino acid active protease
or is disclosed in SEQ ID NO:l and SEQ ID NO:2. The active protease
yl~ is an active HSV-2 protease which is processed by autocleavage at an
internal protease cleavage site between amino acid residues 247 and 248 to produce
a 247 amino acid protein referred to as mature protease. Purified active protease
yi~;u~.Ol, mature protease and active fragment~ thereof may be produced by routine
peptide synthesis methods or by using recombinant DNA technology using the
info~ tion provided in SEQ ID NO: 1. Using standard procedures and readily
available starting materials, one having ordinary skill in the art can produce HSV-2
protease. Furthe~ , using standard procedures and readily available starting
m~teri~l~, one having ordinary skill in the art can determine whether a fragmentand/or derivative of the active protease precursor or mature protease is an active
f~gm~nt
Assays for de~e,llli-ling whether or not a protein or peptide is c~p~hle of
cleaving a specific substrate is disclosed herein. To determine if ar~ HSV-2 protease
fragment has proteolytic activity, one having ordinary skill in the art can y~lrc,l.ll
protease activity assays as desc~ibe~l herein without test compounds and using the
fragment or derivative of the ylutease instead of the protease iclçntir~l to SEQ ID
NO:2. If the fragment or derivative cleaves the substrate, it is active, i.e. the
fragment or derivative possesses proteolytic activity. Thus, one having ordinaryskill in the art can routinely determine if a fragment or derivative of the protease is
an active fragment or derivative.
The present invention relates to nucleotide sequences that encode HSV-2
protease and to nucleotide sequences that encode HSV-2 capsid protein. The UL26
gene including a nucleotide sequence which encodes HSV-2 protease and a
precursor form of HSV-2 capsid protein is disclosed in SEQ ID NO:l. The UL26.5
gene including a nucleotide sequence which encodes HSV-2 capsid protein is also
disclosed in SEQ ID NO:l. One having ordinary skill in the art can, using standard
techniques and readily available starting materials, use the information disclosed
herein incl~lrling SEQ ID NO: 1 to obtain or synthesize a nucleic acid molecule that
-7 -
W0 95/06055 ~ PCT/US94/09303
F~ncoclçsHSV-2 protease or a nucleic acid molecule that encodes HSV-2 capsid
protein. Further, using standard techniques, readily available starting materials and
the in~....~tinn disclosed herein including SEQ ID NO:l, one having ordinary skill
in the art can produce essçnti~lly pure HSV-2 protease including, active precursor
5 protease, mature protease or active HSV-2 protease fr~gmFnt~. Likewise, using
standard techniques, readily available starting m~t~ri~l~ and the illfollllationdisclosed herein including SEQ ID NO: 1, one having ordinary skill in the art can
produce essçnti~lly pure HSV-2 capsid protein inclll~ling capsid precursor, mature
capsid, or HSV-2 capsid fr~m~nt~ capable of assembly functional fr~mellts. One
10 having ordinary skill in the art can, using standard techniques and readily available
star~ng m~t~ri~l~, use the information disclosed herein includin'g SEQ ID NO:l to
obtain or synthesi7F a nucleic acid molecule that encodes HSV-2 protease or HSV-2
capsid protein using codons which provide C~)Lill~um protein production in a given
host cell used in an e~l.,s~ion system.
Nucleic acid molecules encoding HSV-2 protease or HSV-2 capsid protein
may be g~.lel~ed by those having ordinary skill in the art without undue
. ;n.F .~l~tion using a variety of techniques. Using, for eY~mple, Polymerase
Chain Reaction (PCR) methodology, primers may be designed and used to produce
multiple copies of the nucleotide sequences that encode the HSV-2 protease or
HSV-2 capsid protein. The entire nucleotide sequence encoding active protease
~r~;ul~ol may be obtained routinely by amplifying the viral DNA. Similarly, the
nucleotide sequence enroAing mature protease may be obtained routinely by
amplifying the~viral DNA. Likewise, the nucleotide sequence encoding an active
HSV-2 protease fragment may be obtained routinely by amplifying the viral DNA.
In a similar manner, the entire nucleotide sequence encoding capsid pl~cul~ol,
mature capsid or functional fr~gmentc thereof may be obtained routinely by
amplifying the viral DNA. ~ltern~tively, using restriction enzymes, DNA encodingHSV-2 protease, including the active protease precursor, the mature protease, oractive fr~gment~ thereof or HSV-2 capsid protein including capsid precursor, mature
30 capsid or functional fr~gm.ont~ thereof may be obtained from viral DNA cloned into
vectors and iclentified by hybridization using probes cle~igned from the disclosed
nucleotide sequence. Moreover, nucleic acid molecules that encode the HSV-2
protease or the HSV-2 capsid protein may also be synthesi7~A using techniques well
known to those having ordinary skill in the art. Codons which encode HSV-2
35 protease or HSV-2 capsid protein may be selected to optimize protein production in
a host cell selected for recombinant production of the HSV-2 protease or HSV-2
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21~7~
WO 95/06055 : . PCT/US94/09303
capsid protein. The HSV-2 genome is highly rich in G+C nucleotides. This is
particularly true for the UL26 gene which encodes HSV-2 protease. Such high G+C
character poses a problem in ovG~ ssing genes in E. coli because of codon usage
and an increased chance of fra ne-shift mutations. In an effort to improve
5 e,~,es~ion of UL26 in E. coli, the UL26 gene and fr~gment~ thereof were ch~n~
to provide codons ~ltÇe,l~d in E. coli yet m~int~ining the authentic amino acid
sequence of the protease. The reference for pr~rell~d codon usage is: Wada et al.,
(1992) "Codon Usage T~b~ ted from the GenR~nk Genetic Sequence Data",
Nucleic Acid Research, Vol. 20 Supplement, pages 2111-2 118, which is
10 incorporated herein by reference. Optimi7~tion of codon usage is well known and
can be employed to design nucleic acid molecules according to the present invention
which can be e~ ,ssed at an improved level of efficiency in a selected host.
One having ordinary skill in the art can, using well known techniques, insert
such DNA molecules into vectors such as colllnlel.;ially available expression vectors
15 for use in well known expression systems. For example, col~ elcially available
pl~mirl~ such as pSE420 (Invitrogen, San Diego, CA) or pET-16(b) (Novagen,
Madison W.I.j may be used for production of HSV-2 protease in E. coli. The
col~ el~ially available plasmid pYES2 (lnvitrogen, San Diego, CA) may, for
example, be used for production in S. cerevisiae strains of yeast. The commercially
20 available MAXBAC~ complete baculovirus e~r~;,sion system (Invitrogen, San
Diego, CA) may, for example, be used for production in insect cells. The
co.~l..,e.~ially available plasmid pcDNA I (Invitrogen, San Diego, CA) may, for
example, be used for production in ~ n cells such as Clhinese Hamster
Ovary cells. One having ordinary skill in the art can use these cc,m~ l.,ial
25 expression vectors and systems or others to produce the HSV-2 protease or HSV-2
capsid protein using routine techniques and readily available starting materials. (See
e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold
Spring Harbor Press (1989) which is incorporated herein by reference.) Thus, thedesired proteins can be ~ d in both prokaryotic and eukaryotic systems,
30 resulting in a spectrum of processed forms of the protein.
The particulars for the construction of expression systems suitable for
desired hosts are known to those in the art. Briefly, for recombin~nt production of
the protein, the DNA encoding the polypeptide is suitably ligated into the expression
- vector of choice. The DNA is operably linked to all regulatory elements which are
35 neces~. y for expression of the DNA in the selected host. One having ordinary skill
WO 95/060S~; PCT/US9~/09303
in the art can, using well known techniques, prepare expression vectors for
recomhin~nt production of the polypeptide.
The expression vector including the DNA that encodes the HSV-2 protease
or HSV-2 capsid protein is used to transform or transfect the compatible host which
is then cultured and ~ t~;l-~l under conditions wherein expression of the foreign
DNA takes place. The protein of the present invention thus produced is recovered- from the culture, either by lysing the cells or from the culture meclillm as
a~r~,;ate and known to those in the art. One having ordinary skill in the art can,
using well known techniques, isolate the protein that is produced using such
~l,lcssion ~ysh.lls.
According to one embodiment of the invention, protein may be produced
and purified as follows. A DNA molecule that comprises a nucleotide sequence that
encodes the HSV-2 protease or the HSV-2 capsid protein is produced which
in~lufles a nucleotide sequence that encodes multiple histidine residues at a Ic,m,nal
portion of the protein. This DNA molecule is incorporated into an expression vector
which is introduced into suitable host cells. The DNA is expressed and the protein,
incl~ ing the terminal hi~tirline resicl~les, which are referred to herein as the histidine
tag or His-tag, is procluce-l The cells are collected and m~int~ined on ice in
phosphate l~urf~.~,d saline at pH 8.5. The cells are then lysed by sonication. The
sonic~te~l cellular material is centrifuged at 30,000 x g. The supernatant is then
filtered through a .2 micron filter. The filtered supernatant is incubated with a metal
chel~tin~ resin (e.g., a nitrilo tri~retic acid nickel resin is one of many such resins
useful for such a purpose) for 2 hours at room temperature, after which time theresin is separated from unbound material by centrifugation. The resin is then
packed into a colurnn and washed with 50 mM imi~l~7ole to elimin~te non
specifi~lly bound proteins. The His-tagged protease is then eluted from the Ni
column with 150 mM imi-l~701e buffer. The eluate from the column is further
purified by column chromatography using Pharmacia Superdex 75 sizing column in
phosph~te ~urr~,~ saline.
The DNA molecule may be engin~ered to include a specific cleavage site
bc~ ,en the hi~ti~ine tag and authentic HSV-2 protease to enable removal of the
hi~ti~line tag from the e~ ssed protein. Removal of the histidine tag may be
~cornplished as follows: The (asparte)4 lysine sequence can be engineered to
follow the hi~ti(line tag and precede the HSV-2 sequence when the histine tag isplaced at the amino-terminus of the HSV-2 protease. Enterokinase specifically
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WO 95/060SS i ~ ~ ~ PCT/US94/09303
cleaves after the (aspartate)41ysine sequence thereby generating authentic HSV-2protease.
In addition to producing these proteins by recombinant techniques,
o..-~led peptide ~,yl,LLcsizers may also be employed to produce the HSV-2
protease or the HSV-2 capsid protein. Such techniques are well known to those
having ordinary skill in the art.
The present invention provides essentially pure subsL-~es for HSV-2
protease cleavage activity including synthetic substrates. An HSV-2 protease
s~lbsh~t~ is a peptide which can be cleaved at least into two separate peptides by
HSV-~ protease meAi~t~d proteolysis. In some embo-lim~-nt~, the size dirftl~,ntial
~lweell cleaved and uncleaved substrates may be used to detect whether or not the
protease is active. In some embodilllen~s, the substrates of the present invention are
labelled so that they may be detected. In some embodiments, the substrates are
fixed to a solid phase. In some embo~ pl~t~ of the invention, either the substrate or
a proteolytic product has a biologically or ch~ornir~l activity not present in the other
which can be used to tli~ting~ h one from the other. Examples of biological
activities include enzyme activity and the ability to bind with specific antibodies.
Two amino acid sequences are contained in UL26 that have been identif
as natural cleavage sites. The first is LQAS (SEQ ID NO:3) wherein the HSV-2
protease cleaves the peptide between the A and the S. The second is VNAS (SEQ
ID NO:4) whc~ the HSV-2 protease cleaves the peptide between the A and the S.
Natural or synthetic substrates may be produced which contain either of these two
cleavage sites. Accordingly, a substrate according to the present invention haveeither the formula
R, - SEQ ID NO:3 - R2
or the formula
Rl - SEQ ID NO:4 - R2
wherein Rl and R2 are, independently, hydrogen or one or more amino acids. In
some embo~ , the substrate is the UL26 gene product which contains two
protease cleavage sites: one comprising SEQ ID NO:3 and one comprising SEQ ID
NO:4. In some embodiments, the substrate is the UL26.5 gene product which
contains a protease cleavage sites comprising SEQ ID NO:4. In some embodiments,
Rl is preferably 1-20 amino acids, more preferably 1-10, and most preferably 3, 4,
- 5, 6, 7, 8 or 9 amino acids. In some embo li ~ -t~, R2 is preferably 1-20 amino
acids, more preferably 1-10, and most preferably 3, 4, 5, 6, 7, 8 or 9 amino acids.
wo 95/060s5 216 9~ ~ PCT/US94/09303 --
One having ordinary skill in the art can readily design substrates according to the --
above formula. The following peptides have been designed as substrates.
1. Peptides inrl~lcling the internal cleavage site SEQ ID NO:3
(LQA*S):
AHTYLQA*S~ K SEQ ID NO:5
AGIAGHTYLQA*~K~-K SEQ ID NO:6
GIAGHTYLQA*~ ~ ~K SEQ ID NO:7
IAGHTYLQA*SI;.~K SEQ ID NO:8
GHTYLQA*S~Kl~K SEQ ID NO:9
HTYLQA*S~K~K~I SEQ ID NO:10
HTYLQA*~;~K~W SEQ ID NO:11
HTYLQA*~K~WG SEQ ID NO:12
HTYLQA*~ K~WGA SEQ ID NO:13
HTYLQA*SEKFKMWGAE SEQ ID NO:14
2. Peptides including the terminal cleavage site SEQ ID NO:4
(VNA*S):
ALVNA*SSAAHVDVD SEQ ID NO:15
The asterisk (*) infiir~tes the scissile bond where cleavage by HSV-2 ~rolease
occurs.
The substrates may be obtained from proteolytic cleavage of the UL26 or
UL26.5 protein product. They may be produced recombinantly by Gx~ ,ssion of
UL26 or UL26.5 gene or fr~gm~nt thereof cont~ining the cleavage site or may be
made by m~n~ of synthetic organic ch~--mic~l means using standard peptide
synthetic procedures well known in the art such as ~errifiel-l synthesis.
One having Ol'dill~Uy skill in the art can readily design assays using the HSV-
2 protease and substrate to identify compounds that modulate HSV-2 protease
activity. As used herein, the term "test assay" refers to assays that include a l~~iXIUlG
of HSV-2 protease, substrate and test compound; and the term "control assay" refers
to assays that include a mixture of HSV-2 protease and substrate without test
cc,lll~ound. To ~leterrnine whether or not a test compound modulates HSV-2
protease activity, the level of HSV-2 protease activity in a test assay may be
colllpdled to the level of HSV-2 protease activity in a control assay.
In some embo~1iment~ of the present invention, the size dirrelGiltial between
cleaved and uncleaved substrate is used to determine whether or not substrates are
cleaved when contacted with HSV-2 protease in the presence of a test compound.
In some embo l;,..~ , an HPLC assay is performed. Sample containing protease is
-12-
W0 95/06055 ~ 7 k ~ PCT/US94/093n3
inrubptf~ with a substrate, for example HTYLQAS~ IWGAE (SEQ ID
NO:14), for 4 hrs at 37 C in phosphate buffered saline after which the reaction is
1~. "~;n~tecl with trifluoroacetic acid. The reaction is then run on an HPLC column,
showing activity manifested by the peptide cleavage products.
In some embodiments of the present invention, immunoassays are used to
detect whether or not subst~.~tes are cleaved when contacted with HSV-2 protease in
the presence of a test compound. In some embo lim~nts, antibodies are provided
which specifically bind to uncleaved substrate but not HSV-2 protease cleavage
products. Such antibodies are referred to herein as "substrate-specific antibodies".
In some embo~ .t~ ntibotlies are provided which specifically bind to HSV-2
plub:ase cleavage products but not uncleaved substrate. Such antibodies are referred
to herein as "product-specific antibodies". Antibodies which react to either a
product or a substrate but not both (i.e. substrate-specific antibodies and product-
specific antibodies collectively) are referred to herein as "non-crossreactive
antibodies". In some embo l;~e~t~ antibodies are fixed to a solid phase. In someembofli.,...~ , antibodies are labelled.
For example, a Illix.Lulc; containing HSV-2 protease, substrate and test
compound is m~int~ine~ under al)p,~iate conditions and for a s.lfficient amount of
time to allow the proteolytic reaction to occur unless the test compound affects the
reaction. The ~ u-e can be added to a container which has non-crossreactive
~ntibo-lies ~tt~f he~ to the inner surface. If the non-crossreactive are substrate-
specifif, antibodies, any uncleaved substrate rçm~ining in the lliixLule will bind to
the antibodies. If the substrate is l~h~llerl, the contained may be rinsed and the
amount of label present may be ~letf'~t~ The level of HSV-2 protease activity isdete.lllincd accordingly. If the non-crossreactive are product-specific antibodies,
any HSV-2 protease products in the nli~ule will bind to the antibodies. If the
substrate is labelled at a portion which is liberated as the product, the contained may
be rinsed and the amount of label present may be detected. The level of HSV-2
protease activity is determined accordingly.
ICP35 antibodies (Catalog No.: 13-118-100; Rivers Park, 9108 Gulford Rd.
Col-lmhia, Maryland) may be used to detect cleaved substrate. Such antibodies are
product specific and only bind to capsid protein after it has been proteolytically
processed by the HSV-2 protease.
Alternatively, instead of using labelled substrates, the exemplified
immllno~S~ys may be modified as sandwich assays in which antibodies specific forthe bound antigen complex are detected. Such antibodies are referred to herein as
-13-
wo 95/06055 2 ~ 6 9 ~ 4 ~ PCT/US94/Q9303 ~
complex-specific antibodies. The container is again rinsed and sufficient time is
allowed for the binding of the complex specific antibody to any complex present.The level of complex specific antibody is ietect~d and indicative of the level of
HSV-2 protease activity.
S In some embo~ of immunoassays, unlabelled substrate is used in the
reaction mixture. After the reaction mib~LLIlc is added to a conLainel comprising a
non-~ir~ ssleactive antibody and m~int~ineA for a sufficient time for the non-
.;lusslcactive antibody to bind to either substrate or product, either labelled substrate
or l~helle~ product, respectively, is added and will bind to any non-crossreactive
antibody not bound with substrate or product from the reaction ~ . Detecting
the ~mo-lnt of l~hell~d substrate or l~helle l product indi~tçs the level of proteolytic
cleavage.
In some emboL.IlellL~, the substrate is labeled and the label is released when
the substrate is converted to proteolytic products. Detecting the release of the label,
which in~lic~tes the HSV-2 protease activity, may be accomplished by a variety of
well known means. In some emb~im~ont~, labelled substrate is fixed to a solid
phase. Upon cleavage by HSV-2 protease, the label ~tt~çd to the portion of the
suhstr~te that bec. l-lcs an unattached product, is rele~ecl ~omp~ring the level of
label present before and after the reaction n~i~Lu,c in~licat~s how much label is
released and thus the level of HSV-2 protease activity. ~ltern~tively, detecting the
amount of label freed from the solid phase indicates the level of HSV-2 proteaseactivity.
In another emb~liment, metho lc of detecting HSV-2 protease activity
include fluorescence liberation assays in which substrate contains fluorescent label
~dj~ e~t to the scissile bond. At such a location, the label is not detect~ble in
uncleaved substrate. However, when the substrate is cleaved by HSV-2 protease atth~e cleavage site, the fluorescent group becomes exposed and the fluorescence
becc,..l~s cletect~ble. Thus, the level of proteolytic activity may be measured by
,l,ca~uling detect~hle fluoresce~ce after contacting the substrate with HSV-2
30 protease in the presence of a test compound.
In another embodiment, methods of detecting HSV-2 protease activity
include scintill~tion proximity assays in which radiolahelled substrate is conjugated
to solid beads which, when in close pro~ .y to the radiolabel, are excited and
become detectable by scintill~tion. When the substrate is cleaved, the radiolahel is
35 no longer in close ~lo~illlity to the beads and the beads are not excited and not
detectable by scintillation. Thus, the level of proteolytic activity may be measured
-14-
~ Wo 95/060s5 ~3 69 7~ ~ pcTlusg4lo93n3
by measuring the excitation of the beads by scintillation after contacting the --
conjugated substrate with HSV-2 protease in the presence of a test compound.
In addition to these embodiments, one having ordinary skill in the art can
apply well known techniques to devise other methods of identifying compounds that
S mo~lnl~te HSV-2 protease activity using various means of detecting the HSV-2
~t~asc cleavage or the lack thereof.
The present invention relates to kits for identifying compounds that
m~lul~te HSV-2 protease activity. Such kits include s~u~te cont~iners which
comrri~e HSV-2 protease, substrate, and optionally, antibodies or other reagents for
10 fletec~ing HSV-2 protease activity or distinguishing bf L~ ,n uncleaved substrate and
products. The substrate or antibodies may be fixed to the inner surface of a
cont~iner. The substrate or antibodies may be labelled.
Some embo lim~nt~ of the present invention also provide methods of
identifying compounds which inhibit or otherwise modulate HSV-2 capsid assembly
15 using a m~llr;~f~ aLion assay. The present invention provides methods of
ide,lLirying compounds useful as anti-HSV-2 agents since capsid assembly is
essenti~l for viral replication and infectivity. According to the present invention,
chimeric genes are provided which comprise either a sequence including the HSV-2~lL26.5 gene or a portion thereof which encodes an HSV-2 capsid protein linked to
20 a sequence encoding the yeast GAL~ DNA-binding protein or a sequence including
the HSV-2 UL26.5 gene or a portion thereof which encodes an HSV-2 capsid
protein linked to a sequence encoding the yeast GA~ activation protein. While it is
preferred that the portion of the chimeric gene that encodes the HSV-2 capsid
protein encodes the mature capsid, the capsid precursor protein may also be usefully
25 employed. Chimeric genes are inserted into Saccharomyces cerevisiae pl~mi~l~ and
the plasmids are introduced in S. cerevisiae which contains an integrated GAL~-
responsive lacZ inllic~tQr gene. When the chimeric genes on the pl~cmi~s are
e,~ ,sscd, fusion proteins are produced. The portions of the fusion proteins
comprising the HSV-2 capsid protein will, under selected condition bind to each
30 other and thereby bring together the DNA-binding domain and activation domain of
GALa. When the two GAL~ ~lom~in~ which are in close proximity interact with the
GAL,4-responsive lacZ in~lic~tQr gene, the in~lic~tor gene is expressed and, under the
prop~ conditions a detectable blue color is observed. If the fusion proteins arepreveneed from binding, the two GALa domains will not be present in pl~ ity to
35 each other and the intlic~tor gene will not be activated. Thus, no blue color will be
- present to observe.
-15-
woss/06oss ~ 91 4 PCTIUS9~/09303 --
Thus, this yeast system provides a rapid and specific assay for the inter~ction
of HSV-2 capsid proteins that occur during virion assembly. In the presence of
compounds which in~ upl or inhibit HSV-2 capsid protein interaction, the GAL~
dom~in~ in the fusion proteins produced by expression of the chimeric genes will5 not associate and thereby will not activate the ~acZ gene in the yeast system.Accordingly, c~ ou.lds may be identif~ by the absence of activation of the lacZ
gene in t-~lsrol-llcd yeast which inhibit HSV-2 capsid assembly and therefore
possess anti-viral p~ ies.
Some emb~im~onts of the present invention provides methods of
10 ~ ting~ hing ~ ..e~n samples cont~iningHsv-l DNA and samples con~ g
HSV-2 DNA or samples co~ -g HSV-l proteins and samples containing HSV-2
proteins. Accordingly, the present invention provides a method of diagnosing
whether an individual is infected with HSV-l and/or HSV-2. Methods are disclosedfor identifying whether an individual is infected with HSV-l and/or HSV-2 wherein
HSV-l infection can be disting~ hed from HSV-2 infection.
According to some embodim~ont~ of the invention, PCR technology is used to
distinguish beLweell samples co~ it-g HSV-l DNA and samples cont~inin~ HSV-
2 DNA. Such methods provide a means for distinguishing between HSV-l and
HSV-2 infections and allow for the tii~gnosis of the type of HSV infection an
20 individual has. Specific p.i--lcl~ are designed that will provide for amplification of
HSV-l DNA but not HSV-2 DNA and/or HSV-2 DNA but not HSV-l DNA.
Accordingly, by ~,.ru-ming amplification techniques using such ~ l,cl~ with
biological samples taken from individuals such as cell, serum or tissue samples,especi~lly s~mrles taken at sites where blisters or other manifestations of viral
~hedding are observed, one can determine whether or not the DNA in the sample isderived from HSV-l or HSV-2 and therefore whether the individual from which the
sample was taken is infected with HSV-l or HSV-2.
The nucleotide sequence of the UL26 gene including the nucleotide
sequence which encodes the HSV-2 protease and the HSV-2 capsid protein is
30 disclosed in SEQ ID NO: 1. The nucleotide sequence encoding HSV-l protease and
HSV-l capsid protein are disclosed in SEQ ID NO: 16. A set of PCR primers were
desi~ned which amplify HSV-2 sequences but not HSV-1 sequences. Thus,
detection of ampli~led DNA indicates that HSV-2is present. Similarly, a set of
PCR primers were designe(l which amplify HSV-l sequences but not HSV-2
sequences. Thus, detection of amplified DNA in~ tes that HSV-l is present. It ispreferred that both sets of primers are provided and used in separate amplification
-16-
/ WO 95/060S5 2~ 6~ 7~ PCT/US94/09303
protocols with m~teri~l from the same sample in order to provide an additional
control. Other opdonal controls include positive controls which contain DNA
sequences that will be ~mrlifie~l and/or negative controls that cannot be amplified
by the ~ ,..,. Amplified DNA may be ~letected by running the material on an
5 electrophoresis gel after the amplifif ~tion protocol is complete. DNA molecules of
the expected length of an amplification product may be provided as size ~ uk~s.
Present invention also relates to kits for flietinglliehing whether a sample
cont~ine DNA from HSV-1 or HSV-2. The kits of the present invention are useful
to ~ gnose whether an individual is infected with HSV- 1 and/or HSV-2. The kits
10 contain cQ~t~in~rs which conlrriee primers that will amplify HSV-1 DNA but not
HSV-2 DNA or col~t~in~o-rs that will amplify HSV-2 DNA but not HSV-1 DNA.
Kits may optionally contain both sets of primers in separate containers for running
s~Le ~mplific~tion procedures using difrtlGnt portions of the same sample. Kits
may optionally contain positive and/or negative controls in separatG containers. Kits
15 may optionally contain DNA molecules in a separate container which can serve as a
size marker. The DNA molecule may be of the expected length of a DNA molecule
amplified using the ~
According to some embo l;...P~.tx of the invention, immllno~ee~ys are used to
~lietin~lieh bc~ en s~mple~e co,.t;~h.;. g HSV-1 protein and samples con~ g
20 HSV-2 protein. The immunoassays are used to distinguish be~ - HSV-l and
HSV-2 infections and diagnose the type of HSV infection an individual has. Such
immllno~ee~ys are based upon dirrclc~lces bC~GGn UL26 gene products of HSV-l
and HSV-2 or b~ en UL26.5 gene products of HSV-l and HSV-2.
Immunoassays may be based upon differences in proteases and/or capsid proteins of
25 HSV-l and HSV-2. Specific antibodies are provided which selectively bind to
~iLo~cs on HSV-1 antigens not present on HSV-2 antigens or which selectively
bind to .,~ilo~cs on HSV-2 antigens not present on HSV-1 antigens. For example,
specific antibodies are provided which selectively bind to HSV-l protease but not
HSV-2 protease or which selectively bind to HSV-2 protease but not HSV-l
30 protease. Likewise, specific antibodies are provided which selectively bind to HSV-
1 capsid but not HSV-2 capsid or which selectively bind to HSV-2 capsid but not
HSV-1 capsid.
Accordingly, by perforrning antibody binding assays, using specific
antibodies with biological samples taken from individuals such as cell, serum or35 tissue s~mples, especially samples taken from sites where blisters or other
manifestations of viral shecltling are observed, one can determine whether or not the
-17-
wo 95/0605s 2 1~ ~ ~ 4 ~ YCT~Ss4/os3n3 -
HSV-l-speci~lc antibodies or the HSV-2-specific antibodies bind to proteins in the
sample and therefore whether the individual from which the sample was taken is
infected with HSV-1 and/or HSV-2. The amino acid sequence of HSV-2 active
protease ~l~c~ or spans amino acids 1-638 in SEQ ID NO:l and SEQ ID NO:2.
5 The amino acid sequence of HSV-2 mature protease spans amino acids 1-247 of
SEQ ID NO:l and SEQ ID NO:2. The amino acid sequence of HSV-2 capsid
precursor spans amino acids 310-638 in SEQ ~ NO:l and sEQnDNo:2. The
amino acid sequence of HSV-2 mature capsid spans amino acids 310-613 of SEQ
ID NO:1 and SEQ ID NO:2. The amino acid sequence of HSV-l protease and
10 capsid are ~ close l in SEQ ID NO:17. The amino acid sequence of HSV-1 active protease p~ or spans amino acids 1-635 in SEQ ID NO:17. The amino acid
sequence of HSV-l mature protease spans amino acids 1-247 of SEQ ID NO:17.
The amino acid sequence of HSV-l capsid precursor spans arnino acids 307-635 in
SEQ ID NO:17. The amino acid sequence of HSV-l mature capsid spans amino
15 acids 307-610 of SEQ nDNO:17.
Antibodies which specifi~ally bind to HSV-2 protease but not HSV-l
protease may be produced by those having ordinary skill in the art using routinemtqtho~s and widely available starting materials. Likewise, antibodies which
specific~lly bind to HSV-2 capsid but not HSV-l capsid may be produced by those
having ordinary skill in the art using routine m.othorlc and widely available starting
materials. Either of these HSV-2 specific antibodies are used to detect HSV-2 in an
immllno~cs~y which can distinguish HSV-1 from HSV-2. Similarly, antibodies
which specifically bind to HSV-l protease but not HSV-2 protease may be producedby those having ordin~ skill in the art using routine methods and widely available
starting materials. Likewise, antibodies which specifically bind to HSV-l capsidbut not HSV-2 capsid may be produced by those having ordinary skill in the art
using routine methods and widely available starting m~teri~l~ These HSV-l
specific antibodies are used to detect HSV-1 in an immunoassay which can
distinguish HSV-l from HSV-2. It is ~lcre,l~,d that both immunoassays be
30 pclru~ cd using m~t~n~l from the same sample in order to provide an additional
control. Other optional controls include positive controls which include peptides
which will bind to the antibody used in the immunoassay and/or negative controlswhich include peptides which will not bind to the antibody used in the
immllnnassay. Antibodies may be labelled. Alternatively, an antibody that
35 specific~lly binds to the HSV specific antibodies may be used. One having ordinary
~ WO 9S/060SS 2 1 ~ ~ 7~ ~ PCTIUS94/09303
skill in the art can readily produce immunoassays including all necessary reagents
using the inrcllllaLion provided herein.
HSV-1 protease antibody produced by Serotech as Antibody 45KD and
cc.l~ lcl.;ially available from Bioproducts for Science Inc. as catalog number
S MCA406 (P.O. Box 29176, Tn~ n~polis, IN) can be used in imm~no~s~ys to
distinguish HSV-2 from HSV-1. The Serotech antibody binds to HSV-1 precursor
or mature capsid protein but not HSV-2 lJlG~ 0l or mature capsid protein.
Accordingly, an immnno~s~y using the Serotech antibodies may be ~elrc~ ed to
delelllline if a sample contains HSV-1 or HSV-2 and thus if the individual from
whom the sample was taken is infected with HSV- 1 or HSV-2.
Present invention also relates to kits for diagnosing whether an individual is
infected with HSV-1 or HSV-2. The kits of the present invention may comprise a
CO~ ;.lf-l comprising antibodies which bind to HSV-1 protease but not HSV-2
protease and/or a cont~iner comprising antibodies which bind to HSV-2 protease but
not HSV-1 protease. It is ~lGfe,led that the kit comprises both types of antibodies in
se~ ; con~iners. Antibodies used in the kits may be l~belleA The kits contain
all other reagents and materials for p~lrolll~ing an immnnoa~s~y with the antibodies.
Kits may optionally contain positive and/or negative controls in separate containers.
Kits may optionally contain means to detect the antibody including, for example a
second antibody which specifically binds to the anti-HSV protease antibody. The
kits of the present invention may comprise a container comprising antibodies which
bind to HSV-1 capsid but not HSV-2 capsid and/or a container comprising
antibodies which bind to HSV-2 capsid but not HSV-1 capsid. It is ~ie~ll~d that
the kit comrri~es both types of antibodies in Se~ai~le containers. Antibodies used in
the kits may be labelled. The kits contain all other reagents and materials for
~lrolll~ng an immnno~cs~y with the antibodies. Kits may optionally contain
positive and/or negative controls in separate containers. Kits may optionally contain
means to detect the antibody including, for example a second antibody which
specifically binds to the anti-HSV capsid antibody. Kits may comprise the Serotech
antibody.
Another aspect of the present invention relates to the HSV-2 protease
~rumol~ and/or enh~ncer elements and their uses. The HSV-2 protease promoter
may be synthesi7~ or isolated and linked to coding sequences which encode
proteins other than HSV-2 protease. Accordingly, the present invention relates to
recomkin~nt DNA molecules which comprise at least a portion of the nucleotide
- sequence between nucleotides 1-534 of SEQ ID NO: 1 operably linked to a
-19-
wo ss/06055 ~ 1~ 9 ~ 4 ~ PCT/US94/09303--
nucleotide sequence that encodes a protein other than HSV-2 protease. The present
invention relates to cells which comprise DNA molecules which comprise at least a
portion of the nucleotide sequence be~,.eel1 1 and 534 of SEQ ID NO:l operably
linked to a nucleotide sequence that en~odes a protein other than HSV-2 protease.
Another aspect of the invention applies to bacteriophage lambda clones
which harbor HSV-2 UL26 gene (SEQ. I.D. No.:1) and sequences up~can~ and
downstream of the gene. Accordingly, the linked sequences can be used to screen
for UL26 promoter regulatory and/or enh~ncer regions.
Another aspect of the present invention relates to the HSV-2 capsid protein
~ro-l,ol." and its uses. The HSV-2 capsid protein promoter is located u~sllc~ ofnucleotide 1461 of SEQ ID NO: 1. It may be syntheci7.~-1 or isolated and linked to
coding sequences which encode proteins other than HSV-2 capsid protein.
Accordingly, the present invention relates to recombinant DNA molecules which
comprise at least a portion of the nucleotide sequence upstream of nucleotide 1461
of SEQ ID NO:1 operably linked to a nucleotide sequence that encodes a protein
other than HSV-2 capsid protein. The present invention relates to cells which
compri~e DNA molec-lles which comprise at least a portion of the nucleotide
sequence upstream of nucleotide 1461 of SEQ ID NO:1 operably linked to a
nucleotide sequence that encodes a protein other than HSV-2 capsid protein.
Nucleotides 1191 to 1461 (SEQ ID NO:1), for eY~mple, were linked to the
chloramphenicol acetyl tr~ncferace gene and shown to possess signific~nt P1`0l1101~L
activity when transfected into VERO cells
EXAMPLES
FY~mvle 1
A proteolytic activity essenti~l to the virion maturation of herpes viruses has
been characterized for HSV-2. The HSV-2 protease, also referred to as HSV-2
~L26, has a molec~ r weight (Apparent) of about 67,028 Da and a pI = 6.94. The
HSV-2 protease can be employed using molecular and biochemi~l technology in in
vilTo assays identify inhibitors of this activity by rational design and screening and
to test these inhibitors for antiviral activity in infected cells.
The HSV-2 UL26 gene was cloned as an NcoI-EcoRI fragment (1938 base
pairs) which contained the start codon, the entire open reading frame, the stop
codon, and 22 base pairs of 3'-untr~n~l~ted sequence. Full-length HSV-2 ~JL26 was
expressed in E. coli using the pOTS vector system in which the gene is inserted
downstream of the strong and tightly regulated PL promoter from bacteriophage
-20-
~ wo 9510605s 1~9 7~ ~ PCTIUS94/09303
lambda of the pOTS-207 vector. Tight regulation of the promoter is ecsenti~l when
expressing genes that are likely to be toxic to the cells, such as proteases. The 27
KD ~loLease domain corresponding to one of the autoproteolytic products derived
from the HSV-2 UL26 pl;~ y translation product was produced in E. coli using theS tightly regulated e"~r~,;,sion vector pET-16(b) (Novagen, Madison W.I.) which
co~ inc the 'rl p~ olc,,.
Each construct was decigned to include six histidine codons and the
(a~le)41ysine codons prececling the HSV-2 UL26 start codon so that the
e~ ,ssed protein will contain a cleavable hictidine tag at the N-terminus for
10 pl)rific~tion of the protein on Nickel columns. Other chelating columns may be
used. The His-tagged protein is eluted from the column by addition of imi~ ole
Alternàtively, it can be eluted by other means such as pH change. Columns and
technical protocols useful to purify protein may be obtained from coll~nlelcially
available sources such as Qiagen.
For the PL prulllo~l vectors, the recombin~nt constructs are then introduced
into E. coli AR120 (nalidixic acid inducible strain) and E. coli AR58 (heat inducible
strain) for e"~re~sion and processing/ purification studies. For the T7 promotervectors, the recombin~nt constructs are introduced into E. coli BL21, an IPTG
inducible strain. The ploleills can be readily purified by chromatography on nickel
20 chelate column.
The p27 protease fragment is active as shown by its ability to remove the last
25 amino acids from a construct comprising most of the UL26.5 coding region.
FY~mDle 2
The p27 protease gene was synthesi7e~1 to contain codons characteristic of
highly expressed ~. coli genes, yet m~int~ining the amino acid sequence of p27
protease. The synthesized gene was placed downstream of the tightly regulated T7promoter in the expression vector pET-16(b). Following IPTG (lmM) induction the
30 27 k Da protein domain was highly expressed in ~. coli.
F~mDIe 3
The above HSV-2 UL26 gene (Ncol-EcoRI fragment) and the p27 protease
is cloned into the insect cell expression vector pVL1392. The recombinant
35 construct is then introduced into insect cells derived from Spodoplera frugiperda.
wo 9s/06055 2 ~ 6 ~ 7 4~ : PCT/US94/09303 ~
High titer viral stocks are then ~ u~,d for protease activity analysis and
subsequent scale up for protein production.
FY~nlDIe 4
S Oligonucleotide PCR primers were tlçcign--cl to the DNA region of HSV-l
UL26 gene and HSV-2 UL26 gene that shared the least amount of identity to ensurethe specificity of the assay. Such a region can easily be viewed by c(~ ul~.
analysis co~ g the two DNA sequences disclosed in SEQ ID NO: 1 and SEQ ID
NO:16, ~ ely. The region of least identity between the two homolo~s lies
within the UL26.5 dom~in, i.e. the portion of the gene that encodes the capsid. The
following provides the sequences of the ~ ~.. used and the locations of the
~)fi~ ,7 are given based on the nucleotide numbers given in the nucleotide sequence
cc,lll~ison provided in the enclosed computer analysis. As shown below it is
helpful to design a system to generate HSV- 1 and HSV-2 specific products of
15 different sizes to improve the analysis.
5'-PCR ~ .. (sense-strand sequence):
SEQ ID NO:18 HSV-l: 5'-CCGGTGCCCAATCGTCCGT-3' (#864-882)
SEQ ID NO:l9 HSV-2: 5'-GTCCGTGCGCGTCAAGTCG-3' (#1397-1416)
3'-PCR primers (~nti~en~e-strand sequence):
SEQ ID NO:20 HSV-l: 5'-TTCCGGCTCCCCCACCTGA-3' (#1560-1542)
SEQ ID NO:21 HSV-2: 5'-ATTCGGATCCTGGAGGCGA-3' (#2470-2452)
Expected PCR product sizes using these sets of primers:
HSV-l: 696 base pairs
HSV-2: 1073 base pairs.
Separate PCR arnplification protocols are l,. .rc,lllled on samples suspected ofCO. .~ g either HSV-l or HSV-2 DNA using SEQ ID NO: 18 and SEQ ID NO:20
in the HSV-l assay or SEQ ID NO:l9 and SEQ ID NO:21 in the HSV-2 assay. If a
DNA fragment of 696 base pairs is generated in the HSV-l assay, the presence of
HSV-l DNA in the sample is indicated. To detect the presence of a 696 base pair
fr~gm~nt, the amplification product is migrated through an electrophoresis matrix.
A size marker of DNA of about 696 base pairs is run through the same matrix
~imlllt~neously. If a DNA fragment of 1073 base pairs is generated in the HSV-2
assay, the presence of HSV-2 DNA in the sample is indicated. To detect the
-22-
Woss/06~55 ~9 7~ PCT/US94/09303
presence of a 1073 base pair fra~m~nt, the amplification product is migrated
through an electrophoresis matrix. A size marker of DNA of about 1073 base pairsis run through the same matrix Cimlllt~neously~
A kit is provided which compri~es a cont~iner comprising SEQ ID NO: 18
and SEQ ID NO:20 in the HSV-1 assay. A kit is provided which comprises a
con~ compri~ing SEQ ID NO:19 and SEQ ID NO:21 in the HSV-2 assay. A kit
is provided which comprises both a container comprising SEQ ID NO: 18 and SEQ
ID NO:20 in the HSV-1 assay and a container comprising SEQ ID NO:19 and SEQ
ID NO:21 in the HSV-2 assay. Size marker DNA may optionally be provided. In
some kits, a size marker of 696 base pairs is provided. In some kits, a size marker
of 1073 base pairs is provided. In some kits, a size marker of 696 base pairs and a
size marker of 1073 base pairs are provided.
F.x~ le 5
A region of the putative HSV-2 UL26.5 ~lolllotcr contained in the HSV-2
UL26 gene was cloned to test for ~lolllolcl activity. The 256 base pair region that
was analyzed spanned nucleotides ~1191 to # 1447 of SEQ ID NO:1. The DNA
fragment was cloned by the polymerase chain reaction using the sense strand primer
(5'-AACATGAGCTGCGTGACC-3') ~anning nucleotide # 1191 to # 1209 of SEQ
ID NO: 1 and the ~nti~en~e strand primer (5'-AAAGAAGAAGAAGAAGAC-3')
sp~nning nucleotides #1447 to # 1429 of SEQ ID NO: 1 Promoter activity is testedby cloning the 256 base pair PCR fragment U~ 3111 of the chloramphenicol
acetyltransferase (CAT) reporter gene in the colllll.cl~;ially available vector pCAT
Basic (Promega). The res--lting construct can then be introduced into a suitable,..~....~.~li~n cell line, e.g., Vero cells~ to test for promoter activity by analyzing the
levels of CAT activity. The cell line is devoid of endogenous CAT activity; hence,
after introducing the promoter construct into such a cell line, the levels of CAT
activity is a direct measure of HSV-2 UL26.5 promoter activity.
Vero cells were grown in DMEM+10% FCS containing Gent~micine
(lOug/ml). 15 micograms of the HSV-2 UL26.5/pCAT construct was electroporated
into 5 million Vero cells using standard protocols. 48 hrs after electroporation cells
were harvested in 100 microliters of 0.25 M Tris buffer pH 8Ø Cells were Iysed by
repeated freeze-thaw, spun down at 15,000 rpm and the supernatants were
transferred to fresh tubes. Total protein concentration was determined using Bio-
Rad Protein Assay Dye Reagent Kit (Cat. # 500-0006). 5 Microliters of D-
-23-
wo ss/060ss 21 ~ 9 7 ~ ~ PCT/US91/09303 ~
Threo[dichloroacetyl~ 4~C] Chloramphenicol (Amersham, 56 mCi/mmol) and 5
rnicroliters of n-Butyral CoA (5 mg/ml) was added to an aliquot of cell extract
surem~t~nt in a 100 microliter final volume to assay for CAT activity by ethyl
acetate eYtr~tion followed by thin layer cl-.ulma~ography.
In addition to the above construct, the 256 base pair HSV-2 UL 26.5
fragment was also cloned uysL-~;alll of the CAT l~ull~l gene in the pCAT Enhancer
vector, which co~ an SV40 enhancer çlc.-~ t This construct was also tested for
CAT activity in Vero cells by the same methods described above.
The control vector pCAT control (contains the SV40 pro.lloL~,. and
enh~nctor) was used as a cc,l . .p~ o~ of HSV-2 UL26.5 promoter strength.
Figure 2 s,l....--i~l ;7es the results of four eAp~illlents. (~olllmn 1 is a negative
control and rc~l~scnls CAT expression in the absense of ~rollloLcl and enh~n~er
transcriptional control elements. Column 2, a positive control, employs SV40
llloLel and SV40 enhancer element~ to drive CAT gene expression. Cohlmn 3
15 l~ sent~ CAT gene expression driven by UL26.5 promoter alone and Column 4
csenls CAT gene expression when the llL26.5 p,~",ote~ is used in combination
with the SV40 enh~n~er elc.ll- .-s
Having est~hli~he~l a basal UL26.5 expression level (Column 3), ~ litic-n~l
fr~gmlontc of the gene sequence within ffgure 1 can he used to identify the UL26.5
20 enh~nr~er eleTn~ont~ merely by isolating fragments of convenient length u~5LI~,alll
from nucleotide 1191 back to nucleotide 1, introducing the fr~gm-o.nt~ into the basal
cApres~ion construct oriented operatively with respect to the promoter region and
testing their ability to çnh~nce CAT expression over the basal level.
The ~r~ll.otel descrihed here are useful for regulating the expression of
25 heterologous genes when operably linked thereto.
-24-
~ W O 9~/0605~ 21 6~ 7~ PCTrUS94/09303
SEQUENCE LISTING . --
(1) GENERAL INFORMATION:
(i) APPLICANT: DiLella, Anthony G.
Debouck, Christine
(ii) TITLE OF INVENTION: Novel Gene
(iii) NUMBER OF SEQUENCES: 21
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKl;ne Beecham Corporation
Corporate Patents - US UW2220
(B) STREET: P.O. Box 1539
(C) CITY: King of Prussia
(D) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19406-0939
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(c) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25, mmd
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jervis, Herbert H.
(B) REGISTRATION NUMBER: 31,171
(C) REFERENCE/DOCKET NUMBER: P50188
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-270-5019
(B) TELEFAX: 215-270-5090
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2472 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 534..2447
(ix) FEATURE:
(A) NAME/KEY: misc_feature
60 (B) LOCATION: 1461.. 2447
W O 95/06055 2 l ~ 9 7 4~ PCTrUS9~/09303 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GTCr~ACGA~ CGCGlG~lGG ATATGTCGTC GGGCGCCCGC CAGGCGGCGC TCGTGCGCCT 60
CACCGCGClG GAGCTCATCA ACCGCACCCG cArAAArAcc ACCC~ GG GGGAGATTAT 120
TAACGCCCAC GATGCCTTGG GGATACAATA cr~AArAGGGc CTGGGGCTGC TCGCCCAGCA 180
GGr.ArGcATc GG~llGGCGT CGAACGCCAA GCGATTCGCC ACGTTCAACG TGGGCAGCGA 240
CTACGACCTG TTGTACTTTT l~l~.ClCGG GTTCATTCCC CAGTACCTGT CCGTGGCCTA 300
GGGAAGGGTG GGGGlG~lGG TGGTGGGGTG 11 L 1 1~1~'1 1 ~1 1 U 1 1 U-l 1 1 CTGGTCCGCC 360
TGGTCACAAA AGGCACGGCG CCCC~AAACG CGGGCTTTAG TCCCGGCCCG GAC~lCGGCG 420
rArAr,ArAAC AACGGCGGGC CCC~lGG~lG GGTAAGTTGG TTCGGGGGCA TCGCTGTATT 480
CCCTTGCCCG CTTCCACCCC CCCllCCCGT TTG~lllull lulGCGGGTG CCC ATG 536
Met
GCG TCG GCG GAA ATG CGC GAG CGG TTG GAG GCG CCT CTG CCC GAC CGG S84
Ala Ser Ala Glu Met Arg Glu Arg Leu Glu Ala Pro Leu Pro Asp Arg
5 10 15
GCG GTG CCC ATC TAC GTG GCC GGG TTT TTG GCC CTG TAC GAC AGC GGG 632
Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser Gly
20 25 30
GAC CCG GGC GAG CTG GCC CTG GAC CCA GAC ACG GTG CGT GCG GCC CTG 680
Asp Pro Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala Leu
35 40 45
35 CCT CCG GAG AAC CCC CTG CCG ATC AAC GTA GAC CAC CGC GCT CGG TGC 728
Pro Pro Glu Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Arg Cys
50 55 60 65
GAG GTG GGC CGG GTG CTC GCC GTG GTC AAC GAC CCT CGG GGG CCG.TTT 776
40 Glu Val Gly Arg Val Leu Ala Val Val Asn Asp Pro Arg Gly Pro Phe
70 75 80
TTT GTG GGG CTG ATC GCG TGC GTG CAG CTG GAG CGC GTC CTC GAG ACG 824
Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu Thr
85 90 95
GCC GCC AGC GCC GCT ATT TTT GAG CGC CGC GGA CCC GCG CTC TCC CGG 872
Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Ala Leu Ser Arg
100 105 110
GAG GAG CGT CTG CTG TAC CTG ATC ACC AAC TAC CTG CCA TCG GTC TCG 920
Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val Ser
115 120 125
55 CTG TCC ACA AAA CGC CGG GGG GAC GAG GTT CCG CCC GAC CGC ACC CTG 968
Leu Ser Thr Lys Arg Arg Gly Asp Glu Val Pro Pro Asp Arg Thr Leu
130 135 140 145
TTT GCG CAC GTG GCC CTG TGC GCC ATC GGG CGG CGC CTT GGA ACC ATC 1016
60 Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr Ile
150 155 160
-26-
W O95/060S5 t 69 7~ ~ PCT~US94/09303
GTC ACC TAC GAC ACC AGC CTA GAC GCG GCC ATC GCT CCG TTT CGC CAC 1064
Val Thr Tyr Asp Thr Ser Leu Asp Ala Ala Ile Ala Pro Phe Arg His
165 170 175
5 CTG GAC CCG GCG ACG CGC GAG GGG GTG CGA CGC GAG GCC GCC GAG GCC 1112
Leu Asp Pro Ala Thr Arg Glu Gly Val Arg Arg Glu Ala Ala Glu Ala
180 185 190
GAG CTC GCG CTG GCC GGG CGC ACC TGG GCC CCC GGC GTG GAG GCG CTC 1160
0 Glu Leu Ala Leu Ala Gly Arg Thr Trp Ala Pro Gly Val Glu Ala Leu
195 200 205
ACA CAC ACG CTG CTC TCC ACC GCC GTC AAC AAC ATG ATG CTG CGT GAC 1208
Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg Asp
5210 215 220 225
CGC TGG AGC CTC GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC GGA 1256
Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala Gly
230 235 240
CAC ACG TAC CTT CAG GCG AGC GAA AAA TTT AAA ATA TGG GGG GCG GAG 1304
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Ile Trp Gly Ala Glu
245 250 255
25 TCT GCC CCT GCG CCG GAG CGT GGG TAT AAA ACC GGC GCC CCG GGT GCC 1352
Ser Ala Pro Ala Pro Glu Arg Gly Tyr Lys Thr Gly Ala Pro Gly Ala
260 265 270
ATG GAC ACA TCC CCC GCC GCG AGC GTT CCC GCG CCG CAG GTC GCC GTC 1400
30 Met Asp Thr Ser Pro Ala Ala Ser Val Pro Ala Pro Gln Val Ala Val
275 280 285
CGT GCG CGT CAA GTC GCG TCG TCG TCG TCT TCT TCT TCT TCT TTT CCG 1448
Arg Ala Arg Gln Val Ala Ser Ser Ser Ser Ser Ser Ser Ser Phe Pro
35290 295 300 305
GCA CCG GCC GAT ATG AAC CCC GTT TCG GCA TCG GGC GCC CCG GCC CCT 1496
Ala Pro Ala Asp Met Asn Pro Val Ser Ala Ser Gly Ala Pro Ala Pro
310 315 320
CCG CCG CCC GGC GAC GGG AGT TAT TTG TGG ATC CCC GCC TCT CAT TAC 1544
Pro Pro Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr
325 330 335
45 AAT CAG CTC GTC ACC GGG CAA TCC GCG CCC CGC CAC CCG CCG CTG ACC 1592
Asn Gln Leu Val Thr Gly Gln Ser Ala Pro Arg His Pro Pro Leu Thr
340 345 350
GCG TGC GGC CTG CCG GCC GCG GGG ACG GTG GCC TAC GGA CAC CCC GGC 1640
50 Ala Cys Gly Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly His Pro Gly
355 360 365
GCC GGC CCG TCC CCG CAC TAC CCG CCT CCT CCC GCC CAC CCG TAC CCG ' 1688
Ala Gly Pro Ser Pro His Tyr Pro Pro Pro Pro Ala His Pro Tyr Pro
55370 375 380 385
GGT ATG CTG TTC GCG GGC CCC AGT CCC CTG GAG GCC CAG ATC GCC GCG 1736
Gly Met Leu Phe Ala Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala Ala
390 395 400
CTG GTG GGG GCC ATC GCC GCC GAC CGC CAG GCG GGT GGG CTT CCG GCG 1784
Leu Val Gly Ala Ile Ala Ala Asp Arg Gln Ala Gly Gly Leu Pro Ala
405 410 415
-27-
W 095/06055 216 ~ 7 4 8 PCTrUS9~109303 -
GCC GCC GGA GAC CAC GGG ATC CGG GGG TCG GCG AAG CGC CGC CGA CAC 1832
Ala Ala Gly Asp His Gly Ile Arg Gly Ser Ala Lys Arg Arg Arg His
420 425 430
5 GAG GTG GAG CAG CCG GAG TAC GAC TGC GGC CGT GAC GAG CCG GAC CGG 1880
Glu Val Glu Gln Pro Glu Tyr Asp Cys Gly Arg Asp Glu Pro Asp Arg
435 440 445
GAC TTC CCG TAT TAC CCG GGC GAG GCC CGC CCC GAG CCG CGC CCG GTC 1928
0 Asp Phe Pro Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro Val
450 ~55 460 465
GAC TCC CGG CGC GCC GCG CGC CAG GCT TCC GGG CCC CAC GAA ACC ATC 1976
Asp Ser Arg Arg Ala Ala Arg Gln Ala Ser Gly Pro His Glu Thr Ile
470 475 480
ACG GCG CTG GTG GGG GCG GTG ACG TCC CTG CAG CAG GAA CTG GCG CAC 2024
Thr Ala Leu Val Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala His
485 490 495
ATG CGC GCG CGT ACC CAC GCC CCC TAC GGG CCG TAT CCG CCG GTG GGG 2072
Met Arg Ala Arg Thr His Ala Pro Tyr Gly Pro Tyr Pro Pro Val Gly
500 505 510
25 CCC TAC CAC CAC CCC CAC GCA GAC ACG GAG ACC CCC GCC CAA CCA CCC 2120
Pro Tyr His His Pro His Ala Asp Thr Glu Thr Pro Ala Gln Pro Pro
515 520 . 525
CGC TAC CCC GCC GAG GCC GTC TAT CTG CCG CCG CCG CAC ATC GCC CCC 2168
30 Arg Tyr Pro Ala Glu Ala Val Tyr Leu Pro Pro Pro His Ile Ala Pro
530 535 540 545
CCG GGG CCT CCT CTA TCC GGG GCG GTC CCC CCA CCC TCG TAT CCC CCA 2216
Pro Gly Pro Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro Pro
550 555 560
GTT GCG GTT ACC CCC GGT CCC GCT CCC CCG CTA CAT CAG CCC TCC CCC 2264
Val Ala Val Thr Pro Gly Pro Ala Pro Pro Leu His Gln Pro Ser Pro
565 570 575
GCA CAC GCC CAC CCC CCT CCG CCG CCG CCG GGA CCC ACG CCT CCC CCC 2312
Ala His Ala His Pro Pro Pro Pro Pro Pro Gly Pro Thr Pro Pro Pro
580 585 590
45 GCC GCG AGC TTA CCC CAA CCC GAG GCG CCC GGC GCG GAG GCC GGC GCC 2360
Ala Ala Ser Leu Pro Gln Pro Glu Ala Pro Gly Ala Glu Ala Gly Ala
595 600 605
TTA GTT AAC GCC AGC AGC GCG GCC CAC GTG AAC GTG GAC ACG GCC CGG 2408
50 Leu Val Asn Ala Ser Ser Ala Ala His Val Asn Val Asp Thr Ala Arg
610 615 620 625
GCC GCC GAT CTG TTT GTG TCA CAG ATG ATG GGG TCC CGC TAACTCGCCT 2457
Ala Ala Asp Leu Phe Val Ser Gln Met Met Gly Ser Arg
630 635
CCAGGATCCG AATTC 2472
-28-
~ W 095/06055 2 t 69 718 pcTruss4los3n3
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERI ST ICS:
(A) LENGTH: 638 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ser Ala Glu Met Arg Glu Arg Leu Glu Ala Pro Leu Pro Asp
1 5 10 15
Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
20 25 30
Gly Asp Pro Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala
35 40 45
Leu Pro Pro Glu Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Arg
50 55 60
Cys Glu Val Gly Arg Val Leu Ala Val Val Asn Asp Pro Arg Gly Pro
65 70 75 80
Phe Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu
~ 95
Thr Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Ala Leu Ser
100 105 110
Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val
115 120 125
Ser Leu Ser Thr Lys Arg Arg Gly Asp Glu Val Pro Pro Asp Arg Thr
130 135 140
Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr
145 150 155 160
Ile Val Thr Tyr Asp Thr Ser Leu Asp Ala Ala Ile Ala Pro Phe Arg
165 170 175
His Leu Asp Pro Ala Thr Arg Glu Gly Val Arg Arg Glu Ala Ala Glu
180 185 190
Ala Glu Leu Ala Leu Ala Gly Arg Thr Trp Ala Pro Gly Val Glu Ala
195 200 205
Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg
210 215 220
Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala
225 230 235 240
Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Ile Trp Gly Ala
245 250 255
Glu Ser Ala Pro Ala Pro Glu Arg Gly Tyr Lys Thr Gly Ala Pro Gly
260 265 270
Ala Met Asp Thr Ser Pro Ala Ala Ser Val Pro Ala Pro Gln Val Ala
275 280 285
-29-
W 095/06055 21~ 9 ~ ~ ~ PcTrusg~/093n3 ~
Val Arg Ala Arg Gln Val Ala Ser Ser Ser Ser Ser Ser Ser Ser Phe
290 295 300
5 Pro Ala Pro Ala Asp Met Asn Pro Val Ser Ala Ser Gly Ala Pro Ala
305 310 315 320
Pro Pro Pro ,Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His
325 330 335
Tyr Asn Gln Leu Val Thr Gly Gln Ser Ala Pro Arg His Pro Pro Leu
340 345 350
Thr Ala Cys Gly Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly His Pro
355 360 365
Gly Ala Gly Pro Ser Pro His Tyr Pro Pro Pro Pro Ala His Pro Tyr
370 375 380
20 Pro Gly Met Leu Phe Ala Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala
385 390 395 400
Ala Leu Val Gly Ala Ile Ala Ala Asp Arg Gln Ala Gly Gly Leu Pro
405 410 415
ALa Ala Ala Gly Asp His Gly Ile Arg Gly Ser Ala Lys Arg Arg Arg
420 425 430
His Glu Val Glu Gln Pro Glu Tyr Asp Cys Gly Arg Asp Glu Pro Asp
435 440 445
Arg Asp Phe Pro Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro
450 455 460
Val Asp Ser Arg Arg Ala Ala Arg Gln Ala Ser Gly Pro His Glu Thr
465 470 475 480
Ile Thr Ala Leu Val Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala
485 490 495
His Met Arg Ala Arg Thr His Ala Pro Tyr Gly Pro Tyr Pro Pro Val
500 505 510
Gly Pro Tyr His His Pro His Ala Asp Thr Glu Thr Pro Ala Gln Pro
515 520 525
Pro Arg Tyr Pro Ala Glu Ala Val Tyr Leu Pro Pro Pro His Ile Ala
530 535 540
Pro Pro Gly Pro Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro
545 550 555 560
Pro Val Ala Val Thr Pro Gly Pro Ala Pro Pro Leu His Gln Pro Ser
565 570 575
Pro Ala His Ala His Pro Pro Pro Pro Pro Pro Gly Pro Thr Pro Pro
580 585 590
Pro Ala Ala Ser Leu Pro Gln Pro Glu Ala Pro Gly Ala Glu Ala Gly
595 600 605
Ala Leu Val Asn Ala Ser Ser Ala Ala His Val Asn Val Asp Thr Ala
610 615 620
-30-
W 095/06055 æl 6~7~ PCT~US94/09303
Arg Ala Ala Asp Leu Phe Val Ser Gln Met Met Gly Ser Arg
625 630 635
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Leu Gln Ala Ser
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Val Asn Ala Ser
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ala His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
-31-
W 0 95/06055 2~9~ 4 PCT~US9~/09303 -
txi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
5 Ala Gly Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys
1 5 10 15
t2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 14 amino acids
tB) TYPE: amino acid
tD) TOPOLOGY: linear
tii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~:
Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
ti) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 12 aminc acids
tB) TYPE: amino acid
tD) TOPOLOGY: linear
tii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys
1 5 10
-32-
~ W O9S/06055 ~ 7 ~ 8 pcTrus94lo93n3
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met
1 5 10
(2) IN~ORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp
1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii~ MOLECULE TYPE: pepti-de
-33-
W O 95/06055 ~ 1 ~ 97 48 PCT~US9~/093~3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala
1 5 10 15
s
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ala Leu Val Asn Ala Ser Ser Ala Ala His Val Asp Val Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1908 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
tD) TOPOLOGY: linear
(ii~ MOLECULE TYPE: cDNA
(ix) FEATURE:
~A) NAME/KEY: CDS
(B) LOCATION: 1..1908
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 919..1908
-34-
W 095/06055 1 a ~ ~ ~ 8 PCT~US9~/09303
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATG GCA GCC GAT GCC CCG GGA GAC CGG ATG GAG GAG CCC CTG CCC GAC 48
Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp
S 1 5 10 15
AGG GCC GTG CCC ATT TAC GTG GCT GGG TTT TTG GCC CTG TAT GAC AGC 96
Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
20 25 30
GGG GAC TCG GGC GAG TTG GCA TTG GAT CCG GAT ACG GTG CGG GCG GCC 144
Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala
35 40 45
lS CTG CCT CCG GAT AAC CCA CTC CCG ATT AAC GTG GAC CAC CGC GCT GGC 192
Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly
50 55 60
TGC GAG GTG GGG CGG GTG CTG GCC GTG GTC GAC GAC CCC CGC GGG CCG 240
20 Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro
TTT TTT GTG GGG CTG ATC GCC TGC GTG CAG CTG GAG CGC GTC CTC GAG 288
Phe Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu
85 90 95
ACG GCC GCC AGC GCT GCG ATT TTC GAG CGC CGC GGG CCG CCG CTC TCC 336
Thr Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser
100 105 110
CGG GAG GAG CGC CTG TTG TAC CTG ATC ACC AAC TAC CTG CCC TCG GTC 384
Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val
115 120 125
TCC CTG GCC ACA AAA CGC CTG GGG GGC GAG GCG CAC CCC GAT CGC ACG 432
Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg. Thr
130 135 140
CTG TTC GCG CAC GTC GCG CTG TGC GCG ATC GGG CGG CGC CTC GGC ACT 480
Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr
145 150 155 160
ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC 528
Ile Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg
165 170 175
CAC CTG TCG CCG GCG TCT CGC GAG GGG GCG CGG CGA CTG GCC GCC GAG 576
His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu
180 185 190
GCC GAG CTC GCG CTG TCC GGG CGC ACC TGG GCG CCC GGC GTG GAG GCG 624
Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala
195 200 205
SS CTG ACC CAC ACG CTG CTT TCC ACC GCC GTT AAC AAC ATG ATG CTG CGG 672
Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg
210 215 220
GAC CGC TGG AGC CTG GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC 720
Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala
225 230 235 240
-35-
W 095/06055 ~ 1 ~ 9 ~ ~ $ PCTrUS94/09303 -
GGA CAC ACC TAC CTC CAG GCG AGC GAA AAA TTC AAA ATG TGG GGG GCG 768
Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala
245 250 255
5 GAG CCT GTT TCC GCG CCG GCG CGC GGG TAT AAG AAC GGG GCC CCG GAG 816
Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu
260 265 270
TCC ACG GAC ATA CCG CCC GGC TCG ATC GCT GCC GCG CCG CAG GGT GAC .864
0 Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp
275 280 285
CGG TGC CCA ATC GTC CGT CAG CGC GGG GTC GCC TTG TCC CCG GTA CTG 912
Arg Cys Pro Ile Val Arg Gln Arg Gly Val Ala Leu Ser Pro Val Leu
290 295 300
CCC CCC ATG AAC CCC GTT CCG ACA TCG GGC ACC CCG GCC CCC GCG CCG 960
Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala.Pro
305 310 315 320
CCC GGC GAC GGG AGC TAC CTG TGG ATC CCG GCC TCC CAT TAC AAC CAG 1008
Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln
325 330 335
25 CTC GTC GCC GGC CAT GCC GCG CCC CAA CCC CAG CCG CAT TCC GCG TTT 1056
Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser Ala Phe
340 345 350
GGT TTC CCG GCT GCG GCG GGG TCC GTG GCC TAT GGG CCT CAC GGT GCG 1104
30 Gly Phe Pro Ala Ala Ala Gly Ser Val Ala Tyr Gly Pro His Gly Ala
355 360 365
GGT CTT TCC CAG CAT TAC CCT CCC CAC GTC GCC CAT CAG TAT CCC GGG 1152
Gly Leu Ser Gln His Tyr Pro Pro His Val Ala His Gln Tyr Pro Gly
370 375 380
GTG CTG TTC TCG GGA CCC AGC CCA CTC GAG GCG CAG ATA GCC GCG TTG 1200
Val Leu Phe Ser Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala Ala Leu
385 390 395 400
GTG GGG GCC ATA GCC GCG GAC CGC CAG GCG GGC GGT CAG CCG GCC GCG 1248
Val Gly Ala Ile Ala Ala Asp Arg Gln Ala Gly Gly Gln Pro Ala Ala
405 410 415
45 GGA GAC CCT GGG GTC CGG GGG TCG GGA AAG CGT CGC CGG TAC GAG GCG 1296
Gly Asp Pro Gly Val Arg Gly Ser Gly Lys Arg Arg Arg Tyr Glu Ala
420 425 430
GGG CCG TCG GAG TCC TAC TGC GAC CAG GAC GAA CCG GAC GCG GAC TAC 1344
50 Gly Pro Ser Glu Ser Tyr Cys Asp Gln Asp Glu Pro Asp Ala Asp Tyr
435 440 445
CCG TAC TAC CCC GGG GAG GCT CGA GGC GCG CCG CGC GGG GTC GAC TCC 1392
Pro Tyr Tyr Pro Gly Glu Ala Arg Gly Ala Pro Arg Gly Val Asp Ser
450 455 460
CGG CGC GCG GCC CGC CAT TCT CCC GGG ACC AAC GAG ACC ATC ACG GCG 1440
Arg Arg Ala Ala Arg His Ser Pro Gly Thr Asn Glu Thr Ile Thr Ala
465 470 475 480
CTG ATG GGG GCG GTG ACG TCT CTG CAG CAG GAA CTG GCG CAC ATG CGG 1488
Leu Met Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala His Met Arg
485 490 495
-36-
W O 95/06055 ~ 8 PCT~US9~/09303
GCT CGG ACC AGC GCC CCC TAT GGA ATG TAC ACG CCG GTG GCG CAC TAT 1536
Ala Arg Thr Ser Ala Pro Tyr Gly Met Tyr Thr Pro Val Ala His Tyr
500 505 510
5 CGC CCT CAG GTG GGG GAG CCG GAA CCA ACA ACG ACC CAC CCG GCC CTT 1584
Arg Pro Gln Val Gly Glu Pro Glu Pro Thr Thr Thr His Pro Ala Leu
515 520 525
TGT CCC CCG GAG GCC GTG TAT CGC CCC CCA CCA CAC AGC GCC CCC TAC 1632
10 Cys Pro Pro Glu Ala Val Tyr Arg Pro Pro Pro His Ser Ala Pro Tyr
530 535 540
GGT CCT CCC CAG GGT CCG GCG TCC CAT GCC CCC ACT CCC CCG TAT GCC 1680
Gly Pro Pro Gln Gly Pro Ala Ser His Ala Pro Thr Pro Pro Tyr Ala
545 550 555 560
CCA GCT GCC TGC CCG CCA GGC CCG CCA CCG CCC CCA TGT CCT TCC ACC 1728
Pro Ala Ala Cys Pro Pro Gly Pro Pro Pro Pro Pro Cys Pro Ser Thr
565 570 575
CAG ACG CGC GCC CCT CTA CCG ACG GAG CCC GCG TTC CCC CCC GCC GCC 1776
Gln Thr Arg Ala Pro Leu Pro Thr Glu Pro Ala Phe Pro Pro Ala Ala
580 585 590
25 ACC GGA TCC CAA CCG GAG GCA TCC AAC GCG GAG GCC GGG GCC CTT GTC 1824
Thr Gly Ser Gln Pro Glu Ala Ser Asn Ala Glu Ala Gly Ala Leu Val
595 600 605
AAC GCC AGC AGC GCA GCA CAC GTG GAC GTT GAC ACG GCC CGC GCC GCC 1872
30 Asn Ala Ser Ser Ala Ala His Val Asp Val Asp Thr Ala Arg Ala Ala
610 615 620
GAT TTG TTC GTC TCT CAG ATG ATG GGG GCC CGC TGA 1908
Asp Leu Phe Val Ser Gln Met Met Gly Ala Arg
625 630 635
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 635 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp
1 5 10 15
Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala
35 40 45
Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly
50 55 60
- 60 .
Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro
65 70 75 80
-37-
W 095/06055 2 ~ ~ 9 7 ~ ~ PCTrUS9~109303 -
Phe Phe Val Gly ~eu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu
85 90 95
Thr Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser
100 105 110
Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val
115 120 125
0 Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr
130 135 140
Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr
145 - 150 155 160
Ile Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg
165 170 175
His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu
180 185 190
Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala
195 200 205
25 Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg
210 215 220
Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala
225 230 235 - 240
Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala
245 250 255
Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu
260 265 270
Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp
275 280 285
Arg Cys Pro Ile Val Arg Gln Arg Gly Val Ala Leu Ser Pro Val Leu
290 295 300
Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro
305 310 315 320
Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln
325 330 335
Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser Ala Phe
340 345 350
Gly Phe Pro Ala Ala Ala Gly Ser Val Ala Tyr Gly Pro His Gly Ala
355 360 365
55 Gly Leu Ser Gln His Tyr Pro Pro His Val Ala His Gln Tyr Pro Gly
370 375 380
Val Leu Phe Ser Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala Ala Leu
385 390 395 400
-
Val Gly Ala Ile Ala Ala Asp Arg Gln Ala Gly Gly Gln Pro Ala Ala
405 410 415
-38-
~ W O 95/06055 21 ~ 7~ ~ PCTrUS94/09303
Gly Asp Pro Gly Val Arg Gly Ser Gly Lys Arg Arg Arg Tyr Glu Ala
420 425 430
Gly Pro Ser Glu Ser Tyr Cys Asp Gln Asp Glu Pro Asp Ala Asp Tyr
435 440 445
Pro Tyr Tyr Pro Gly Glu Ala Arg Gly Ala Pro Arg Gly Val Asp Ser
450 455 460
0 Arg Arg Ala Ala Arg His Ser Pro Gly Thr Asn Glu Thr Ile Thr Ala
465 470 475 480
Leu Met Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala His Met Arg
485 490 495
Ala Ar~ Thr Ser Ala Pro Tyr Gly Met Tyr Thr Pro Val Ala His Tyr
500 505 510
Arg Pro Gln Val Gly Glu Pro Glu Pro Thr Thr Thr His Pro Ala Leu
515 520 525
Cys Pro Pro Glu Ala Val Tyr Arg Pro Pro Pro His Ser Ala Pro Tyr
530 535 540
Gly Pro Pro Gln Gly Pro Ala Ser His Ala Pro Thr Pro Pro Tyr Ala
545 550 555 560
Pro Ala Ala Cys Pro Pro Gly Pro Pro Pro Pro Pro Cys Pro Ser Thr
565 570 575
Gln Thr Arg Ala Pro Leu Pro Thr Glu Pro Ala Phe Pro Pro Ala Ala
580 585 590
Thr Gly Ser Gln Pro Glu Ala Ser Asn Ala Glu Ala Gly Ala Leu Val
595 600 605
Asn Ala Ser Ser Ala Ala His Val Asp Val Asp Thr Ala Arg Ala Ala
610 615 620
40 Asp Leu Phe Val Ser Gln Met Met Gly Ala Arg
625 630 635
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
- 60 CCGGTGCCCA ATCGTCCGT 19
-39-
W 095/06055 21~ ~7 ~ PCT~US9~/09303 ~
(2) INFORMATION FOR SEQ ID NO:I9: _.
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 19 base pairs
~B) TYPE: nucleic acid
C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
5 GTCCGTGCGC GTCAAGTGG 19
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TTCCGGCTCC CCCACCTGA 19
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ATTCGGATCC TGGAGGCGA 19
-40-
