U.S. patent application number 10/745718 was filed with the patent office on 2004-09-30 for pharmaceutical compositions comprising herpes virus entry receptor protein.
This patent application is currently assigned to NORTHWESTERN UNIVERSITY. Invention is credited to Montgomery, Rebecca I., Spear, Patricia G..
Application Number | 20040191257 10/745718 |
Document ID | / |
Family ID | 24024996 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040191257 |
Kind Code |
A1 |
Spear, Patricia G. ; et
al. |
September 30, 2004 |
Pharmaceutical compositions comprising herpes virus entry receptor
protein
Abstract
The present invention provides isolated and purified
polynucleotides that encode HVEM of mammalian origin, expression
vectors containing those polynucleotides, host cells transformed
with those expression vectors, a process of making HVEM using those
polynucleotides and vectors, and isolated and purified HVEM.
Inventors: |
Spear, Patricia G.;
(Chicago, IL) ; Montgomery, Rebecca I.; (Hinsdale,
IL) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
NORTHWESTERN UNIVERSITY
|
Family ID: |
24024996 |
Appl. No.: |
10/745718 |
Filed: |
December 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10745718 |
Dec 24, 2003 |
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09924231 |
Aug 8, 2001 |
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09924231 |
Aug 8, 2001 |
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09333279 |
Jun 15, 1999 |
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6303336 |
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09333279 |
Jun 15, 1999 |
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08509024 |
Jul 28, 1995 |
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6291207 |
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Current U.S.
Class: |
424/145.1 |
Current CPC
Class: |
C07K 14/71 20130101;
A61K 38/00 20130101; C07K 14/7151 20130101 |
Class at
Publication: |
424/145.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0002] This invention was make in part using funds obtained from
the U.S. Government (National Institutes of Health Grant Nos. RO1
AI36293 and F32HI09022) and the U.S. Government may have certain
rights in the invention.
Claims
What is claimed is:
1. A pharmaceutical composition comprising a recombinant human HVEM
polypeptide, wherein said polypeptide is encoded by a cDNA
contained within the plasmid pBEC580, designated as ATCC No. 97236,
and a physiologically acceptable diluent.
2. The pharmaceutical composition of claim 1, wherein said HVEM
polypeptide comprises amino acids 1-185 of human HVEM.
3. The pharmaceutical composition of claim 1, wherein said HVEM
polypeptide is soluble.
4. The pharmaceutical composition of claim 3, wherein said soluble
HVEM polypeptide does not comprise a rabbit immunoglobulin heavy
chain amino acid sequence.
5. The pharmaceutical composition of claim 1, wherein said cDNA is
the sequence of SEQ ID NO:1 from nucleotide position 294 to
nucleotide position 1142.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/333,279, which is a divisional of U.S. application Ser. No.
08/509,024, filed on Jul. 28, 1995, both of which are herein
incorporated by reference in their entireties.
TECHNICAL FIELD OF THE INVENTION
[0003] The field of this invention is a herpes virus entry receptor
(HVEM). More particularly, the field of the present invention is
recombinant mammalian HVEM, polynucleotides encoding that HVEM, and
methods of making recombinant HVEM.
BACKGROUND OF THE INVENTION
[0004] Glycosaminoglycan chains on cell surface proteoglycans serve
as receptors for the binding of herpes simplex virus types 1 and 2
(HSV-1 and HSV-2) to cells. Binding is not sufficient for entry,
however: other cell surface components are necessary for virus
entry, which occurs by fusion of the virion envelope with a cell
membrane. For example, Chinese hamster ovary (CHO) cells express
glycosaminoglycan chains to which HSV-1 and HSV-2 can bind, but are
resistant to the entry of some HSV strains, particularly
HSV-1(KOS).
[0005] The present invention is directed to a newly discovered
protein that enables herpes simplex virus (HSV) to penetrate into
cells and is a previously undiscovered member of the family of
receptors designated the tumor necrosis factor receptor/nerve
growth factor receptor (TNFR/NGFR) family. Members of this family
have characteristic repeats of amino acid sequence containing
multiple cysteines and serve as receptors for a variety of specific
ligands, including but not limited to cytokines. The protein is
designated herpes virus entry receptor protein or HVEM.
[0006] By identifying the gene that encodes HVEM, by showing that
HVEM can mediate the entry of HSV into cells and by performing
experiments to define viral and cell factors that influence the
ability of HVEM to mediate HSV entry, the inventors have provided
the knowledge and biological material required (i) to develop
antiviral drugs that can act to block HSV (and perhaps other
herpesvirus) entry into cells; (ii) to identify other members of
the TNFR/NGFR family (or other cell surface molecules) that can
serve as receptors for HSV-1, HSV-2 or other herpesviruses; (iii)
to identify the natural ligand for the receptor, and (iv) to
develop therapeutic approaches for enhancing or inhibiting action
of the ligand on the receptor, depending on the pathologic or,
beneficial consequences of this action.
BRIEF SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides an isolated
and purified polynucleotide comprising a nucleotide sequence
consisting essentially of the nucleotide of SEQ ID NO:1 from about
nucleotide position 294 to about nucleotide position 1142; (b)
sequences that are complementary to the sequences of (a), and (c)
sequences that, when expressed, encode a polypeptide encoded by a
sequence of (a). A preferred polynucleotide is a DNA molecule. In
another embodiment, the polynucleotide is an RNA molecule. A
preferred polynucleotide is SEQ ID NO:1.
[0008] In another embodiment, a DNA molecule of the present
invention is contained in an expression vector. The expression
vector preferably further comprises an enhancer-promoter
operatively linked to the polynucleotide. In an especially
preferred embodiment, the DNA molecule has the nucleotide sequence
of SEQ ID NO:1 from about nucleotide position 294 to about
nucleotide position 1142.
[0009] In another aspect, the present invention provides an
oligonucleotide of from about 15 to about 50 nucleotides containing
a nucleotide sequence of at least 15 nucleotides that is identical
or complementary to a contiguous sequence of a polynucleotide of
this invention. A preferred oligonucleotide is an antisense
oligonucleotide that is complementary to a portion of the
polyrnicleotide of SEQ ID NO:1.
[0010] The present invention also provides a pharmaceutical
composition comprising a polypeptide or an antisense
oligonucleotide of this invention and a physiologically acceptable
diluent.
[0011] In another aspect, the present invention provides an HVEM
polypeptide of mammalian origin. In one embodiment, that HVEM is an
isolated and purified polypeptide of about 300 amino acid residues
and comprises the amino acid residue sequence of SEQ ID NO:2. More
preferably, an HVEM of the present invention is a recombinant human
HVEM.
[0012] In another aspect, the present invention provides a process
of making HVEM comprising transforming a host cell with an
expression vector that comprises a polynucleotide of the present
invention, maintaining the transformed cell for a period of time
sufficient for expression of the HVEM and recovering the HVEM.
Preferably, the host cell is an eukaryotic host cell such as a
mammalian cell or a bacterial cell. An especially preferred host
cell is a mammalian ovarian cell. The present invention also
provides an HVEM made by a process of this invention. A preferred
such HVEM is recombinant human HVEM.
[0013] The present invention still further provides for a host cell
transformed with a polynucleotide or expression vector of this
invention. Preferably, the host cell is a mammalian cell such as an
ovarian cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In the drawings, which form a portion of the
specification:
[0015] FIG. 1 shows a map of the plasmid (pBEC580) cloned from the
cDNA library on the basis of its ability to convert resistant
CHO-K1 cells to susceptibility to HSV-1(KOS) infection. The cDNA
insert prepared as described in the text was ligated between BstXI
and Not I sites in the polylinker region of pcDNAI (shown in the
inset).
[0016] FIG. 2A-2B shows the nucleotide sequence of HVEM cDNA (SEQ
ID NO:1) and amino acid sequence (SEQ ID NO:2) of the open reading
frame. The nucleotide sequence of the cDNA insert of pBEC580 was
determined as shown. Analysis of the sequence revealed the presence
of a single open reading frame, the translation of which is shown
below the nucleotide sequences. Features of the 283 amino acid open
reading frame include a signal peptide (dashed underline), two
potential sites for the addition of N-linked carbohydrate (white
underline) and a hydrophobic region predicted to be a membrane
spanning domain (black underline). Also indicated by the shaded
amino acid sequences are the three complete and one partial
cysteine rich repeats characteristic of members of the TNF/NGF
receptor family.
[0017] FIG. 3 shows a map of the plasmid (PBEC 10) produced by
transferring the cDNA insert of pBEC580 to the vector, pcDNA3
(shown in the inset). The cDNA insert was excised from pBEC580 by
cutting with HindIII and XhoI and was ligated to pcDNA3 that had
also been cut with HindIII and XhoI. The position of the
cytomegalovirus promoter (P-CMV) is shown and also the position of
the selectable marker Neo, along with upstream and downstream
sequences required for its expression in eukaryotic cells.
[0018] FIG. 4A-4B shows susceptibility of HeLa cells and various
CHO cell lines to infection by HSV-1(KOS). The values reported are
the optical density at 410 nm. Each point represents the mean of
triplicate (FIG. 4A) or quadruplicate (FIG. 4B) determinations. The
individual values were within 10% of the mean. FIG. 4A, HeLa cells
(open circles) and CHO-K1 cells (closed circles). FIG. 4B, CHO
cells lines stably transfected with pBEC10, which carries the HVEM
cDNA [CHO-A3 (closed triangles); CHO-A12 (open squares); CHO-B3
(open triangles); CHO-B9 (closed squares); CHO-B11 (open circles)]
and a control cell line stably transfected with the vector pcDNA3
[CHO-C8 (closed circles)].
[0019] FIG. 5 shows replication of three HSV strains in CHO cells
stably transfected with HVEM and in control CHO cells and HeLa
cells. Cells plated in 6-well plates at about 5 .times.10.sup.6
cells per well were inoculated with the virus indicated at 10.sup.8
PFU per well, to ensure that all susceptible cells were
synchronously infected. After allowing 2 hr for virus binding and
entry, the cells were washed and treated with citrate buffer, pH3,
to inactivate input virus that bound to cells but failed to
initiate infection. Culture medium was added and one set of
cultures harvested immediately (2 hr after addition of the virus
inoculum) for quantitation of infectious virus by plaque assay on
Vero cells, to determine the baseline viral titer prior to the
appearance of progeny virus (black bars). The remainder of the
cultures were harvested at 31 hr for quantitation of viral progeny
(diagonal-hatched bars). The values presented represent half the
yield from each culture.
[0020] FIG. 6A-6B shows CHO-A12 cells that express HSV-1 or HSV-2
gD are resistant to HSV-1(KOS) infection. The results shown are for
the amount of plasmid DNA giving maximal interference (1.5 .mu.g
per well for the gD-1 expressing plasmid and 2.0 .mu.g per well for
the gD-2 expressing plasmid). The control plasmid was used at 1.5
.mu.g per well and the CHO-K1 cells were not transfected. The
values given are the means of quadruplicate determinations.
[0021] FIG. 7 shows a map of the plasmid (pBL58) expressing the
HVEM-Fc-hybrid protein.
[0022] FIG. 8A-8C shows the nucleotide sequence (SEQ ID NO:6) of
pBL58 and the amino acid sequence (SEQ ID NO:7) of the open reading
frame encoding HVEM-Fc. Features of the HVEM ectodomain that were
described in FIG. 2A-2B are shown here along with the site at which
the HVEM sequence is fused to the rabbit IgG heavy chain sequence
(the boxed residues are three amino acids inserted at the fusion
site due to the EcoRI linker added). The two potential sites for
the addition of N-linked glycans in HVEM are underlined along with
a third site in the IgG sequence.
[0023] FIG. 9 shows a schematic drawing of human HVEM. The protein
has characteristics of a typical type I membrane glycoprotein,
including an N-terrninal signal sequence (diagonal-hatched box) and
a membrane-spanning region (cross-hatched box). The protein also
has the cysteine-rich repeats characteristic of the TNFR/NGFR
family of cell surface receptors. Each repeat has 4-6 cysteine
residues (represented by vertical lines).
[0024] FIG. 10 shows the relative susceptibilities of A12 cells
transfected with various gD-expressing plasmids.
DETAILED DESCRIPTION OF THE INVENTION
I. The Invention
[0025] The present invention provides isolated and purified
polynucleotides that encode HVEM of mammalian origin, expression
vectors containing those polynucleotides, host cells transformed
with those expression vectors, a process of making HVEM using those
polynucleotides and vectors, and isolated and purified HVEM.
II. HVEM Polynucleotides
[0026] In one aspect, the present invention provides an isolated
and purified polynucleotide that encodes an HVEM polypeptide of
mammalian origin.
[0027] A polynucleotide of the present invention that encodes HVEM
is an isolated and purified polynucleotide that comprises a
nucleotide sequence consisting essentially of the nucleotide
sequence of SEQ ID NO:1 from about nucleotide position 294 to about
nucleotide position 1142 of SEQ ID NO:1, (b) sequences that are
complementary to the sequences of (a), and (c) sequences that, when
expressed, encode a polypeptide encoded by the sequences of (a). A
preferred polynucleotide is a DNA molecule. In another embodiment,
the polynucleotide is an RNA molecule.
[0028] A nucleotide sequence and deduced amino acid residue
sequence of human HVEM are set forth in FIG. 2A-2B. The nucleotide
sequence of SEQ ID NO:1 in FIG. 2A-2B is a full length DNA clone of
human HVEM. SEQ ID NO:2 in FIG. 2A-2B is the deduced amino acid
residue sequence of that clone.
[0029] The present invention also contemplates DNA sequences which
hybridize under stringent hybridization conditions to the DNA
sequences set forth above. Stringent hybridization conditions are
well known in the art and define a degree of sequence identity
greater than about 70%-80%. The present invention also contemplates
naturally occurring allelic variations and mutations of the DNA
sequences set forth above so long as those variations and mutations
code, on expression, for an HVEM of this invention as set forth
hereinafter.
[0030] As set forth above, SEQ ID NO:1, is a full length cDNA clone
of human HVEM. As is well known in the art, because of the
degeneracy of the genetic code, there are numerous other DNA and
RNA molecules that can code for the same polypeptide as those
encoded by SEQ ID NO:1. The present invention, therefore,
contemplates those other DNA and RNA molecules which, on
expression, encode for the polypeptide encoded by SEQ ID NO:1.
Having identified the amino acid residue sequence of HVEM, and with
knowledge of all triplet codons for each particular amino acid
residue, it is possible to describe all such encoding RNA and DNA
sequences. DNA and RNA molecules other than those specifically
disclosed herein and, which molecules are characterized simply by a
change in a codon for a particular amino acid are within the scope
of this invention.
[0031] A Table of codons representing particular amino acids is set
forth below in Table 1.
1 TABLE 1 First Third position position (5' end) Second Position
(3' end) T/U T/U C A G Phe Ser Tyr Cys T/U Phe Ser Tyr Cys C Leu
Ser Stop Stop A Leu Ser Stop Trp G C Leu Pro His Arg T/U Leu Pro
His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser T/U
Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp
Gly T/U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G
[0032] A simple change in a codon for the same amino acid residue
within a polynucleotide will not change the structure of the
encoded polypeptide. By way of example, it can be seen from SEQ D
NO:1(see FIG. 2A-2B) that a CCT codon for proline exists at
nucleotide positions 300-302. It can also be seen from that same
sequence, however, that proline can be encoded by a CCC codon (see
e.g., nucleotide positions 324-326). Substitution of the latter CCC
codon for proline with the CCT codon for proline, or vice versa,
does not substantially alter the DNA sequence of SEQ ID NO:1 and
results in expression of the same polypeptide. In a similar manner,
substitutions of codons for other amino acid residues can be made
in a like manner without departing from the true scope of the
present invention.
[0033] A polynucleotide of the present invention can also be an RNA
molecule. A RNA molecule contemplated by the present invention is
complementary to or hybridizes under stringent conditions to any of
the DNA sequences set forth above. As is well known in the art,
such a RNA molecule is characterized by the base uracil in place of
thymidine. Exemplary and preferred RNA molecules are mRNA molecules
that encode an HVEM of this invention.
[0034] The present invention also contemplates oligonucleotides
from about 15 to about 50 nudeotides in length, which
oligonucleotides serve as primers and hybridization probes for the
screening of DNA libraries and the identification of DNA or RNA
molecules that encode HVEM. Such primers and probes are
characterized in that they will hybridize to polynucleotide
sequences encoding HVEM or related receptor proteins. An
oligonucleotide probe or primer contains a nucleotide sequence of
at least 15 nucleotides that is identical to or complementary to a
contiguous sequence of an HVEM polynucleotide of the present
invention. Thus, where an oligonucleotide probe is 25 nucleotides
in length, at least 15 of those nucleotides are identical or
complementary to a sequence of contiguous nucleotides of an HVEM
polynucleotide of the present invention. Exemplary HVEM
polynucleotides of the present invention are set forth above.
[0035] A preferred oligonucleotide is an antisense oligonucleotide.
The present invention provides a synthetic antisense
oligonucleotide of less than about 50 nucleotides, preferably less
than about 35 nucleotides, more preferably less than about 25
nucleotides and most preferably less than about 20 nucleotides. An
antisense oligonucleotide of the present invention is directed
against a DNA or RNA molecule that encodes HVEM.
[0036] Preferably, the antisense oligonucleotide is directed
against the protein translational initiation site or the
transcriptional start site. In accordance with this preferred
embodiment, an antisense molecule is directed against a region of
SEQ. ID NO:1 from about nucleotide position 254 to about nucleotide
position 334. It is understood by one of ordinary skill in the art
that an antisense oligonucleotide can be directed either against a
DNA or RNA sequence that encodes a specific target. Thus, an
antisense oligonucleotide of the present invention can also be
directed against polynucleotides that are complementary to those
shown in SEQ.ID NO:1 as well as the equivalent RNA molecules.
[0037] Preferably, the nucleotides of an antisense oligonucleotide
are linked by pseudophosphate bonds that are resistant to cleavage
by exonuclease or endonuclease enzymes. Preferably the
pseudophosphate bonds are phosphorothioate bonds. By replacing a
phosphodiester bond with one that is resistant to the action of
exo-and/or endonuclease, the stability of the nucleic acid in the
presence of those enzymes is increased. As used herein,
pseudophosphate bonds include, but are not limited to,
methylphosphonate, phosphomorpholidate, phosphorothioate,
phosphorodithioate and phosphoroselenoate bonds.
[0038] An oligonucleotide primer or probe, as well as an antisense
oligonucleotide of the present invention can be prepared using
standard procedures well known in the art. A preferred method of
polynucleotide synthesis is via cyanoethyl phosphoramidite
chemistry. A detailed description of the preparation, isolation and
purification of polynucleotides encoding human HVEM is set forth
below.
[0039] Briefly, CHO-K1 cells are resistant to the entry of
HSV1(KOS). The present invention discloses an assay to screen for
human cDNAs encoding proteins capable of conferring susceptibility
to HSV1(KOS) infection on the CHO-K1 cells. Control and transfected
CHO-K1 cells were exposed to a strain of HSV-1 (KOS) that had been
modified to express E. coli beta-galactosidase, under control of a
human cytomegalovirus promoter, immediately after viral entry into
a cell. Any transfected cells that became susceptible to HSV-1(KOS)
entry expressed beta-galactosidase after infection. Addition of the
appropriate beta-galactosidase substrate (X-gal) caused the
infected cells to turn blue. The high level of resistance of the
CHO-K1 cells to HSV-1(KOS) infection made it possible to detect
very small numbers of cells rendered susceptible to infection by
transfection of the human cDNAs.
[0040] A commercially obtained unidirectional cDNA library prepared
from human HeLa cell mRNA was used for the transfections. The
plasmids in this libraiy express human proteins under control of
the human cytomegalovirus promoter, after transfection into
eukaryotic cells. The cDNA library was purchased from Invitrogen
Corp (3985 B Sorrento Valley Blvd., San Diego, Calif. 92121):
2 catalog no. A950-10 mRNA source HeLa cells (a human cell line
derived from a carcinoma) primer oligo dT(Not I) vector pcDNAI
[0041] This library was constructed using materials produced by
Invitrogen according to the following protocol:
[0042] mRNA was isolated from the HeLa cells using the Invitrogen
FastTrack.RTM. mRNA Isolation Kit. The mRNA was copied by AMV
reverse transcriptase, using an oligo dT(NotI) primer, to produce
the first strand of DNA. The sequence of this primer is 5'- d
PO.sub.4[AACCCGGCTCGAGCGGCCGCT.sub.18]-3' (SEQ ID NO:3). The
underlined sequence is the NotI site used in a later step for
cleavage of the cDNA and its insertion into the vector in a
directional fashion.
[0043] The product was converted to double-stranded DNA by DNA
polymerase in combination with RNaseH, and E. coli DNA ligase. Any
sticky (single-stranded) ends were made blunt (filled in) by use of
T4 polymerase. A BxtXI/EcoRI adapter was added to the ends by
blunt-end ligation. The sequence of the adapter is:
GAATTCCACCACACTTAAGGTG (SEQ ID NO.:4). The cDNA was cut with BstXI
and NotI and cloned directionally by sticky-end ligation into
pcDNAI, which had also been cut with BstXI and Not I.
[0044] The plasmids were used to transform E. coli strain
MC1061/P3. The number of primaiy recombinant plasmids was estimated
to be about 1.5.times.10.sup.6. The number of colonies in the
amplified library was 4.5.times.10.sup.7 per ml. The estimated size
range of the inserts was 0.9 kb to 1.6 kb.
[0045] 1.5.times.10.sup.7 bacteria were plated (1.5.times.10.sup.5
bacteria per large Petri plate for a total of 100 plates) to allow
the growth of bacterial colonies. The colonies were scraped from
each plate to yield one pool of bacteria from each plate. Samples
of the 100 bacterial pools were mixed to yield 10 mixtures of 10
pools each. Plasmid DNA was extracted from each mixture of pools by
standard means.
[0046] Each plasmid DNA mixture was prepared with LipofectAMINE.TM.
(GibcoBRL), according to the manufacturer's directions, for
transfection into Chinese hamster ovary cells, strain K1
(CHO-K1).
[0047] To determine whether any of the transfected cells became
susceptible to HSV-1(KOS) infection, the transfected cells and
control cells (untransfected or transfected with irrelevant DNA)
were exposed to a mutant form of HSV-1(KOS) at an input dose
sufficiently high to infect all susceptible cells. This mutant is
deleted for one of the essential glycoproteins, gL, and must be
propagated on gL-expressing Vero cells. The virus produced on the
gL-expressing cells is fully infectious but can undergo only one
round of replication because defective virus is produced in
non-complementing cells. The gL open reading frame was replaced by
the E. coli lacZ gene, downstream of the strong cytomegalovirus
promoter. The lacZ gene encodes beta-galactosidase.
[0048] After exposure to virus for several hours, the transfected
CHO-K1 cells were fixed and incubated with the beta-galactosidase
substrate, X-gal. Susceptible cells were readily identified by
their blue color resulting from conversion of the substrate to an
insoluble blue preceipitate by the beta-galactosidase expressed
after entry of the mutant HSV-1(KOS).
[0049] DNA from one mixture of ten pools was found to be positive
for ability to convert some of the transfected CHO-K1 cells to
susceptibility. Each of the ten bacterial pools in this mixture was
tested separately by extracting plasmid DNA and repeating steps set
forth above. Pool 82 was found to be positive.
[0050] Bacterial pool 82 was itself divided into 100 subpools as
described above. It was found that subpool 53 was positive. The
bacteria in subpool 53 were plated and 900 individual clones were
picked and grown up. Plasmids DNAs were extracted from each of the
clones for testing. Clone 580 was found to be positive. Clone 580
was designated pBECS80. A map of this plasmid is shown in FIG.
1.
[0051] The nucleotide sequence of the cDNA insert of pBECS80 was
determined by use of the Sequenase.RTM. Kit (US Biochemical Corp)
according to the manufacturer's instructions.
[0052] The PCgene suite of software from Intelligenetics, Inc. was
used to analyze the nucleotide sequence. As shown in FIG. 2A-2B,
one open reading frame was found in the correct orientation. The
protein encoded in this open reading frame was designated a
herpesvirus entry receptor protein (HVEM) and was found by sequence
analysis to have properties of a type I membrane glycoprotein.
Shown in FIG. 2A-2B are 1) the predicted signal peptide that could
direct translocation of the nascent peptide across membranes of the
rough endoplasmic reticulum; 2) two sites that are signals for the
addition of N-linked carbohydrate; and 3) a hydrophobic region that
is predicted to be a membrane-spanning region, adjacent to a very
basic region which could serve to anchor the protein in a
membrane.
[0053] The blastp and blastn programs were used to search databases
maintained by the National Center for Biotechnology Information
(NCBI), National Library of Medicine, National Institutes of
Health, in Bethesda, Md., for proteins or nucleotide sequences that
might be identical to, or related to, those of the cDNA insert. The
blastp program was used to search for HVEM-related protein
sequences in the database updated daily that contains non-redundant
protein sequences from five component databases (Brookhaven Protein
Data Bank, the SWISS-PROT database, The PIR database, the coding
sequence translations from the GenBank databases and two other
databases that contain cumulative weekly or daily updates,
respectively, of the SWISS-PROT database and the translations from
Gen Bank).
[0054] This search failed to detect any closely related proteins,
indicating that HVEM has not been previously described. The blastp
program identified about 30 proteins that share a characteristic
sequence motif with HVEM, namely three or more cysteine-rich
repeats with a characteristic pattern of 6 cysteine residues. These
other proteins that are related to HVEM by this motif are all
members of the TNF/NGF receptor family. They encode membrane
receptors that can be triggered by the binding of specific ligands
to activate specific pathways important to cell survival, apoptosis
or induced protective responses against infectious agents or
trauma.
[0055] The blastn program identified two entries in the DNA
database (the combined non-redundant database consisting of
nucleotide sequence entries from the Brookhaven Protein Data Bank,
GenBank, the EMEL Data Library and cumulative daily updates of the
GenBank and EMBL databases) that provide partial nucleotide
sequence information for cDNAs that are very closely related to the
cDNA encoding HVEM. One entry (locus HSC0BG042) provides partial
sequence that is closely related to sequence in the 3' non-coding
region of the HVEM cDNA. The other entry (locus HSC0BG041) provides
partial sequence that is closely related to sequence in the 5'
non-coding region and extending 43 amino acids into the N-terminal
region of the HVEM open reading frame, but not extending into the
TNF/NGF receptor motifs.
[0056] The cDNA insert was transferred to another vector, pcDNA3,
which carries a selectable marker (the neomycin gene) that can be
used to isolate cell lines stably carrying the plasmid. Cells that
carry and express this gene are resistant to the toxic effects of a
drug called G418. The cDNA insert of pBEC580 was excised by cutting
with HindIII and Xhol and the insert was ligated to pcDNA3, which
had also been cut with HindIII and XhoI, to produce the new plasmid
called pBEC10. A map of pBEC10 is shown in FIG. 3.
[0057] CHO-K1 cells were transfected with pBEC10 or pcDNA3 and,
after about 48 hours, incubated with medium containing G418. Only
cells carrying the plasmid (with the Neo marker) were able to
survive.
[0058] Several stably transformed colonies of cells were isolated
after transfection with each plasmid and were cloned. None of the
clones obtained with pcDNA3 were susceptible to HSV-1(KOS)
infection. About half of the clones obtained with pBEC10 were
susceptible (the resistant clones may not have been able to express
the protein encoded in the cDNA insert). Cells plated in 96-well
dishes, at densities ranging from 10.sup.4 to 5.times.10.sup.4
cells per well, were exposed to HSV-1(KOS)gL86 in the quantities
indicated. At 6 hr after the addition of virus, the cells were
solubilized with detergent and beta-galactosidase substrate added
to assess the efficiency of viral entry. Expression of
beta-galactosidase signals that the virus has entered a cell and
the amount of enzyme produced is proportional to the number of
infected cells, at least until plateau values of beta-galactosidase
activity are achieved. FIGS. 4A-4B and 5 show that CHO-K1 parental
cells and CHO cells transfected with the control plasmid, pcDNA3,
are resistant to HSV1(KOS) infection whereas the cells transfected
with, and stably cariying pBEC10, are susceptible to HSV-1(KOS)
infection.
[0059] Although the cells transfected with the HVEM cDNA are fully
susceptible to infection by HSV-1(KOS), they are resistant to
infection by a mutant of HSV-1(KOS), designated HSV-1(KOS)rid1,
that differs from parental virus only by an amino acid substitution
in the viral envelope glycoprotein gD. This indicates that gD, at
least in part, determines the ability of virus to use HVEM for
entry. Because HSV-1(KOS) expressing the mutant form of gD can
infect human cells almost as efficiently as parental HSV-1(KOS),
there must be cell surface molecules expressed in human cells, in
addition to HVEM, that can be used for entry.
[0060] CHO-A12 cells in 6-well plates were transfected with
plasmids that express HSV-1 gD (pRE4) or HSV-2 gD (pWW65) under
control of the Rous sarcoma virus promoter or with a control
plasmid consisting of the vector with no insert (pdH). These
plasmids were obtained from G. Cohen and R. Eisenberg (Univ. of
Pennsylvania).
[0061] Transfection was done with the LipofectAMINE.TM. reagent
(GibcoBRL) using plasmid quantities ranging from 0.5 to 2.5 .mu.g
per well. At 24 hr after transfection, the cells were replated in
96-well plates and, 12 hr later, were exposed to HSV-1(KOS)gL86 to
assess the susceptibility of the cells to infection.
[0062] Transfection of HVEM-expressing CHO cells with a plasmid
that expresses wild-type gD (either the HSV-1 or HSV-2 forms of gD)
confers resistance to infection by HSV-1(KOS) (See FIG. 6). This is
an interference activity of gD that has been previously described.
When gD is expressed by the cell, it can render a susceptible cell
resistant to HSV-1 infection, possibly by sequestering a cell
surface receptor needed for HSV-1 entry. The fact that gD
expression renders the HVEM-expressing cells resistant to
HSV-1(KOS) infection suggests that there may be a direct physical
interaction between gD (both the HSV-1 and HSV-2 forms) and
HVEM.
3TABLE 2 below lists the cell lines obtained and summarizes some of
their properties: Plasmid used for Susceptible to infection by:
Cell line transfection HSV-1(KOS) HSV-1(KOS)rid1 HSV-1(F)
HSV-2(333) Cell lines obtained from others or from culture
collections: HeLa (human) None Yes Yes Yes Yes Hep-2 (human) None
Yes Yes Yes Yes CHO-K1 (hamster) None No No Partially Yes New cell
lines CHO-A3 pBEC10 Yes N.T..sup.a N.T..sup.a N.T..sup.a
CHO-A12.sup.b pBEC10 Yes No.sup.c Yes Yes CHO-B3 pBEC10 partially
N.T..sup.a N.T..sup.a N.T..sup.a CHO-B9 pBEC10 Yes N.T..sup.a
N.T..sup.a N.T..sup.a CHO-B11 pBEC10 Yes N.T..sup.a N.T..sup.a
N.T..sup.a CHO-C8 pcDNA3 No No.sup.c N.T..sup.a N.T..sup.a
.sup.aN.T.--Not tested. .sup.bWhen the A12 cells were trausfected
with plasmids expressing HSV-1 or HSV-2 gD, they became resistant
to HSV-1(KOS) infection. .sup.cCHO-K1 cells are slightly more
susceptible to infection by HSV-1(KOS)rid1 than by parental
HSV-1(KOS) but the expression of HVEM in the transfected cells does
not enhance susceptibility of the cells to HSV-1(KOS)rid1, in
marked contrast to the results obtained with HSV-1(KOS).
[0063] Southern blots were done with digests of DNA from three
human cell lines (Hep-2, HeLa and HT1080), one monkey cell line
(Vero), the Chinese hamster overy cell line used for cloning HVEM
(CHO-K1) and two of the CHO cell lines stably tranfected with
pBEC10 (CHO-A12 and CHO-B9). The probes used to detect DNA
fragments with homology to HVEM were an EcoRI fragment of the HVEM
cDNA insert that includes most of the insert and a smaller PvuII
fragment that includes only the 3' end of the HVEM open reading
frame and some of the on-coding sequence downstream. The results
showed that: (i) all three human cell lines contain DNA homologous
to HVEM with fragment sizes that are the same for all three cell
lines in a single digest (different digests yield hybridizable
bands of different sizes but the DNAs from three cell lines are
indistinguishable); (ii) only a subset of the human DNA fragments
that hybridize to the larger EcoRI fragment also hybridize to the
smaller PvuII fragment; (iii) the monkey cells contain weakly
hybridizable DNA fragments of different sizes from those found in
the human DNAs; (iv) the parental CHO-K1 cells contain no
hybridizable DNA fragments; (v) the stably transfected cell lines
(CHO-A12 and CHO-B9) contain DNA homologous to HVEM as
predicted.
[0064] The results obtained with the human, monkey and Chinese
hamster DNAs confirm that HVEM is encoded by a human cDNA and
indicate that the human HVEM gene is probably a single-copy gene
with multiple introns and exons, perhaps extending over a large
stretch of DNA. The results also indicate that monkey cells have a
gene related to human HVEM. If Chinese hamster cells have an HVEM
gene, its sequence has diverged too much to be detected by a human
HVEM probe.
[0065] Poly-adenylated RNAs extracted from various human tissues
(heart, brain, placenta, lung, liver, skeletal muscle, kidney, and
pancreas) were obtained from Clontech, Inc., as samples that had
already been fractionated by electrophoresis and transferred to a
membrane. The membrane was used for hybridization with the larger
EcoRl probe mentioned above (almost the entire HVEM cDNA insert).
The results showed that there were variable amounts of RNA
homologous to HVEM in all the samples. The largest amounts were
found in lung and kidney. The sizes of the bands were about 1.8 and
3.8 kb.
[0066] The HVEM cDNA insert claimed in the application is about 1.8
kb.
III. HVEM Polypeptides
[0067] In another aspect, the present invention provides an HVEM
polypeptide of mammalian origin. An HVEM of the present invention
is a polypeptide of about 300 amino acid residues. Preferably, an
HVEM is a human HVEM. A human form of HVEM is shown in SEQ ID NO:2.
Thus, human HVEM can be defined as a polypeptide of about 293 or
less amino acid residues comprising the amino acid residue sequence
of SEQ ID NO:2.
[0068] The present invention also contemplates amino acid residue
sequences that are substantially duplicative of the sequences set
forth herein such that those sequences demonstrate like biological
activity to disclosed sequences. Such contemplated sequences
include those sequences characterized by a minimal change in amino
acid residue sequence or type (e.g., conservatively substituted
sequences) which insubstantial change does not alter the basic
nature and biological activity of HVEM.
[0069] It is well known in the art that modifications and changes
can be made in the structure of a polypeptide without substantially
altering the biological function of that peptide. For example,
certain amino acids can be substituted for other amino acids in a
given polypeptide without any appreciable loss of function. In
making such changes, substitutions of like amino acid residues can
be made on the basis of relative similarity of side-chain
substituents, for example, their size, charge, hydrophobicity,
hydrophilicity, and the like.
[0070] As detailed in U.S. Pat. No. 4,554,101, incorporated herein
by reference, the following hydrophilicity values have been
assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp
(+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2);Gly (0); Pro
(-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3);
Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp
(-3.4). It is understood that an amino acid residue can be
substituted for another having a similar hydrophilicity value
(e.g., within a value of plus or minus 2.0) and still obtain a
biologically equivalent polypeptide.
[0071] In a similar manner, substitutions can be made on the basis
of similarity in hydropathic index. Each amino acid residue has
been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. Those hydropathic index
values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys
(+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8);
Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln
(-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In
making a substitution based on the hydropathic index, a value of
within plus or minus 2.0 is preferred.
[0072] A comparison of the amino acid sequence SEQ ID No.2 with the
protein databases maintained at the National Library of Medicine
(NIH) and with a computer program designed to detect functional
motifs in proteins revealed that HVEM has not previously been
described, that it is not closely related to other proteins in the
database, but that it has three copies of a cysteine-rich motif
found in members of the TNFR/NGFR family. heavy chain fragment.
This latter fragment was prepared for the ligation by using PCR
technology to insert an EcoRI site just upstream of the following
rabbit sequence (ACAAGACCGTTGCACCCTC) (SEQ ID NO:5). Cleavage at
this EcoRI site, followed by filling-in, permitted blunt-end
ligation to the PvuII site of HVEM so that the two open reading
frames were joined in the same reading frame. The 3' end of the
rabbit cDNA insert was cut with PstI and joined to the HindIII site
of pGEM4 by blunt-end ligation (the PstI cut end was trimmed and
the HindIII cut end was filled in). The vector sequences are from
pGEM4 and include sequences extending to a unique NheI site that
was joined by sticky-end ligation to an SpeI site adjacent to the
cytomegalovirus promoter (P-CMV) from pcDNAneo. The other end of
the P-CMV region was cut with HindIII and is joined to the HindIII
site at the top of the map (FIG. 7).
[0073] Expression of the hybrid protein has been demonstrated both
in Vero cells and in CHO-K1 cells. The protein is secreted into the
medium, as predicted, since it should have a signal sequence for
translocation into the cell's secretory pathway but has no
membrane-spanning region to anchor it to a membrane. The hybrid
protein is readily detected on Western blots by use of commercially
available antibodies specific for the constant regions of rabbit
IgG.
[0074] Treatment of the hybrid protein with various glycosidases
(endoH, endoF and endoO) has revealed that the protein carries
N-linked glycans of the complex type, which is characteristic for
secreted proteins, and also carries O-linked glycans, as predicted.
This hybrid protein is used to screen for mouse monoclonal
antibodies specific for HVEM and to identify HSV proteins with
which it might interact.
[0075] The hybrid gene is contained in two different expression
plasmids, the latter of which contains a selectable marker for
obtaining transformed cells that stably carry the plasmid.
Transfection of these including pGEM3, pGEM4 and pcDNAneo. Starting
from the HindIII site of pBL58, part of the polylinker from pGEM3
(HindIII site to XbaI site) was linked to a sticky end created by
cutting the HVEM insert with NheI about 37 nucleotides upstream of
the HVEM start codon. Another cleavage of the HVEM insert at a
PvuII site within the open reading frame created a blunt end that
was blunt-end ligated to the rabbit IgG heavy chain fragment. This
latter fragment was prepared for the ligation by using PCR
technology to insert an EcoRI site just upstream of the following
rabbit sequence (ACAAGACCGTTGCACCCTC) (SEQ ID NO:5). Cleavage at
this EcoRI site, followed by filling-in, permitted blunt-end
ligation to the PvuII site of HVEM so that the two open reading
frames were joined in the same reading frame. The 3' end of the
rabbit cDNA-insert was cut with PstI and joined to the HindIII site
of pGEM4 by blunt-end ligation (the PstI cut end was trimmed and
the HindIII cut end was filled in). The vector sequences are from
pGEM4 and include sequences extending to a unique NbeI site that
was joined by sticky-end ligation to an SpeI site adjacent to the
cytomegalovirus promoter (P-CMV) from pcDNAneo. The other end of
the P-CMV region was cut with HindIII and is joined to the HindIII
site at the top of the map (See SEQ ID NO:6, FIGS. 8A and 8B).
[0076] Expression of the hybrid protein (SEQ. ID. NO:7, FIGS. 8A
and 8B) has been demonstrated both in Vero cells and in CHO-K 1
cells. The protein is secreted into the medium, as predicted, since
it should have a signal sequence for translocation into the cell's
secretory pathway but has no membrane-spanning region to anchor it
to a membrane. The hybrid protein is readily detected on Western
blots by use of commercially available antibodies specific for the
constant regions of rabbit IgG.
[0077] Treatment of the hybrid protein with various glycosidases
(endoH, endoF and endoO) has revealed that the protein carries
N-linked glycans of the complex type, which is characteristic for
secreted proteins, and also carries O-linked glycans, as predicted.
This hybrid protein is used to screen for mouse monoclonal
antibodies specific for HVEM and to identify HSV proteins with
which it might interact.
[0078] The hybrid gene is contained in two different expression
plasmids, the latter of which contains a selectable marker for
obtaining transformed cells that stably carry the plasmid.
Transfection of these plasmids into cells has revealed expression
of a hybrid polypeptide of molecular weight approximately 60,000
after dissociation into its component chains.
[0079] This hybrid polypeptide, designated HVEM/Fc, carries
N-linked glycans and is expressed as a dimer held together by
disulfide bonds (this is characteristic of hybrid proteins prepared
with IgG domains that can dimerize to form the Fc region).
[0080] Commercially available antibodies specific for rabbit IgG
were used to detect HVEM/Pc in Western blots and in ELISA
assays.
[0081] The observed apparent size of the hybrid protein is similar
to the size predicted, provided the predicted molecular weight
includes about 10,000 for the added carbohydrate.
[0082] Evidence has been obtained that HVEM is only one of several
cell surface receptors that can mediate the entry of HSV-1 and
HSV-2 into cells and that functional use of HVEM (and perhaps other
receptors) is determined by the structure of the virion envelope
glycoprotein gD. A mutant of HSV-1(KOS), designated HSV-1(KOS)rid1
has a single amino acid substitution in gD that confers resistance
to gD-mediated interference with HSV infection and alters slightly
the ability of this virus, relative to the wild-type parental
strain, to penetrate into various cell types including CHO cells
and human cells. By use of a mutant strain of HSV-1(KOS) that is
deleted for gD and complemented by replication in cells expressing
either the wild-type or mutant form of gD it has been shown that
HVEM expression renders CHO cells fully susceptible to infection by
virus carrying wild-type gD but not to infection by virus carrying
mutant gD, despite the fact that both viruses could infect human
cells with nearly equal efficiency. The implications of this result
are several-fold.
[0083] First, the result shows that the structure of gD determines
whether HVEM can be used as a receptor for entry, raising the
possibility of a direct physical interaction. This is consistent
with knowledge that gD is one of at least four envelope
glycoproteins required for HSV entry. Second, although HVEM is
expressed in cultured human cells, such as HeLa cells (the cDNA
library used was prepared from HeLa cells), there must be other
receptors expressed in human cells that can facilitate the entry of
HSV-1(KOS) carrying the mutant form of gD. Third, because CHO-K1
cells are so resistant to HSV-1(KOS) carrying the rid1 form of gD,
it is possible to use the gD-negative mutant of HSV-1(KOS), which
expresses beta-galactosidase and can be complemented with the rid1
form of gD, to screen for expression of the human gene or genes
that can facilitate the entry of HSV-1(KOS)rid1 into CHO-K1
cells.
[0084] The possibility exists that several members of the TNFR/NGFR
family can serve as receptors for entry of HSV-1, HSV-2 or other
herpesviruses, and that the particular receptor favored by a given
herpesvirus or strain is determined at least in part by the
structure of gD.
[0085] Expression of HVEM in CHO-K1 cells significantly enhances
the entry of at least two HSV-1 strains. Because the original CHO
cells are fully susceptible to entry of the HSV-2 strains tested,
it is not possible to assess directly whether HVEM has any effect
on HSV-2 entry into CHO-K1 cells. Cells expressing HSV gD become
resistant, however, to HSV-1 and HSV-2 infection, and also to
infection with related alphaherpesviruses, because of a block to
penetration (binding is unimpaired by gD expression). This
phenomenon has been called gD-mediated interference.
[0086] The fact that HSV-1 gD can interfere with infection by
HSV-1, HSV-2or other herpesviruses implies that all the
herpesviruses may use an overlapping set of receptors for entry.
Transient expression of gD in CHO cells already expressing HVEM
renders the cells resistant to HSV-1(KOS) entry. Both the HSV-2 and
HSV-1 forms of wild-type gD are able to interfere with ability of
HSV-1(KOS) to use HVEM for entry, suggesting that both forms can
interact with HVEM for interference and possibly also for entry. As
predicted from the hypothesis about the mechanism of interference,
the rid1 form of gD is impaired in ability to mediate interference
in HVEM-expressing CHO cells (consistent with the finding that
virus carrying the rid1 form of gD is impaired in ability to enter
HVEM-expressing CHO cells).
[0087] The interference activity of gD can be quantitated by
transfecting gD-expressin gplasmids into HVEM-expressing CHO cells
and then exposing the cells to HSV-1(KOS)gL86 to determine whether
the cells are susceptible or resistant to viral entry. This
provides an assay for testing the interference activity of various
fD mutants, in order to define the structural features of fD that
are required for interference. A number of mutant forms of gD
produced by others (G. Cohen and R. Eisenberg of the Univ. of
Pennsylvania) have already been tested by others to determine
whether the mutations alter gD function in the virion (function
required for viral entry into cells). These same mutant forms of gD
(provided by G. Cohen and R. Eisenberg) are being tested for their
interference activity. Results obtained to date are summarized in
FIG. 10. CHO-A12 cells, which stably express HVEM, were plated on
6-well dishes and transfected with one of several plasmids that
express different forms of gD. At 24 hr after transfection, the
cells were replated in 96-well plates and 12 hr later they were
exposed to HSV-1(KOS)gL86 at several concentrations. At 6 hr after
adding virus, the cells were solubilized and .beta.-galactosidase
substrate was added.
[0088] The colored product was quantitated by spectromophotometry.
Selecting the values obtained (OD410) at a dose of virus where the
amount of virus added was directly proportional to the amount of
.beta.-galactosidase detected, the data were normalized for
comparison by dividing the b-values obtained for cells transfected
with a gD-expressing plasmid by the value obtained for cells
transfected with a control plasmid (control). The forms of gD
expressed by the various plasmids were wild-type gD-1 (pRE4), which
is 369 amino acids in length, wild-type gD-2 (pWW65), mutant gD-I
deleted for amino acids 196-207 (pWW13), mutant gD-1 deleted for
amino acids 234-244 (pWW17), mutant gD-1 deleted for amino acids
194-287 (pWW49), mutant gD-1 deleted for amino acids 234-287
(pWW52), mutant gD-1 deleted for amino acids 208-287 (pWW61),
mutant gD-1 with a substitution that replaces Glu with Asp at
position 63 (pWW62), mutant gD-1 deleted for amino acids 338-369
(pWW63) and mutant gD-1 with a substitution that replaces Gln with
Pro at position 27 (pMW13). Low galactosidase activity implies that
the transfected gD had interference activity; high activity
indicates that the transfected gD had reduced or no activity. All
plasmids used except pMW13 were obtained from G. Cohen and R.
Eisenberg (Univ. of Pennsylvania). The results indicate that
deletions or alterations of gD between the middle and
membrane-spanning region of the molecule eliminate interference
activity whereas deletion of the cytoplasmic tail of gD and an
amino acid substitution at position 63 are without effect. An amino
acid substitution at position 27 (the rid1 mutation) reduces, but
does not eliminate, interference activity. From the results
obtained to date, it appears that alterations affecting the
function of gD in infectivity also affect its function in
interference. This is consistent with the hypothesis that gD
interference results from competition between cell-associated gD
and virion-associated gD for a common target, possibly HVEM.
[0089] An HVEM polypeptide of the present invention has numerous
uses. By way of example, such a polypeptide can be used in a
screening assay for the identification of drugs or compounds that
inhibit or augment the action of HVEM (e.g., agonist and antagonist
to HSV entry into a cell). A screening assay for the identification
of such compound, therefore, can be established whereby the ability
of a compound to alter the action of HVEM can be determined by
exposing cells to HSV in the presence of a polypeptide of the
present invention and varying amounts of compounds suspected of
inhibiting the activity of HVEM.
[0090] The hybrid protein HVEM/Fc is being used to immunize rabbits
for the production of polyclonal antisera specific for the HVEM
portion of the molecule. In addition the hybrid protein is used to
screen for hybridomas secreting antibodies specific for the HVEM
portion (the mice were immunized with HVEM-expressing CHO cells).
The hybrid protein is used to determine whether a physical
interaction between the hybrid protein and gD or other viral
proteins can be detected. The hybrid protein also has use in
screening expression cDNA libraries for natural ligands of HVEM and
screening compounds for inhibitors of the interaction between HSV
virions and HVEM.
[0091] In addition, an HVEM polypeptide of the present invention
can be used to produce antibodies that immunoreact specifically
with HVEM. Means for producing antibodies are well known in the
art. An antibody directed against HVEM can be a--polyclonal or a
monoclonal antibody.
[0092] Antibodies against HVEM can be prepared by immunizing an
animal with an HVEM polypeptide of the present invention. Means for
immunizing animals for the production of antibodies are well known
in the art. By way of example, a mammal can be injected with an
inoculum that includes a polypeptide as described herein above. The
polypeptide can be included in an inoculum alone or conjugated to a
carrier protein such as keyhole limpet hemocyanin (KLH). The
polypeptide can be suspended, as is well known in the art, in an
adjuvant to enhance the immunogenicity of the polypeptide. Sera
containing immunologically active antibodies are then produced from
the blood of such immunized animals using standard procedures well
known in the art.
[0093] The identification of antibodies that immunoreact
specifically with HVEM is made by exposing sera suspected of
containing such antibodies to a polypeptide of the present
invention to form a conjugate between antibodies and the
polypeptide. The existence of the conjugate is then determined
using standard procedures well known in the art.
[0094] An HVEM polypeptide of the present invention can also be
used to prepare monoclonal antibodies against HVEM and used as a
screening assay to identify such monoclonal antibodies. Monoclonal
antibodies are produced from hybridomas prepared in accordance with
standard techniques such as that described by Kohler et al.
(Nature, 256:495, 1975). Briefly, a suitable mammal (e.g., BALB/c
mouse) is immunized by injection with a polypeptide of the present
invention. After a predetermined period of time, splenocytes are
removed from the mouse and suspended in a cell culture medium. The
splenocytes are then fused with an immortal cell line to form a
hybridoma. The formed hybridomas are grown in cell culture and
screened for their ability to produce a monoclonal antibody against
HVEM. Screening of the cell culture medium is made with a
polypeptide of the present invention.
IV. Method of Making HVEM
[0095] In another, aspect, the present invention provides a process
of making HVEM. In accordance with that process, a suitable host
cell is transformed with a polynucleotide of the present invention.
The transformed cell is maintained for a period of time sufficient
for expression of the HVEM. The formed HVEM is then recovered.
[0096] Means for transforming host cells in a manner such that
those cells produce recombinant polypeptides are well known in the
art. Briefly, a polynucleotide that encodes the desired polypeptide
is placed into an expression vector suitable for a given host cell.
That vector can be a viral vector, phage or plasmid. In a preferred
embodiment, a host cell used to produce HVEM is an eukaryotic host
cell and an expression vector is an eukaryotic expression vector
(i.e., a vector capable of directing expression in a eukaryotic
cell). Such eukaryotic expression vectors are well known in the
art.
[0097] In another embodiment, the host cell is a bacterial cell. An
especially preferred bacterial cell is an E. coli. Thus, a
preferred expression vector is a vector capable of directing
expression in E. coli.
[0098] A polynucleotide of an expression vector of the present
invention is preferably operatively associated or linked with an
enhancer-promoter. A promoter is a region of a DNA molecule
typically within about 100 nucleotide pairs in front of (upstream
of) the point at which transcription begins. That region typically
contains several types of DNA sequence elements that are located in
similar relative positions in different genes. As used herein, the
term "promoter" includes what is referred to in the art as an
upstream promoter region or a promoter of a generalized RNA
polymerase transcription unit.
[0099] Another type of transcription regulatory sequence element is
an enhancer. An An enhancer provides specificity of time, location
and expression level for a particular encoding region (e.g., gene).
A major function of an enhancer is to increase the level of
transcription of a coding sequence in a cell that contains one or
more transcription factors that bind to that enhancer. Unlike a
promoter, an enhancer can function when located at variable
distances from a transcription start site so long as the promoter
is present.
[0100] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" or its grammatical equivalent means that a
regulatory sequence element (e.g. an enhancer-promoter or
transcription terminating region) is connected to a coding sequence
in such a way that the transcription of that coding sequence is
controlled and regulated by that enhancer-promoter. Means for
operatively linking an enhancer-promoter to a coding sequence are
well known in the art.
[0101] An enhancer-promoter used in an expression vector of the
present invention can be any enhancer-promoter that drives
expression in a host cell. By employing an enhancer-promoter with
well known properties, the level of expression can be optimized.
For example, selection of an enhancer-promoter that is active in
specifically transformed cells permits tissue or cell specific
expression of the desired product. Still further, selection of an
enhancer-promoter that is regulated in response to a specific
physiological signal can permit inducible expression.
[0102] A coding sequence of an expression vector is operatively
linked to a transcription terminating region. RNA polymerase
transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few
hundred base pairs downstream of the polyadenylation site serve to
terminate transcription. Those DNA sequences are referred to herein
as transcription-termination regions. Those regions are required
for efficient polyadenylation of transcribed messenger RNA (mRNA).
Enhancer-promoters and transcription-terminating regions are well
known in the art. The selection of a particular enhancer-promoter
or transcription-terminating region will depend, as is also well
known in the art, on the cell to be transformed.
[0103] A clone of the human form of HVEM was identified by DNA
sequence analysis as set forth above. This clone was used in all
subsequent expression studies. HVEM was expressed in CHO-K1 cells
under the control of a human cytomegalovirus promoter.
[0104] Expression vectors containing the encoding DNA sequence for
all or a portion of human HVEM are designated pBEC580, pBEC10, and
pBL58. Both vectors were deposited, under the terms of the Budapest
Treaty, on Jul. 28,1995 in the American Type Culture Collection,
12301 Parklawn Drive, Rockville, Md. 20852, and have been assigned
ATCC Accession Nos:97236 (pBEC580), 97235 (pBEC 10), and 97237
(pBL58).
[0105] The present invention also contemplates a host cell
transformed with a polynucleotide or expression vector of this
invention. Means for transforming cells and polynucleotides and
expression vectors used to transform host cells are set forth
above.
[0106] Preferably, the host cell is an eukaryotic host cell such as
a mammalian cell or a prokaryotic cell such as an E. coli.
V. Pharmaceutical Compositions
[0107] The present invention also provides a pharmaceutical
composition comprising a polypeptide or a polynucleotide of this
invention and a physiologically acceptable diluent.
[0108] In a preferred embodiment, the present invention includes
one or more antisense oligonucleotides or polypeptides, as set
forth above, formulated into compositions together with one or more
non-toxic physiologically tolerable or acceptable diluents,
carriers, adjuvants or vehicles that are collectively referred to
herein as diluents, for parentarel injection, for oral
administration in solid or liquid form, for rectal or topical
administration, or the like.
[0109] The compositions can be administered to humans and animals
either orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, locally, or as a buccal or nasal
spray.
[0110] Compositions suitable for parenteral administration can
comprise physiologically acceptable sterile aqueous or non-aqueous
solutions, dispersions, suspensions or emulsions and sterile
powders for reconstitution into such sterile solutions or
dispersions. Examples of suitable diluents include water, ethanol,
polyols, suitable mixtures thereof, vegetable oils and injectable
organic esters such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersions and by the use of surfactants.
[0111] Compositions can also contain adjuvants such as preserving,
wetting, emulsifying, and dispensing agents. Prevention of the
action of microorganisms can be insured by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, and the like. It may also be desirable to
include isotonic agents, for example, sugars, sodium chloride and
the like. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0112] Besides such inert diluents, the composition can also
include sweetening, flavoring and perfuming agents. Suspensions, in
addition to the active compounds, may contain suspending agents, as
for example, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonit, agar-agar and tragacanth, or mixtures of
these substances, and the like.
[0113] The invention has been described in terms of preferred
embodiments. One of ordinary skill in the art readily appreciates
that changes and modifications can be made to those embodiments
without departing from the true scope of this invention.
Deposit
[0114] Under the terms of the Budapest Treaty on the international
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure, deposit of the plasmids pBEC10, pBEC580 and pBL58
has been made on Jul. 28, 1995, with the American Type Culture
Collection (ATCC) of Rockville, Md. USA, where the deposits were
given ATCC accession Numbers ATCC 97235, ATCC 97236 and ATCC 97237,
respectively.
[0115] Applicant's assignee, Northwestern University, represents
that the ATCC is a depository afforded permanence of the deposit
and ready accessibility thereto by the public if a patent is
granted. All restrictions on the availability to the public of the
material so deposited will be irrevocably removed upon granting of
a patent. The material will be readily available during the
pendency of the patent application to one determined by the
Commissioner to be entitled thereto under 37 C.F.R. .sctn. 1.14 and
35 U.S.C. .sctn. 122. The deposited material will be maintained
with all the care necessary to keep it viable and uncontaminated
for a period of at least five years after the most recent request
for the furnishing of a sample of the deposited material, and in
any case, for a period of at least thirty (30) years after the date
of the deposit or for the enforceable life of the patent, whichever
period is longer. Applicant's assignee acknowledges its duty to
replace the deposit should the depository be unable to furnish a
sample when requested due to the condition of the deposit.
Sequence CWU 1
1
7 1 1724 DNA Homo sapiens 1 ccttcatacc ggcccttccc ctcggctttg
cctggacagc tcctgcctcc cgcagggccc 60 acctgtgtcc cccagcgccg
ctccacccag caggcctgag cccctctctg ctgccagaca 120 ccccctgctg
cccactctcc tgctgctcgg gttctgaggc acagcttgtc acaccgaggc 180
ggattctctt tctctttctc ttctggccca cagccgcagc aatggcgctg agttcctctg
240 ctggagttca tcctgctagc tgggttcccg agctgccggt ctgagcctga
ggcatggagc 300 ctcctggaga ctgggggcct cctccctgga gatccacccc
cagaaccgac gtcttgaggc 360 tggtgctgta tctcaccttc ctgggagccc
cctgctacgc cccagctctg ccgtcctgca 420 aggaggacga gtacccagtg
ggctccgagt gctgccccaa gtgcagtcca ggttatcgtg 480 tgaaggaggc
ctgcggggag ctgacgggca cagtgtgtga accctgccct ccaggcacct 540
acattgccca cctcaatggc ctaagcaagt gtctgcagtg ccaaatgtgt gacccagcca
600 tgggcctgcg cgcgagccgg aactgctcca ggacagagaa cgccgtgtgt
ggctgcagcc 660 caggccactt ctgcatcgtc caggacgggg accactgcgc
cgcgtgccgc gcttacgcca 720 cctccagccc gggccagagg gtgcagaagg
gaggcaccga gagtcaggac accctgtgtc 780 agaactgccc cccggggacc
ttctctccca atgggaccct ggaggaatgt cagcaccaga 840 ccaagtgcag
ctggctggtg acgaaggccg gagctgggac cagcagctcc cactgggtat 900
ggtggtttct ctcagggagc ctcgtcatcg tcattgtttg ctccacagtt ggcctaatca
960 tatgtgtgaa aagaagaaag ccaaggggtg atgtagtcaa ggtgatcgtc
tccgtccagc 1020 ggaaaagaca ggaggcagaa ggtgaggcca cagtcattga
ggccctgcag gcccctccgg 1080 acgtcaccac ggtggccgtg gaggagacaa
taccctcatt cacggggagg agcccaaacc 1140 actgacccac agactctgca
ccccgacgcc agagatacct ggagcgacgg ctgctgaaag 1200 aggctgtcca
cctggcgaaa ccaccggagc ccggaggctt gggggctccg ccctgggctg 1260
gcttccgtct cctccagtgg agggagaggt ggggcccctg ctggggtaga gctggggacg
1320 ccacgtgcca ttcccatggg ccagtgaggg cctggggcct ctgttctgct
gtggcctgag 1380 ctccccagag tcctgaggag gagcgccagt tgcccctcgc
tcacagacca cacacccagc 1440 cctcctgggc cagcccagag ggcccttcag
accccagctg tctgcgcgtc tgactcttgt 1500 ggcctcagca ggacaggccc
cgggcactgc ctcacagcca aggctggact gggttggctg 1560 cagtgtggtg
tttagtggat accacatcgg aagtgatttt ctaaattgga tttgaattcc 1620
ggtcctgtct tctatttgtc atgaaacagt gtatttgggg agatgctgtg ggaggatgta
1680 aatatcttgt ttctcctcaa aaaaaaaaaa aaaaaaaaaa aaaa 1724 2 283
PRT Homo sapiens 2 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp
Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr
Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro
Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys
Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly
Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr
Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95
Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100
105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys
Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr
Ala Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr
Glu Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro Pro Gly
Thr Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln His Gln
Thr Lys Cys Ser Trp Leu Val Thr Lys Ala 180 185 190 Gly Ala Gly Thr
Ser Ser Ser His Trp Val Trp Trp Phe Leu Ser Gly 195 200 205 Ser Leu
Val Ile Val Ile Val Cys Ser Thr Val Gly Leu Ile Ile Cys 210 215 220
Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser 225
230 235 240 Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val
Ile Glu 245 250 255 Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala
Val Glu Glu Thr 260 265 270 Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn
His 275 280 3 21 DNA Homo sapiens 3 aacccggctc gagcggccgc t 21 4 22
DNA Homo sapiens 4 gaattccacc acacttaagg tg 22 5 19 DNA Homo
sapiens 5 acaagaccgt tgcaccctc 19 6 4622 DNA Homo sapiens 6
aagcttgcat gcctgcaggt cgactctagc tgggttcccg agctgccggt ctgagcctga
60 ggcatggagc ctcctggaga ctgggggcct cctccctgga gatccacccc
cagaaccgac 120 gtcttgaggc tggtgctgta tctcaccttc ctgggagccc
cctgctacgc cccagctctg 180 ccgtcctgca aggaggacga gtacccagtg
ggctccgagt gctgccccaa gtgcagtcca 240 ggttatcgtg tgaaggaggc
ctgcggggag ctgacgggca cagtgtgtga accctgccct 300 ccaggcacct
acattgccca cctcaatggc ctaagcaagt gtctgcagtg ccaaatgtgt 360
gacccagcca tgggcctgcg cgcgagccgg aactgctcca ggacagagaa cgccgtgtgt
420 ggctgcagcc caggccactt ctgcatcgtc caggacgggg accactgcgc
cgcgtgccgc 480 gcttacgcca cctccagccc gggccagagg gtgcagaagg
gaggcaccga gagtcaggac 540 accctgtgtc agaactgccc cccggggacc
ttctctccca atgggaccct ggaggaatgt 600 cagcaccaga ccaagtgcag
aattcacaag accgttgcac cctcgacatg cagcaagccc 660 acgtgcccac
cccctgaact cctgggggga ccgtctgtct tcatcttccc cccaaaaccc 720
aaggacaccc tcatgatctc acgcaccccc gaggtcacat gcgtggtggt ggacgtgagc
780 caggatgacc ccgaggtgca gttcacatgg tacataaaca acgagcaggt
gcgcaccgcc 840 cggccgccgc tacgggagca gcagttcaac agcacgatcc
gcgtggtcag caccctcccc 900 atcacgcacc aggactggct gaggggcaag
gagttcaagt gcaaagtcca caacaaggca 960 ctcccggccc ccatcgagaa
aaccatctcc aaagccagag ggcagcccct ggagccgaag 1020 gtctacacca
tgggccctcc ccgggaggag ctgagcagca ggtcggtcag cctgacctgc 1080
atgatcaacg gcttctaccc ttccgacatc tcggtggagt gggagaagaa cgggaaggca
1140 gaggacaact acaagaccac gccggccgtg ctggacagcg acggctccta
cttcctctac 1200 aacaagctct cagtgcccac gagtgagtgg cagcggggcg
acgtcttcac ctgctccgtg 1260 atgcacgagg ccttgcacaa ccactacacg
cagaagtcca tctcccgctc tccgggtaaa 1320 tgagcgctgt gccggcgagc
tgcccctctc cctccccccc acgccgcagc tgtgcacccc 1380 gcacacaaat
aaagcaccca gctctgccct gaacagcttc cggtctccct atagtgagtc 1440
gtattaattt cgataagcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
1500 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
ggtcgttcgg 1560 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
ggttatccac agaatcaggg 1620 gataacgcag gaaagaacat gtgagcaaaa
ggccagcaaa aggccaggaa ccgtaaaaag 1680 gccgcgttgc tggcgttttt
ccataggctc cgcccccctg acgagcatca caaaaatcga 1740 cgctcaagtc
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 1800
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
1860 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
tctcagttcg 1920 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
cccccgttca gcccgaccgc 1980 tgcgccttat ccggtaacta tcgtcttgag
tccaacccgg taagacacga cttatcgcca 2040 ctggcagcag ccactggtaa
caggattagc agagcgaggt atgtaggcgg tgctacagag 2100 ttcttgaagt
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 2160
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
2220 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga 2280 tctcaagaag atcctttgat cttttctacg gggtctgacg
ctcagtggaa cgaaaactca 2340 cgttaaggga ttttggtcat gagattatca
aaaaggatct tcacctagat ccttttaaat 2400 taaaaatgaa gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac 2460 caatgcttaa
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 2520
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
2580 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
aataaaccag 2640 ccagccggaa gggccgagcg cagaagtggt cctgcaactt
tatccgcctc catccagtct 2700 attaattgtt gccgggaagc tagagtaagt
agttcgccag ttaatagttt gcgcaacgtt 2760 gttgccattg ctacaggcat
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 2820 tccggttccc
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 2880
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
2940 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
cttttctgtg 3000 actggtgagt actcaaccaa gtcattctga gaatagtgta
tgcggcgacc gagttgctct 3060 tgcccggcgt caatacggga taataccgcg
ccacatagca gaactttaaa agtgctcatc 3120 attggaaaac gttcttcggg
gcgaaaactc tcaaggatct taccgctgtt gagatccagt 3180 tcgatgtaac
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 3240
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
3300 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
tcagggttat 3360 tgtctcatga gcggatacat atttgaatgt atttagaaaa
ataaacaaat aggggttccg 3420 cgcacatttc cccgaaaagt gccacctgac
gtctaagaaa ccattattat catgacatta 3480 acctataaaa ataggcgtat
cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 3540 gaaaacctct
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 3600
gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
3660 aactatgcgg catcagagca gattgtactg agagtgcacc atatcgacgc
tctcccttat 3720 gcgactcctg cattaggaag cagcccagta gtaggttgag
gccgttgagc accgccgccg 3780 caaggaatgg tgcaaggaga tggcgcccaa
cagtcccccg gccacggggc ctgccaccat 3840 acccacgccg aaacaagcgc
tcatgagccc gaagtggcga gcccgatctt ccccatcggt 3900 gatgtcggcg
atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat 3960
gcgtccggcg tagaggatct ggctagttat taatagtaat caattacggg gtcattagtt
4020 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
gcctggctga 4080 ccgcccaacg acccccgccc attgacgtca ataatgacgt
atgttcccat agtaacgcca 4140 atagggactt tccattgacg tcaatgggtg
gactatttac ggtaaactgc ccacttggca 4200 gtacatcaag tgtatcatat
gccaagtacg ccccctattg acgtcaatga cggtaaatgg 4260 cccgcctggc
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 4320
tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
4380 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
caatgggagt 4440 ttgttttggc accaaaatca acgggacttt ccaaaatgtc
gtaacaactc cgccccattg 4500 acgcaaatgg gcggtaggcg tgtacggtgg
gaggtctata taagcagagc tctctggcta 4560 actagagaac ccactgctta
actggcttat cgaaattaat acgactcact atagggagac 4620 cc 4622 7 419 PRT
Homo sapiens 7 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg
Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu
Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser
Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro
Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu
Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr
Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln
Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105
110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile
115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala
Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu
Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro Pro Gly Thr
Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln His Gln Thr
Lys Cys Arg Ile His Lys Thr Val Ala 180 185 190 Pro Ser Thr Cys Ser
Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly 195 200 205 Gly Pro Ser
Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 210 215 220 Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 225 230
235 240 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
Val 245 250 255 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn
Ser Thr Ile 260 265 270 Arg Val Val Ser Thr Leu Pro Ile Thr His Gln
Asp Trp Leu Arg Gly 275 280 285 Lys Glu Phe Lys Cys Lys Val His Asn
Lys Ala Leu Pro Ala Pro Ile 290 295 300 Glu Lys Thr Ile Ser Lys Ala
Arg Gly Gln Pro Leu Glu Pro Lys Val 305 310 315 320 Tyr Thr Met Gly
Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 325 330 335 Leu Thr
Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 340 345 350
Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala 355
360 365 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser
Val 370 375 380 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys
Ser Val Met 385 390 395 400 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Ile Ser Arg Ser 405 410 415 Pro Gly Lys
* * * * *