Note: Descriptions are shown in the official language in which they were submitted.
~o s4ns4s6 2 i 6 ~ 6 01 pcTluss4lo5s2o
HERPES SIMPLEX VIRUS TYPE-2 PROTEASE
This applir~tion is a c~n~ ;on-in-part of U.S. Serial No. 08/073,819,
filed June 8, 1993, which enjoys co~ w~ hil~ and is incc.ll,ul~t~d herein
by l~r~ ce.
Techni~l Field
The present i.,~,~nlion relates to the ~ n~;r~AI;Qn of a new ellL~rll~ in
the area of herpes simplex virology. More pardcularly, it relates to the
i~lt .";r.,~ ~;on of a protease ~x ~ with h~pes simplex virus type-2 (HSV-
10 2) protease, to nucleic acid s ~ fx s CRf,Q~ g such a protease, and to theCA~ SS;On of the pl~tease by a host cell. The present invendon also relates to
the use of the pr~teolytic activity as a target for and-viral th~,~y.
ulld
lS Human ;nr~;o~c with herpes simplex virus (HSV) are ubiqllit~llc
tl~v~J..lu~ the world. The clinical course of HSV infecdons is e.~ ~e,l~
variable and pli~ ~ inr~1i~n is s,~ -f ~1 or mild enough to be U1U~CG~1~1~1
in a .l.ajfJlil~ of cases. The major clinical co~d;~;onc A~:SC~ t"d with HSV
i lr~cti~ns are gingiVosh~ ;c~ keratids and col~ju--r~ ids, vesiG~ r erupdons
20 of the skin, asepdc .~..R;f~jl;C,, e.~r.e~h~liti~, genital tract infe~tionc, and
n~ l herpes.
HSV falls into two disdnct ~)t~,~s, ~esi~ type 1 and ty~pe 2
(HSV-l and HSV-2, ~ .~ti~cly). The HSV-1 e~ no.-~e speeifies an ~
capsid protein (Gibson et al., J. Virol. 13: 155-165 (1974)) and a set of
2s g~ ~,!;r~lly and ;~ -ol~prqlly related viral capsid proteins have been
irl. Rt;r.~d and desig1~qte~1 i,-r~ t~ cell proteins 35 (ICP35) (Braun et al. J.Virol. 49: 142-153 (1984)).
HSV-2, a .. ~ - . .~ - of the alphaherpesviridae subfq-mily of the
herpesviridae family, has a variable host range, a short l~ ~ludu~ e cycle, and
30 the ability to estq~ a latent inÇ~lion in the sacral ganglia (R~ i7mqn et al. Virology, 2nd Ed. New Yo~; Raven Press 65: 1795-1841 (1990). The HSV-
2 virion is co~ ~sc~d of a ~ cl~p.~)tein core ~ullounded by a capsid, b~ ..RI~
and a lipid ..~ --b-..~e~ These ~tluclul~l ~ealul.,s are dl& ~tf ;Ctir, of all herpes
viruses. The HSV-2 ~..~ r, is a~.p~A~ately 150 lcilob~es in size. It
35 con~ of two colll~ lb, the unique long (IJL), and unique short (Us)
regions (Braun et al. J. Virol 49: 142-153 (1984)). The g~nnme encodes at
least 70 l~luleins which are eA~ ssed during a pludu~ e infection; ho..~
216~GOl
WO 94l29456 pcT/us94lo~s2o
only a few are understood biologically (Roi7mqn et al., supra). The
acquisition of HSV-2 infection is usually the conse.luence of trqn~mi~sion via
genital routes. Under these cil.~ nres, virus replicates in the vaginal tract
or on penile skin sites with seeding of the sacral ganglia (Whitley, Virology,
s 2nd Ed. New York: Raven Press 66: 1843-1887 (1990)). As with HSV-1-
in~ ce~ oral lesions, lecullGnl genital i~-f~l;ol~s are the largest reservoir ofherpes simplex virus type 2.
A major .lla~l,ac~ in developing effecdve therapy against HSV-1 and
HSV-2 has been the failure to discover viral-specific replicative ...echqnicm~
o which can be bl~-1~sd by antiviral agents. The ideal antiviral agent should
hlle,lul)l viral replication at an eSc~l;ql step of the life cycle without
~ignific~ntly altering host-cell m~t~holism. While some progress has been
made in controlling HSV infecti~nc through the use of drugs such as 5-
fluorodeo~yul;dine and ac~clo~/~, no satisfactory l,~ .". .t for HSV infecti~nc
s has been found. Con~ve~.lly, improve,ll~,nt for antiviral therapy is needed in
this area.
Maturation of herpes virus particles is believed to occur through the
f~rm~tion of a ~l~,ca~)sid ~llu.;lulc, which ac-lui,~s DNA and an envelope to
becc.~ an infectious virion (Whitley, supra, 1990; E2~i7m~n, supra, 1990).
E~ol.,ases appear to be e~ to the development of the capsid of the virus.
Consequently, inhibitin~ ~lotcase action will lead to disruption of the lytic
cycle of the virus. Thus, inhihitors of pl~t~ase action are desirable targets for
antiviral therapy.
~ol~ases have recenlly been identifisd for HSV-1 by ~c~i7m~n et al.
(EP 514830 publi~hed No.elll~l 25, 1992, which is incol~olaled herein by
reference) and for cy~ lQvirus (CMV) by Gibson, et al. (WO 93/01291
puhlishsd January 21, 1993, which is incoll,vlated herein by IGrtl~,nce). While
there is some homology ~l~.cen these two prot~es, it is well-established that
alteration of even a single amino acid residue in an c,l~yllle can plofoundly
infln-~nr~e its suscep~ibility toward inhihition by a given agent. Therefore,
est~hli~hment of the plhl~ uClul`t for individual herpes proteases is a vital
- step in the discovery of agents which have inhibjtory activity against a specific
v~rus.
Up to the present time, a protease from HSV type 2 had not been
35 i~1entified
21646(~1
WO 941294s6 PCT/US94/05g20
Sul~ of the Il~f.~ n
The present invention provides a HSV type 2 ~lu~ase. The amino acid
se~lu~nce of the plUtiCa~Se and the DNA sequenr,e, or ~e.e.~ e equivalents
s thereof, which e,n''Qdf'S the protease is shown in Figure 1.
The present in~enliol further provides eA~l~s~ion vectors c~p~s of
,ssing HSV type 2 protease in a host cell. In particular, a DNA se~ e~l
e ~ o~ g a HSV type 2 protease or a portion thereof is operably linked to
suiPbl~ re~ t~ry regions in a vector, . h~ the vector is replic~te~l and
o carried by the host cell. R~ host cells carrying said vectors are
$~1ition~11y provided.
DeS~ )tiOIl of the F~lres
Figure 1 shows the .--lclc~ e and ~l~li ,t~d amino acid se~lu~nces of
15 the HSV-2 g~ ;G region co~ g the coding se~-,e-~çes of the p~u~ase
gene and ICP35 protein. The HSV-2 gene COlll~l;~S a DNA sequ~ce
g at .- ,~l~t;~le 211 and c~ g through ~ ck41;~les 951-1120;
this range is flexible and is meant to denote all ~.lions of the s~.,~,nce whichencode for protease activity. The open Ieading frame for the ICP35, the
20 ~u~ dt~, for the plot~ase, comprises the amino acid s~u~,nce ~l~s~n~ in
Figure 1 ~~ ,n ~ I~lP~1;Aes 1124 and 2119.
Figure 2 is a sc~ ,~ ~t; ~ion of the HSV gene showing the
CA~ ~,s~;on ca~cet~,s used in the examples.
Figure 3 shows the SDS-PAGE for the e A~ ;Oll and self-~.ùces~ g
25 of HSV-2 protease-CKS fusion protein in E. coli.
Figure 4 shows the SDS-PAGE for the e.~ ,ss;on and self-l~f~ce~;
of HSV-2 protease translationally coupled to CKS in E. coli.
Figure 5 is a Western Blot of HSV-2 ~lutcase cAp-ess;on and self-
p~ceC~;..g in E. coli.
Figure 6 is a scl.~-.. ";c il~ n of the conslluclion of pl~m;~
pSSPIl as ~es~be~l in Example 13.
Figure 7 shows the SDS-PAGE for the cA~ ;,sion and self-p.~ces~;ng
of HSV-2 proteasellCP35 1 ~ n~lly coup'ed to CKS in E coli.
Figure 8 is a Western Blot of HSV-2 p.~t~ase e,~ ion and self-
35 ~ )GeC` i~ in S cerevisiae.
2164~01
Wo 94/2g456 ~ PCT/US94/05920
Figure 9 shows the SDS-PAGE for the e~"ession and self-processing
of HSV-2 p,ul~ase in Sf9 cells.
Figure 10 is a Western Blot of HSV-2 protease expression and self-
procescing in Sf9 cells.
s Figure 11 shows the SDS-PAGE for the eAlJlcssion and self-pn,cecc;,-g
of HSV-2 ~,otease in Baculovirus-inf~t~d Trichoplusia ni larvae.
Figure 12 is a Western Blot of HSV-2 p,ut~ asc cAlJlcssion and self-
procç~Ccing in Baculovirus-inÇcct~ Trichoplusia ni larvae.
o Detailed Description
Definitions
The following terms are defined as used herein:
"CKS" refers to CTP:CMP-3-deoxy-D-manno-o~nllos~n~tç cytidylyl
sr~ ,ase, also known in the art as CMP-KDO ~y~lhf l~ce or CKS, an el,Ly~,~
derived from Escherichia coli (E. coli), accor~ling to methoAc known in the art."DNA eA~,~ s~ion vector" is any ~ulO~ OuS elçm~nt c~rahl~ of
replicæting in a host intle~ lle~lly of the host's ch,v...oso~e~ after ?~-litio~ql
sequences of DNA have been incul~ulaled into the ~ulol~u~ s rle...~ 's
g4n. ,.... ....~.~
"Gene" is a se~ of DNA, a portion of which codes for a spe~fic
polypeptide or RNA molç~
"ICP35" refers to a set of g~netic~lly and immllnQlogically related viral
capsid proteins i~l~ntified with the HSV-l and -2 gçnom~s
~UlllO~ iS a DNA sf~ur~ce g~ne~lly Lle~ribe~ as the 5' region of a
25 gene, located pl~in,al to the start codûn. At the pç~"~lel region, Il~-sc,i~lion
or t;~ ssion of an ~ ent gene is i~ l This is r~r~ d to as the
transcription initi~tion site. At the plu"~ r region may be a sequence of
nucleotides that inte~ as a control over the eAp,~s~ion of any operably
linked structural gene or genes.
"Operably linked" is a term for the control exerted by the p,~n,otcr
over the initi~tion of e~ s~ion of the polypeptide encocled by a structural
gene.
"Open reading frame" (ORF) is a DNA sequence co..l~-in;ng a series of
triplets coding for amino acids WilllOUI any te~min~tiQn codons. Sequences of
35 this type are potentially tr~ncl~t~ble into a protein.
~_ 1VO 94lW56 216 4 6 ~1 PCT/USg4/05920
"n~ase" refers to a proteolytic acdvity and the co~ ~nding
en~oll;ng nucleic acid s~ Arxs which are capa~le of cleaving a herpes ViTUS
~c~ ."bly protein ~l~cul~r. The HSV-2 protease gene of the present
inventioncc...~ esaDNA s~ ence be.~ ;..gatnllcle~i~e211 and
s e ~ .. l;.,g through .~urle~ s 951-1120; this range is flPYi~e and is meant to
denote all p~ ions of the ~ -nce which encode for plvlease activity-.
"Tl -~ ; n ;n;l ;g~ - site" is a DNA a ~u~ nce of a ~ lllot~. to
which RNA IJoly~.,l~ binds, lh~ ;";~;gl;i~g ~ ;nn of s~ccc~;ng
codons in a 5' to 3' dh~
"T~ c.;l.!;n~ s~" is a DNA s~"c-~rc, at the end of the
transcript that causes RNA pol~rnl, ~ to terminate l- ~n~. ;p! ;nn
"UL26" refers to that part of the HSV-2 e- -~n~e beL~,~ to encode the
protease and the adjoining IC~P35 gene.
;r~ And Mnl~lllqr ~lnnir~ Of Th~- HSV-2 ~.~.t. .~
The present i~l~ relates to the diCo.~ of a new viral protease
~ ~c~e~ by HSV type 2. The g~ -~o..~e of HSV-2isap~lv;n~ ,1ylS0
kilobases in size. Unlike HSV-l, much of the ~'nn~..c DNA of HSV-2 has not
been sc~ ~A And, to date, only a few ansl~ s genes related to HSV-l
have been ide~l;r.ed within the HSV-2 e~ n~ e~ The pl~t,~S ~A~ scd by
these genes which have been id~ to date include thymidine kinase,
cul,luleins C ~ D, DNA pol~w~ and gl1rgline e-o~.-,cle~ce.. To
de ~- ...;~e .. helh~ a novd protease similar to the l"utease e-ncoded by the UL26 gene of HSV-l could be ide ~ e~ within the HSV-2g~o..~.~ a strategy
2s was devised which utilized the nucleic acid se.~,..- nl enr~i~g the HSV-l
protease gene as a tool for d.,tr~ g ~ ?,ocollc ~lu~ s wi~in the HSV-2
g~ .. HSV-2 DNA was i~ t~1 from Vero cells infected wi~ HSV-2
strain G using the ~,~Jce~ of Straus a al. J. Virol. 40: 51~525 (1981).
A1~ J~ ly 10 ~g of DNA was ~ligrst~ with Bal7tHIe~L~ e and S~a.al~d
on a 1% TBE gel. For ~e l,~.;~: A1;~n and Sosth~n blot analysis, the gel
was stained with ethi~ n bromide, photoL,, ~pl~d and placed on a c4~"l,. "
b'~ning al~y~ C for l,~ r to nitrocell~llose. A nick t~nclAted 32p labeled
- probe of the HSV-l protease gene was added to the blot and left to hybridize
oven~i~t After e~.,s;.~e ~.ashing, the blot was ~.lbje~t~d to autoradiGE;,~hy
35 for 30 ~ lt~ S A sing!e band co"~ g to a~lo~ ely 4.0 lcilob~es in
216~
Wo 94/2g456 - PCT/US94/05920
size was vi~u~1i7PA This sf,~;J~If, ~t of DNA co~ f~A. the putative HSV-2
,rvlease coding s~uence. In order to clone the protease gene from the HSV-2
~ nU...P, a shotgun cloning pfocedufe was employed which utilized the
BamHI-digested HSV-2 ge -v~np~ randomly cloned into the plasmid vector
5 pUCl9. Tlal~r~ llf d b~Gte ;~1 colonies from the ligated pUCl9 vector were
scl~ie.lPd by DNA-DNA colony hybriAi7qti~n. Five HSV-2-specific DNA
clones conl;.il-il-g the pfotease gene were identified by hybri-li7ing the nick-trnclqtfA HSV-l ~lvtsase DNA probe to nitfoce11nlose For vefificqtion of the
HSV-2 DNA sequence co~ q-;nPA within pUCl9, positive bacterial col~niec
10 were picked from a replica plate, grvwn~ and plasmid DNA extracted from
them. A BamHI digest of the DNA de-o~ eA that each clone contqin~A the
4.0 kilobases DNA fragment ç..r~l;i~g the HSV-2 protease gene. To further
define a fragment el~r~o~l;n~ the HSV-2 protease, the 4.0 kilobases fragment
was subjected to several dirÇ~.enl restriction digests and Southern blot
15 analyses. A single 1.5 kilob~s Ban~-SalI fragment conl~in;ng the pfot~ase
gene was iA~ntifi~d by Sollth~fn blot hybfi.l;,~ u- and s~lbclQned into pUCl9.
This plqcmi~ has been ~lecigllqt~d pH2Pro which conlA;ns the entire HSV-2
protease coding se lu~nce.
20 DNA Se~lu~ ,~ce of the HSV-2 r~ol~ase Codin~ Sequence
In order to ~ete- ...;ne the DNA se lu~.~ce of the HSV-2 ~rot~ase coding
sequence, the Ban~l-Sall fragment was cloned into M13mpl8 and M13mpl9
phage RF vectors for single-~l.~ ded DNA sequence analysis. Several phage
clones from tran~Çu~ ed mpl8 and mpl9 plates were picked and sequenced
25 through the first 100 bases to ~ ~-ln;ne the correct ul;e~ l;Qn for sequence
analysis. One mpl9 clone was ccll~lly ~nted and became the focus of the
DNA se4ue .1-;ng analysis. Applv ;..~ ly 1900 bases of the plol~,ase gene
have been sequenced from the Ban~l-Sall mpl9 clone.
The cv~plfte coding s~ue--re for the HSV-2 protease is shown in Figure 1
30 ~I~ ,n nucleul;~es 211-951. The ~IOlllvt~ region for the HSV-2 protease has
also been j~ and maps ~l-._en ~o~ -c 1-210 as shown in Figure 1. The
coding s~lue~re for ICP35 is ~l-.~n nucleotides 112~2119.
The se~luence can be derived from pl~cmi~ pH2proA (which conl~ the
entire HSV-2 plulease coding s~uence along with fl~nking ~rvllluterh~gul~t-~ry
35 sequenres and partial ICP35 coding se~lue -res) or pH2proB (which col.l~;n~ the
2~ 6~6nl
WO 94/29456 PCT/US94/05920
h.i~lg coding sequence for the ~CC~ ~bly protein ICP35). Pl~cm~ pH2proA
and pH2proB were d~po~it~d on April 26, 1993 at the Agricultural Research
Service Patent Culture ~ P~tinnl Peoria, ~linois and have the accession nu
NRRL B-21185 and NRRLB-21186, lcs~li~ely.
s The nucleic acid s~u~ es of the present invention may include a sequenceor portion thereof as ill-~ tr~ in Figure 1. For e~ ple, the sequence may be
either smaller or larger than those ill--~ t~3 in Figure 1 as long as the nucleic acid
se~ nl encodes a fim~tit~n~l equivalent of the pl~tease.
o Protein Se~u~nce of the HSV-2 Protease
The HSV-2 protease coding region enco~lps a polypeptide of a~ x;...Atf,ly
247 amino acids which has the amino acid sequence ~l. s~,nt~,d in Figure 1 be~ en
nucleotide 211 and 951. It is .~ od that the size of the p~l.ase may be
smaller or larger than this range as long as the protein fragment retains its biological
15 or filn~tion~l activity. In r~ tion~ any herpes plVt~ ase co~lA;.~;"g at least 70%
h( molrgy, and preferably 90% k.~...olcg~r, to any conti~loll~ stretch of ten or more
amino acids ~ scnt~d herein which is j~l~te~ from a HSV-2 source is also
in~.n~e~d to be within the scope of the present ih,~ ion. This hom~logy is
.;n~1 by any of the available s~u~nce analysis ~r...~ p~l~s such as
20 that available from DNAstar, ~n~lli~en~tics, the Gen~ti~s C~c...~l-ulel Group of the
Uni~w~ of Wi~on~;.-, and the lilce.
The DNA and d~Juce~l amino acid s~,~-nre of a prercllcd HSV-2
pl~tease and ICP35 are provided in Figure 1. In ~d~1itiol~ to the amino acid
se~quçnce shown, any ...~d;r.. -I;nn of the protein which does not destroy the
25 activity of the protein is spc~ ;r~ ~lly includ~ These m~ylifir~tion~ include,
but are not int~n-l~d to be limited to, ~Yi~lqtion, reduction, and the like. In
~d~1ition, mo~ifir~ion~ to the plill~r shllclul~, itself by d~PlPtion~ ition~ oralteration of the amino acids inccu~lated into the se~uçn~e during tr~nsl~tion
which can be made Wi~ UI de;.ll~ing the activity of the e.l~ll.e fall within
30 the conl~,...plDted scope of the present invention.
Expression of Reco...bh-AI-~ HSV-2 F~ P
- In general terms, the ~l~lu~;!;nn of a lGc~.. hi.~D .t form of HSV-2
typically involves (a) i~l~tinp a DNA that e~co~3es the mature en2~ e, (b)
3s placing the recovered coding sequence in operable linkage with sllit~le
2164601
WO 94/29456 ~ pcTlus94los92o
control sequences in a replicable cA~lcs~ion system; (c) l,ansrollll,llg a suitable
host with the vector; and (d) clllhlfing the tran~r,l,llcd host under conrlitiQnc to
effect the ~ c!;on of the lcccl...bi~ HSV-2 protease.
The control se~lu~,nces, eA~rcssion ~y~t~lllS, and tl~lsÇollnalion methods
s are r1epend~nt on the type of host cell used to express the gene.
Prok~oles are most rl~u.,.lll~ lcplc~enled by various strains of
Escherichia coli. However, other microbial strains may also be used, for
example, Bacillus subdlis, various strains of Pseudomonas, or other b~tefi3l
strains. In such prokaryotic ~tc,ns, pl~cmitl vectors that contain repli.~qti~.no sites and control sequences derived from a species co~ e with the host are
used. For c ~ ple~ E. coli is ty-pically lldn~Çolllled with derivatives of
pBR322, a Fl~cmi~l derived from an E. coli species ~les~ibe~d by Bolivar, et al.,
Gene 2: 95 (1977). Many l~.~o~b;~n~ pl~,t.,;ns have also been eA~ssed in
cultured insect cells by using a baculovirus vector which co~ ;n~ the gene of
lS interest under the control of the polyhe~in pl.,ll~t~r (for review, see Luckow,
V. A. & S~ , M. D. (1988) BioTechnology 6, 47-55). Although the
activity of the polyl,e~in y~ lOt~,~ is quite high in insect cells, it appears to be
even higher in insect larvae (Shieh, T. R. & Bohmf~lk, G. T. (1980)
Biotechnol. Bioeng. 22, 1357-1375). The larval eA~I~ssion system is often an
20 i~ re ~lt~n~tive to the cell culture eA~Ic~sion system for cA~l~ssion ofmilligram quqntities of foreign proteins (E'rice, P. M., Reich~lderfer, C. F.,
Joh~ ol~ B. F., Kilboume, E. D., & Acs, G. (1989) Proc. Natl. Acad. Sci.
86, 1453-1456; Medin, J. A., Hunt, L., Gathy, K., Evans, R. K., & Colem~n,
M. S. (1990) Proc. Natl. Acad. Sci. 87, 2760-2764) because the larval
2s eAl l~,s~ion system circumvents the need for spe~ 7~ large-scale tissue
culture facilities and cA~nsi~e tissue culture media. Infection of insect larvaewith baculovirus can be achieved by injection of the virus into the larval
hemolymph (Medin, et al., 1990, su~ra), or by oral ingestion (Price, et al.,
1989, supra). It has been lepwt~d that infection of larvae by the oral route
30 with re~omhin~nt viruses lacking the polyLe~in protein can be improved by
coinf~;lion with wild-type nuclear polyh~sis virus (Price, et al., 1989,
su~ra.). In some of the eY~mr!es descrihed below, infection of c~bh~g~
looper larvae was achieved using recomhin~n~ virus alone as well as with a
~ ule of rec.ombin~nt and wild type baculoviTuses.
W O 94/29456 21~ 4 6 01 PCT~US94/05920
The present invention also provides suit~ble vectors for the CA~ ,sion
of the HSV-2 p~ .,ase. The co~ .J~;!;on of s~lit~blc vectors CQ~ g the
desired coding and control C~u~nr-es employs standard ligptio~ and restriction
~hniques that are well und~ od in the art. Site-specific DNA cleavage is
5 pclr,~ ed by treating the DNA with the s~lit~ble restriction enzyr;ne under
conAitionc that are generally well-u ~d~ k~oA in the art.
With l~r~. ce to a vector of the present invention, any selecta~lc marker
may be used which is funrtinnsl in E. coli or other sek~te~d host and allows
cells l,~,sÇ~l.lled with a vector of the present invention to be Ai~tin~lic~d from
10 cells not so lla, sÇolllled. A gene that provides a Ao~..;n~n~ ele~t~lr marker for
antibiotic resistPnce in E. coli is such a select~ble maricer. The gene for
P~p:~illin reC;rt~nr,e is espe~ ly I,l.,f~ d. Other DNA se~ -t~ which confer
nce to other antibiotics, inrlnAing a~,l~ll~cin, tylosin, picromycin,
A ~A~ cjl~, viomycin, neo,~cill, tetracycline, chlol~ .h. n;col, h~ olll~n;i
5 and the like, can be used either as l~ ce-n.- nl~ of, or in -~Aitic!n to, the drug
re seg,nrnl Aes~ibed herein.
A ~ Çoll.lil g DNA according to the present invention may include
c4-- ~ ,t~ for its s~l~ l ;on and ~ lir.~.t;r n in bart~i~ espe~lly E. coli,
~.h~.l.,l~ pluJu~-!;on of large 4~ t;l;es of DNA by reFlirqtion in b~rteri~ will20 be f~r-ilitqt~-A In this regard, a ~l~f~llGd DNA of the present invention is a
plq~ which ;nrl--A~s a SG~ Com~ cing the origin of replir~tir~n and
~-np pllin resist~nr~e gene or fr~gTrtent thereof of pl~cmirl pB R322.
Yeast offer an attractive z lt~qtive host system to E. coli. For
e ~..q~lc~ a typical yeast eA~ ion vector will comprise (i) a yeast selecdve
2s marker, (ii) a yeast origin of replir~tinn and (iii) yeast plUIltOt~ and t~ linalo~
s~u~ nees positioneA relative to a unique restriction site in such a way that
G~ ,ssion of HSV-2 protease may be obt~ A For eY~mrle a non-fusion
vector c~cse~e and a fusion vector c~sette col~t~inin~ eleven amino acids from
the amino ~l"lin.-s of the sorbitol deh~d,ug. --~ce (SDH) polypeptide as
describK~d in US. Serial Number 07~998,226 filed Dcc~ Jf.l 30, 1S~92 entitled
"F.nh~nced Yeast Expression Using Re~ ry Control Se lu~,nces From Yeast
Sorbitol Dehy~l~ugen~ce Gene" which is inc~l~latGd by lG~el~,nce herein, may
- be used. Both c~Cse~t~s may be insellGd into a 30 copy yeast plasmid
co~ h~ g the yeast TRPl gene as a select~lc marker and 2 micron origin of
replirqtir,n
216~Q~
WO 94/29456 PCT/US94/05920
- 10-
Any yeast repliration origin known in the art may be used to construct
the vector. For eY~rnrle. the rep1iratiQn region of the natural yeast plasmid 2
micron can be employed. This pl~cm 1 is cryptic in that it confers no readily
Aete~tA~'e phenuly~ and is preænt in about 100 copies per cell.
s For eYA nrle, S. cerevisiae, a con.. ~ n laboratory strain of yeast uæd
for its low toxicity and well known genetic characteristics can be used. This
strain is readily cultivated on a large scale. The ~o.,~hinAI~l DNA en~oAing
the HSV-2 proteaæ of the preænt i~ .,tion is placed under the control of
transcriptional and tr~n~l~tion ~ t;ol and te~ ;n~;on regulatory s~uences
o of the alcohol deh~dlug~-n~ce I gene (as 7Aesc~ibeA in the Example section) and
used to express HSV-2 in any yeast cell capable of l~ Ç ~ n, in~ Aing,
but not limited to, yeast ,~ c that alter reg~ tion, and the like.
The vast majority of yeasts can be culdvated under relatively UlliÇu
c4nA;l;.~nc ~ltili7ing c4.. ~n l~b~ . ~ media and .. ,.,ll.oAc known in the ar~
s As would be ulld~ od by one skilled in the art, the typical growth
~Ui~ n~ of yeast col..~ e an organic carbon co~ A for carbon and
energy, organic or illolganic nillo~n for the s~ l.esis of polypeptides and
nucleic acids, various minf ~lc, and a I~ ul~ of ~ ; "~;nc Such growth
n~ are met by yeast r~ ug~,n baæ (YNB), a cl.e ..:r~lly defined
20 .,.PA;.J~.. which c41.l~;n~ a nu~hr of trace c4 ..-~ l Is~ ;nc, trace ~.-nu-.l~ of
amino acids to stim~ te growth, and the principal minerals pO!Ac~ .- ..
phGsl)h~e, m~.. e~;.. sulfate, sodium chloride, and c~lc;llm chloride. The
I~L ogen source is ;..~ OI~;~J-~ sulfate. The desired carbon source is added at a
c4nce-nl.~l;on of from hh. ~en about 0.5% and ~l~. eel- about 3%. The pH
2s range of the, .~c~ .. " is usually from hl-. een about pH 3.0 and about pH 8.0,
preferably from hl~ n about pH 4.5 and about pH 6.5.
In the examples that follow, se~ of the HSV-2 viral g.-~n~..e
incluAing all or portions of the UL26 gene, believed to encode the protease and
the adjoining ICP35 gene, are cloned into a series of vectors de~ign~A. to give
30 ~,ffi~ient ~ s:,ion in E. coli, S. cerevisiae or insect cells. In each case, the
sel ...--l.l~ to be cloned are ~mrlifieA frûm the viral ~ o...c employing the
polyl..~ ase chain reaction (PCR) as Aessribe~A in US. Patents 4,883,195 and 4,
883,202, the entire Ai~closllres of which are inco.~ cd herein by .ef~ .~ nce.
Since PCR often introduces ch~nges into the prnrlifieA s~u~,nCe, the se~ of
35 UL26 Pmrlifie~A are s~lu~,n~e~ after cloning into the .~sl.ecli~e vectors to
WO 94/2g456 216 ~ 6 01 pcTlus94loss2o
d~ t~ lfille that they match the se~lu~ .-ce obtained from pH2proA and pH2proB,
which carly the ~lnqnlrlified UL26 gene derived from genomic HSV-2 DNA.
Seg,..enl~ of UL26 cloned include the first 247 amino acids, from the N te.lllinus
to the site believed to be cleaved by the HSV-2 p~lease or the first 306 amino
s acids, from the N-lell~ us to the methiQnine residue which is believed to
COll~ ond to the start of the ICP35 gene. Expression of the pl~tease as part of
an operon whelGin the ~lOt~ ase gene is dowll~l,ca,l, of a highly eA~,~ssed gene is
also ~lesc~ibe~ In another eY-q-~nrle, eA~l~s~ion of the entire UL26 gene,
conlrrising the l,lot~,ase and ~1jnCç.~l ICP35 se~ ul~ in yeast is described.
Method of Screenin.~ Canrlidate Antiviral Inhibitor Compounds Usin~ HSV-2
P~t~ ~
The ~lut~ase of the present invention is useful in a s ;l~ning method
for idel-lirying pot~ l herpes viral protease inhibitor co~l)ou"ds, also known
as '~c~u1i~le antiviral inhib;t~)r collll~uu..ds". It is cc.l-le-.~ t~d that this
s~ ening t~hnique will prove useful in the gene~al itlentifir~tion of any
cc--.~ Ac that will serve the ~ul~)ose of inhibiting HSV-2 protease. It is
further c~-~-t~ pl~led that useful co~..pou~ c in this regard will not be limited to
prot~ r~c or peptidyl co...l~ lc but may include s~nllletic organic
20 co~ u~ls which are non-peptidyl in nature and which will be l~CGgl~ and
bound by the p,ut~ase, and serve to inhibit the ellzyllle through a dght bindingor other chpm;~al interaction. The use of such inhibitors to block the action ofthe p 1~ t~,ase will serve to treat or alleviate an HSV-2 infection Tnhibit~rs of
HSV-2 pl~t~ase will be useful by th~mcp~lves or in conjun~;lion with other
2s herpes therapies.
Thus, in these e n~ the present invention is dh~;t~l to a
method for ~1~ t'~ ~--;t~in~ the ability of a c~ndid~te colll~und to inhibit HSV-2
pi~lease, the method comprising: oblaining a con~)osilion comrricing HSV-2
l,lo~ase that is capable of cleaving an a~pl~opliate snbssr~tP in a reaction
30 llliAl~lle, mixing a c~nr~ t~ colll~und with the protease and sllit~'c
substrate; and ~f~h, ...;n;ng whether the c~n~ te cc....l O~ inhibited the
protease from cleaving the substrate.
- An i,ll~ol~nl aspect of the c~nt~ tp~ colll~A~ulld scl~ g assay is the
ability to prepare a ~ tease cc,...l~os;l;on in a relative purified fonn. This is an
35 illlpul~nl aspect of the c~n~ te co...~ n-l s.;l~c.-il-g assay in that without at
2 1 ~
WO 94/29456 PCT/US94/05920
- 12-
least a relatively ~u~irled pl~alalion, one will not be able to assay specifically
for HSV-2 p~lease inhibition, as opl)osed to inhibition by extraneous
s~b~ ces in the assay. In any event, the succescful c A~ ,ssion of the
~co-.-k;-~n~ HSV-2 ~,-Jtcase now allows for the first time the ability to
5 identify new col"~ullds which can be used for inhibiting this herpes-related
protein.
To pc.rO~I" the assay, it will be nr~eS~ y to measure the activity of the
relatively pll~ifi~d HSV-2 l)r~tease in the ~bs~nce of the assayed c~ndid~te
col,l~und relative to the activity in the ~,~ ~nce of the c~nAiA~te colll~ul,d in
order to assess the relative inhibitory c~p~bility of the ç~nAid~te co...l ou -d
F.~ lf.c
Materials and Methods
Standard ~f Ih~: were employed for res~içtion endon~)cle~ce Ai~stion,
15 DNA lig~tion, pl~cmid p,~ ~ ?n E. coli ~ r~,.... ~ ;on and other DNA
manipulation t~çhniques and for SDS-PAGE and Western blotting as desç~ibed
by ~ni~tic et. al., Mt~l,ec~ r ~ning, A La~,alc"y Manual, (2nd Ed.) Cold
Spring Harbor, N.Y. (1989). Plasmid DNAs used for co-L,~,~r~;lion of insect
cell cultures were pl~)àl~ by eqllilihrillm cenhirugation in cesium chln~ide
20 gradients according to Maniatis et. al., supra. DNA fr~gm~ntc were recovered
from low melting te.ll~lalul~ agarose (SeaPlaque Agarose, FMC, Ror~l~rd
ME.). Plasmid plep aLions were done using the Magic DNA p,ep&àlion
S~ llS by Promega. Insertion of DNA fr~gmentc into pUC18 or its
de~ivatives, pKB130, pJO201, etc. often employed the use of X-gal as a color
25 reagent to screen for the p,Gse.lce of inserts.
The s~d~-l PCR ll~xtUIG co~ ;nC the following c~...pone.-tc: 50 ng
HSV-2 gei o... ^ DNA, 20 mM Tris-HCl pH 8.3, 1.5 mM MgC12, 50 mM KCl,
0.2 mM dATP, dCTP, dTTP and dGTP, 0.4 pmoUml each primer, 10%
fc.. ~ e7 2% glycerol and 1 U Taq DNA polymerase in a 50 111 re~tion The
30 ~ndar~ PCR con~liti~nc can be varied to inclu(le 50 ng HSV-2 genomic DNA,
20 mM Tris-HCl, pH 8.8 10 mM KCl, 10 mM ~.. o,.;.~.. sulfate, 6 mM
ma~..e~;V.-- sulfate, and 0.1% Triton X-100, 0.2 mM dATP, dCTP, dTTP and
dGTP, 0.4 pmoVml each primer. The v~ tiQn can also include 0.1 mg/ml
acetylated BSA, 10% r.~ le, and 2% glycerol. A cc .---~ .iially available
35 Illel...os~ polymerase such as Taq DNA polyl,le.ase (Thennus ~ql nr~
216 ~fiOl
VO 94/29456 PCT/US94/05920
Vent DNA polymerase, or Tth DNA polymerase (Thermus thermophilus) must
also be added. Cycling t~ ~.alul~,s and times are described for each
apFlirqtion Growth of E. coli in L broth and l,~,sÇ~ llalion of plasmid DNA
into E. coli is done as described by Maniatis et al., supra.
s General mPtho~c used in the manipulation of yeast are described by
Sherman et al., Methods in Yeast Genetics; A Lal.o~ato. ~ Manual, Cold
Spring Harbor, N.Y. (1983). Minimal ,,,~ .", contain 0.67% yeast nillogen
base and 2% glllcose Amino acids are added accor~ing to Sherman et al., supra.
T~ Ç... ~ n of yeast is ~esç~ibe~l by Percival et al., Anal. Biochem 163:39
o (1987). T~ srwl~ CQ~ i.. gplqcmi~lcderivedfrompVT100-Uandit's
derivatives were grown selectively at 30C for 48 hrs in minimql liquid ,,,.-Ai..."
co~ 2% glucose as the carbon source. For SDS-PAGE, cell pellets from
10 ml cultures were first washed with 3 ml of glass--lictillPA water, l~ ,el-d~PA
in 1 ml of Tris pH 7.4, 2 mM EDTA, 1 mM PMSF and disrupted with glass
s beads by vortexing for 2 min. An aliquot of the lysate was mixed with an equal volume of 2x sample buffer (125mM TRIS pH 6.8, 4% SDS, 20% glycerol,
1.4M ~ a~l loell~q~ol and 0.004% Bromo Phenol Blue). 20111 aliquot were
P--qli7PA on SAS-PAGE. E~l".,s~ion of the HSV-2 Protease is dc t ..~ d by
Western Blot analysis.
To prepare soluble protein eYt~tc of Sfg insect cells, 2 x 106 cells were
plated in 25 cm2 flasks and il~f~t~ with 1 ml of culture fluid col lA;n;.-f~
recomhinqnt virus plus 4 ml of fresh media. Tnfçctinnc were allowed to proceed
for three days, . fter which time cells were harvested by low speed centrifugation
nd w. shed once with ~.ho~hqte burr~ d saline. Cells were then ~ P~ in
1OO ml of h~lonic lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 1.5 mM
MgC12) and in~ub~t~PA on ice with occa~:o~-ql vortexing for 20 ...~ "es. The
extract was then pelleted in a micr~ruge for 2 minut~s to remove any incol~lble
mqt~iql and used for SDS-PAGE.
Cq-bhqg~. looper moths and larvae (Trichoplusia ni ) were reared accol~ing
30 to the method of Guy, R., Leppla, N., Rye, J., Green, C., Barrette, S. &
Hollien, K. (1985) in "Handbook of Insect Rearing, Vol. II", edited by P.
Singh and R. Moore, Elsevier, Al,l~t~,.dalll. Adults were .~ A;n~d in
en~ growth ehA~ at 28 C, 80% relative h~ idity~ with a 14
hour phol~h~ce and fed a 10% sucrose solution Oviposition oc~ d on
3s paper toweling which was Wl~ppUl around the wire mesh cages. The egg
216~60~
WO 94/29456 - PCT/US94/05920
laden toweling was surface sterili7ed with dilute formqlin and rinsed
thoroughly with water. Eggs were in~Ub~qvt~ at 27 C and 50% relative
hnmir1ity in sealed 2-liter plastic contS~;n.,, ~ for two days. Newly hqtchyl
larvae were ~ Ç~ d onto the surface of freshly made, solirlifi~ insect diet
5 (a wheat germ/soy flour based agar diet) in 30 ml plastic cups with lids.
Details l~ g~ding the forml~lqtion and p~ a~ion of semisynthetic diets for
cvbbqge looper larvae can be found in Guy, et al., 1985, and in Medin, et al.,
1990).
o 12~q~ntc and Enz~ues
Media for growth of bacteria and yeast were puç~l.ased from Difco,
Detroit, Mi~hig,qn All e..L~ .es were purchased from New F.nglqn~ BioLabs,
Be.e,le~ hl)settc; ne~h~s~3 Research Labc,lalc,lies (BRL), ('.qithc.~,~...gMaryland. Zymolyase 60T was pu~Lased from Miles Labulalc.ly, ELlchart,
lS TnAiq-lq Nick Tl~--c~ ;o-- kit and other reagents for Nick trqnClptionc were
obtained from Alll.,.~h~ll Cul~ula~ion, ~rlin~on Heights, lllinc~ic
Th~ *~ hl~ l,ol~..}, ~e.s were ~I~l.,ha~ from New Fnglqn~l Biolabs,
13e~e.1e~, MA, Epicentre T~ o'ogies, M~dicon, WI, Ph~lllacia P-L
Bioch~ ... ^alc Milwaukee, WI, and P~u~llega, Inc. Factor Xa was pulcl~ased
20 from Boehring~o-r-Mpnnhpim~ Tnrliqn~l)olic, IN and used accc.LIlg to
manufacturer's ~;;r,~-;,.nc
Host Cells Cultures and Vectors for F~ s~ion.
Vero cells were grown in Dulbecco's Mo-lified Eagle Me~iulll
25 (DMEM) suppl~ ~ with 10% Fetal Calf Serum. HSV-2 strain G was
oblAin~ from the Am~c ~n Type Culture CollectiQrl (ACT VR-734). Viral
stocks were grown and titered on Vero cells.
E. coli strain XL1-Blue and DHSa cc....j~t~ nl cells for ll~ sÇ~l.l~lion
were purchased from Stratagene and BRL, l~ ;,~;li~rely. Saccharomyces
30 cerevisiae strain YJO ( ura3-52 leu2-3,112 gal4~ gal80~) was ob~ined from
Dr. B. Kohorn, Duke Uni~e.~ , Durham, NC. Yeast vector pVT100-U
(which cQI~lA;nc the yeast alcohol dehydrogenqcel [ADHl] eApl~;,sion c~csette,
a yeast URA3 sele~ marker and the yeast 2~ origin of repli~tion; Gene
52, 225-233, 1987) was obl~inr~l from Dr. D. Thomas, Bioterl-nolcgy
3s Research Tnctitllte~ National Research Council of C~n~ Montreal, Que.,
21G~Ol
WO 94/29456 -- - PCT/US94/05920
C~ A The Baculovirus eA~lession system, incl~lAing Baculogold insect
virus, eA~l~ssion vector pVL1392, Sf9 insect cells, and TMN-FH serum-
supp~ ted culture media were ob~ ed from Pl,~...;ngPn, San Diego,
California. ~AnAling of dssue culture cells and proFA~gAtion of l~cc,...bin~
5 virus were ~lÇ~.l"lcd accol~ing to the suppli-Prs sl~c~;l;r I;o~c as i~ uct~,d in
the acco...l.~..ying manual by C~u~nwald et. al., Baculovirus Expression
Vector System: ~)CCdul~,S and Mell,ods Manual, 2nd Ed. Sf9 insect cell
cultures were n~ A;~d in 75 cm2 dssue culture flasks at 27 C. Cells were
fed with fresh TMN-FH media every 2 to 3 days and split 1:5 once per week.
o Cdls were split into fresh cultures 1 or 2 days prior to their use in tran~f~li~ns
or inr~;lions to insure healthy log phase growth. All viral stocks were stored at
4 C. and pl{~ ~t~P~d from c~ to light.
Synthesis and y~ ;r.cat;o~ of ol~on~ Pl~tiAPc
All olig~n~lcleotiAes were ~yl~ A on an Applied Bio~y~t~ model
380A DNA sy..l1~e~ at the Mole~ Biology Services Facility, Abbott
Lab~ .lies. Crude oligonllcl~potiAp~ pl~ nc were purified by HPLC.
DNA Se~u.,nce Analysic
For DNA s~ c;~ of positive colc~nies C4~1Ainine the putative HSV-
2 ~ ,~ase and ICP35 genes, BaniHI-Sal I, BamHI, and Sal I f~grnP-nt~ were
cloned into M13mpl8 and M13mpl9 phage RF vectors for single-~ d~d
DNA s~uen~e _nalysis. Single-stranded DNA se~ culg was done using the
United Biocl-- ---:r~l S~uen~ce Kit~. DNA se~lu~ ;ng was done from
2s plA~iA DNA by primer walking using All~Alinç f~ ;on, the USB
Se~lu.nase kit and prot~ lC~ and 33P-dATP (DuPonl-NEN, Wilmin~Q~, DE.).
In order to resolve sc~lu~ iing Aiffi~ulties due to high G-C DNA, deaza
nucleotides and altered reaction ccnAiti~nc were employed. The 1A1~1;ng
re~ ^t;~nC were done on ice for 5-10 n~;nut~ s and the le~ slio~ re~ti~nC were
done at 42 C.
T~Y~ e 1
ion of HSV-2 nuck~ c and viral DNA
HSV-2 DNA was icl l~t~ from Vero cells in~i~d with HSV-2 (G) at a
35 mnl~ ;rjjly of 0.001 plaque-forming units per cell using the ~ cedu.~i of
216~601
WO 94/2g456 PCT/US94/05920
16 -
Straus et al. supra (1981). Cells were cQllPcteA spun down at low speed
(3000 rpm) and ~ ded in lX lysis buffer [0.5% NP-40, 3.6 mM CaC12, 5
mM m~..ej!i.--.- acetate, 125 mM KCl, 0.5 mM EDTA (pH 7.5), 6 mM ~
,dp~ nnl, 0.5% de~Aycholate]. Cell lysate was extracted one time with
5 freonby sh~in~for 1 minuteandcenhirugingat 1000rpmfor 10.. ;.. -Jt~sat
4C. The aqueous phase was removed and layered onto a diSCo~ uuc
gradient of 5% and 40% glycerol in lX lysis buffer and spun at 33,000 rpm for
45 ...;~ es After srinning~ cdl pellets were taken up in 2X STE [0.lM Tris-
HCl pH 7.5), 20 mM EDTA, 2% SDS], proteinase K was added to a final
10 conce ~ I;a~ of 200 mg/ml and inrub~t~ at 50C for 30 ~ t~,S Viral DNA
was gently e~h~t~d by washing one time with phenol, one time with a phenol-
ChlOlOrCIlm ~ ul`t, and one time with a chl~,luru~ /isoamyl alcohol mi~lul~.
The upper a~lue~us phase co~ ;ning HSV-2 DNA was carefully removed with
a wide-bore pipette and pl~-;p;l;-t~ in three volumes of ethanol at -20C.
F.Y~ny?lc 2
Sollth~rn Blot ~n~lysi~ of HSV-2 DNA
Southern blctting and hybri~ l;nll was done eSs.~ y as ~lescribeA
by Maniatis et al. su~ra (1982). A~l~ ely 10 ~g of HSV-2 DNA was
20 AigesteA with the restriction e.l~,~llle Ba~ and s~d by gel
eleehul)hol~;~is on a 1% TBE agarose gel for 3 hours at 100V. After
ele~hu~h~ is, the gel was stained with ethiAil~tll bromide, photo~hed, and
placed in a South~rn blotting unit for ~l~,Sre~ to nitrocelllllQse~ The DNA was
d~ulh~ated for S ...;.~u~es in a sQluti~n of 0.25N HCl, dena~ d in a solutiQn
2s of 1.5M NaCl and 0.5M NaOH, and neutrali_ed. A nick-tr~n~ ted probe
derived from the HSV-l protease seq~lence was used for the hybridi7~ti~n
The hybri-li7~tiQn was left overnight, then washed in 2X SSC [300 mM NaCl,
0.90 mM sodium citrate, 3 mM EDTA, pH 7.0]/0.1% SDS for a total of 30
.-.;n~ ,s, dried, and ~ l~se~ to X-ray film. For similar blots, a BaniHI-Sal I
30 restriction digest was used to g.~ nF ~e dirr~.~nt restriction digest 1
Generation HSV-2 ~enomic libraries
For the g_..e.alion of an E. coli derived library of HSV-2 DNA
~t~nomi~ nents, HSV-2 DNA was ~.-bj~;t~ to BamHI, Sal I, and Ban~-
Sal I digests, and the res~llting ~ig~sted DNA was cloned into the pUCl9
35 VeCtQr e ,~ ic restriction as des-"ibed in Straus et al. supra (1981). The
2154~01
Vo 94/29456 PCT/US94/05920
ligated DNA was used to l1~ SÇO~1~1 co...~ l JM109 cells and colc.niec were
picked to ge~ç~ e an HSV-2 derived genomi~ library.
DNA-DNA colony hybrifli7AtiQn
In all cases, colony hyb. ;.~ ;nn was used to identify HSV-2-pl~tcase
s ~ clones in pUCl9. Colcnies derived from the genQmiC library were
replica-plated onto nitroce~ se~ The bacterial colcnies were lysed and
p,~al~d as 1es~ibeA in Maniatis et al., supra, (1982). After neutrAli7~ti~n,
the colony blot was ll~.sr~"~ onto 3 MM paper and baked at 80C for 2
hours. Colony blots were ~,~hyl.. ;~i75~1 for 4 hours, then a nick-trAncl~ted
o probe derived from HSV-1 protease strain F was dena~ d and added to th
p,ehylJ~ i7atiQn solutiQn and left to hybridiæ overnight. After washing, the
blot was dried and autoradioL"~l~hed rosili~e colQnies were picked, grown,
and subjected to ~ ;m~ digest analysis.
Fs- ,le 3
T~ent;fi~at;OD AnA MQ1~111~r ~ nir~ of the HSV-2 ~}Ut~ ~C~.
HSV-2 DNA was jcnl~t~l from Vero cells ;-~fc~t~ with HSV-2 strain
G as ~esc~ l in FY~ . 'e 1, above. Ap~ ~ly 10 ~lg of DNA was
ligested with Ba~ e.-L~ and ~c~t~ on a 1% TBE gel. For the
20 hy~ri-li7Ati~n and South~rn blot analysis, the gel was stained with e~
bromide, photo~ h~ and placed on a sou~ ,.. blottine app&alus for
r~ to nit~cçlllllose A nick-~ cl~t~ 32P-labeled probe of the HSV-l(F)
protease gene was added to the blot and left to hyl"i~lize ovo,rnieht After
eA~nsi~e washing, the blot was sul~;ect~d to autoradio~rhy for 30 ..~;nvt~,s
2s A single band cu~ ~n(line to a~,u~ ely 4.0 Irilûb ~es in si_e was
ViC.l~li7P~l This se~ of DNA col-~A;-~d the putative HSV-2 p~t~,ase gene.
The ~"otease gene was cloned from the HSV-2 g.~ employing a
~hotelln cloning ~loce~lul~ utili7ine the BamHI~igested HSV-2 ge~
la.ldon~ly cloned into the plasmid vector pUCl9. T~ Ç~lllled b.~lc ~
30 colonies from the ligated pUCl9 vector were s.,l~ned by DNA-DNA colony
hybri~1i7~tion Five HSV-2-specifi~ DNA clones col.~ the pr~ase gene
were identified by hybridi~ng the nick-trancl~ted HSV-l protease DNA probe
- to nitrocellulose For verifir~ti~n of the HSV-2 DNA sequence conl~ rd
within pUCl9, positive ~ t~i~l colonies were picked from a replica plate,
35 grown, and p~ DNA e,.ll~t~,d from them. A Ban~II digest of the DNA
216~601
WO 94/294s6 PCT~US94/05920
- 18-
d~...o~ t~A that each clone cont~ined the 4.0 kil~ces DNA fragment
encoding the HSV-2 protease gene. To further define a fragment e~co ling the
HSV-2 ~lul~ase, the 4.0 kilo~aces fragment was subjected to several dirr.,,~"-t
~ectriction digests and Southern blot analyses. A single 1.5 kilobases BamHI-
5 Sal I fragment CQI.I;.i,.i,.g the protease gene was i~lf nlil ;fd by South~n blothybri~l;7~l;ol- and ss~ ned into pUCl9 . Further l~ ion analyses also
revealed a pUC19-derived clone co~ inin a 1.9 kilobases Sal I fragment with
ition~l coding s~uences. This p~ d has been desi~t~d pH2ProA
which CQn~ the entire HSV-2 protease coding ~ u~--ce along with fl~nking
o ~ t~ ry s~u~.. ces and partial ICP35 coding se.lucl ccs.
To idendfy the 3' end of the coding sc~luence of the ICP35 ~se .~1~1y
protein, a similar DNA-DNA colony h~lL.. ;~; ,~l ;on was done using eYi~ting
ICP35 s~lue-~ces as a probe to identify the full-length ICP35 gene. Several
positive clones derived from an HSV-2 Ban~ libraIy were id~ntifieA and
lS s~u~"~ced. This p1~cmi~1 has been designated pH2ProB which col-l~il-c the
-g coding se~lu~ ~e for the ~CC~."hl~ protein ICP35. Together, both
pl~cmi~lc pH2ProA and pH2ProB contain the full-length HSV-2 pl~t~ and
~cc~.m~ly protein ICP35.
F Y Z - ~ ~- 4
DNA Se~u~"~ce of the HSV-2 ~.~h ~! Gene
In order to d~,t~ l.ne the DNA s~ ce of the HSV-2 p~tease gene,
the Bar~-Sal I fragment was cloned into M13mpl8 and M13mpl9 phage RF
vectors for single-sh~de~ DNA sc~luc n~e analysis. Several phage clones from
25 h~r~ ~ mpl8 and mpl9 plates were picked and se~uenced tJ~rough the
first 100 bases to dete- --;ne the correct - ;rnlAl;l)n for se~.,e~-ce analysis. One
mpl9 clone was coll~lly ori~nteA and became the focus of the DNA
se~lu~ nc; i-g analysis. In ~dAition, both the 1.9 kilob ces Sall fragment derived
from pH2ProA and the Barri~ frag~ nt derived from pH2ProB were cloned
30 into M13mpl9 for se~lu~ n~ ;ng,
FY~m~ S
Expression of HSV-2 ~ùtease(1-247) as a C~S Fusion Protein
A c~csette system was utilized to clone portions of the UL26 gene for
35 e"~lession of the plotease gene in E. coli as shown in Figure 2. The c~Csettes
2~ fi~01
NO 94/29456 PCT~US94/05920
- 19-
were PCR ~mplified from HSV-2 g~nomi~ DNA utili7ing ~ with non-
homologous t_ils and applo~liate restrictinn sites for cloning Part A con~
the 5' MET ATG of protease up to a unique AccI site at nucleQtide 177. Part B
conlAinc the AccI site at n~lcleoti~es 177 through 741 which enr,ofles the
5 ~lu~ase gene up to the cleavage site at amino acid 247. Part C con~;n~
nuclç4tirles 725 to n~cleoti~es 921 which çncodes the 3' end of the protease
from a unique Aflm site through to the start of the ICP-35 portion of the
mrle nl~, '
Consl~uclion of pl~c~ l pAlB6 was acheived by PCR ~mplifir~tion of
o parts A and B and cloning into the ~A~l~Ssioll vector pJO201 res~llting in the eAp~ssion of C t~,.,l~nally fused HSV-2 ~/tcase(1-247).
PCR was rc~o...l~lic~d using a llli,~lul~ co~-~A;n;.~g 50 ng HSV-2
g~-nnmie DNA, 20 mM Tris-HCl pH 8.8, 2 mM MgS04, 10 mM KCl, 10 mM
(NH4)2S04, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dTTP, 0.2 mM dGTP,
lS 0.4 IlM each oli~o~ clGo1;de primer, 10% fo~ ..ide 2% glycerol and lU
Vent DNA polymerase in a 50 ~ n Vent was added after heating the
llliAIUl~ at 95 C for 1 minute. Five cycles were ~clr~Jlll~d as follows:
d~ n&lu.alion at 95 C for 30 sec; ~nnP~ling at 44 C for 30 sec; eYtçncir~n at
72 C for 45 sec which were followed by 25 cycles as follows: den&lu~on at
20 95 C for 30 sec, ~nnP~ling at 54 C for 30 sec, çytçncion at 72 C for 45 sec.
A final ey~tçn~ion period of 10 min at 72 C completed the l`~lC~;OI~ All PCR
p~lu~;~ were verified by DNA S~u~ g
The 5' (U~ alll) PCR primer for part A was:
S-TAGAlGAATI~ATAGAA(~ w lA~AT~GC~GAAAlWG1~3AA
2s T~GAGGaGCLlWUaa~f'~3'. It has an EcoRI site for cloning and a
Factor Xa cleavage site at the CKSIHSV-2 jvn. I;on The codons for the first
10 amino acids were ~~ 1 in the third position; the following 21
nucleotides are completely homologous for PCR priming. The 3' primer for
part A was: 5' AGTAGI~AGA~I~lA~l~l~l-lW~W~3'.
30 It has an XbaI site for cloning and 28 .~.~ck4!;~1es of homologous se l!.en~
- ending in the AccI site. The PCR ~l~lucl was 213 nucleotides.
The 5' primer for part B was:
- S-AGTAG~:lAGAGTAGAaC~ Cl~Gl~G~GG-3'. Ithasan XbaI
site for cloning and 22 nUcleoti~es of hnm~l~us se~uence starting from the
35 AccI site. The 3' primer for part B was:
216~601
WO 94/29456 PCT/us94los92o
20 -
SAGAI~;CI~GlTACGaCI~}AAGGTAC~lUl~ L~WAT-~. It has a Psd
site for cloning, a TAA stop codon and 27 nl)c4~otiAeS of homologous
se~lu~nce. The PCR ~r~lucl was 591 nUcleQtiAes~
The eA~l~ission vector was pJO201 which carries the ampicillin
reSict~nce gene and kdsB, the gene which codes for CKS, driven by a m~ifi~A
lac pç~ el. CKS (CMP-KDO SynthPt~ce) is an E. coli ellLylllc involved in
cell wall bio~yllthe~is. The enLylne has been ovel~iA~Iessed in E. coli using a
moAifi~A lac promoter and has been used as a fusion partner to ove~,A~ s a
number of genes (BioTec~ ues,Vol.8, No. S [1990]).
o A m-~ltipl~ cloning site was en~ d at the 3' end of kdsB; the
cassettes were cloned as EcoRVPstI restriction fr~m~ntc Parts A and B were
cloned in pJO201 (4.0 kilo~ces) to give pl~C-mid pAlB6 of 4.8 kilobases. The
pl~clniA was lla"sÇ~Illled in E. coli strain XLl-Blue to give strain SSHPl.
The strain was grown in L Broth to an OD600 of 0.5-1.0, ~ luce~ with
1 mM IPTG and grown for an 1Aitionsl ~16 hours. Expression of
CKS/HSV-2 protease was evaluated by SDS-PAGE; a band cc~ d;l g to
the predicted mole Illsr weight for HSV-2 plu~ase fused to CKS (55
kiloAsl~nc) was ob~ned as shown in Pigure 3. The fusion protdn was
cleaved by Aigesti~?n with factor Xa at an er~-l-e to subst~te ratio of 1:200
(W/W) for 12 hours. Cleavage was evaluated by SDS-PAGE and Western Blots
using an antibody against the CKS p.~te;n; ~ xluc~ion of bands cc~ ,~nding
to the predicted m~le Ill~r weight for HSV-2 protease (26.6 kDa) and for CKS
(28.8 kDa) were obl~ine~l
EY~nu?l~ 6
ProAuctioll of an Antibody to HSV-2 P'~u1~c~
Strain SSHPl co..~ g plqcmi~l pAlB6 (~l~,p&-~d as desribed above)
encoding CKSIHSV-2 ~f~t~se(1-247) was grown for eAp~cssion as described
in F cqmple 5. ~cpa.~ e SDS-PAGE was done on qli~uQtc of whole cell
30 lysates. Proteins were vic~lqli7~d with 0.25 M KCVl mM Aithiothreitol and
then destq-ined with 1 mM ~1ithiothreitol. The band co..~s~ l;ng to the
predicted moleclllqr weight for CKS/HSV-2 protease(l-247), 55 kDa, was
excised and used as the ;--~ nGgen for andbody pr~u~;l;on. The protein was
~en~led in Freund crnlplete adjuvant to appl~A;~AIely 0.1 mg/ml and was
35 used to inject the ~qnimqlc Two rabbits received initial i~ ~ions and three
WO 94/29456 2 ~. ~ 4 5 a 1 PCT/US94/05920
boosts at ~ r~ alely monthly intenrals. Two weeks following the ffnal
boost, the qnimqlc were c ~ ;rice~d and a large bleed was obt~ ed as the source
of antibody. The antibody was preabs~,ll,cd with E. coli ~l-Blue lysate for
48 hours and was used for Western Blots at a Aillltion of 1:1000.
s
FY~U~ 7
Consl. uclion of pAlB6C and Expression of HSV-2 notease 1-307 as a CKS
Fusion Protein
The c~sv!le system Aes~ibeA. in FYq-~nple S and Figure 2 was used to
o construct a CKS fusion to HSV-2 E~,tcase (1-307). Part C was a new PCR
pro~lucl, parts A and B were iA~ntirql to that d~ ;bcd in ~Y~ c 5. PCR
cQnAiti~n~ were as Ae~fibeA in Example 5. The 5' primer for part C was:
S-TAGA~ _ ~ -3'. It conl~ an XbaI
site for cloning, the AJ~m site and 26 nl~cleotiAes of homrlcgous s~u~,nce.
lS The 3' primer was: S-AGAACI~AGITAA~3G~GGlr~J~AAAGAA-3'.
It c~ c a Psd site for clc~nin~ a TAA stop codon and 28 ~.ucl~ol;Aes of
homvlo~oll~ s~ e The PCR 1" lu~l was 223 n~rl~,otiA,e~
Parts A, B and C were cloned in pJO201 to give the 5.0 kilQ~ces pl~niA~
pAlB6C3 which was ~ -rv~ A in E. coli strain XLl-Blue to give strain
20 SSHP2. The strain was grown and evaluated for eA~l.,s~ion as described in
Fl~rnr'~ 1. As shown in Figure 4, a band cc,~ ol~il-g to the l,l~ie~
molef Ill~r weight for the fusion protein (61 kiloA~lt7nc) was obselved on SDS-
PAGE. An ~1Aition~ql band c~ ~nl1;ng to the mrlec~ weight of self-
p,vcess~ CKS/HSV-2 p,ut~ase at amino acid 247 cleavage site (55kDa) was
25 also obs~
FY~mrl~ 8
Construction of pAlB6Cmut and EA~"~s~;on of CKS/HSV-2 with 247
Mutation
A mut~tion was made in HSV-2 ~ole~e at amino acid pos;tion nulll~.
247 converting an alanine residue to a glycine residue at the plvt~ase clip sitethereby elimin~ting ælf-p,~ce-c~ g at amino acid 247. The c~csel~ system
was as described in FY~n~rlc 5
Part CmUt was a new PCR product, parts A and B were identir~l to
35 FY~rnplel.The.. ls.l;~l-wasillcl~l~lat~lintothe5~pcRprimerforpart
216~601
WO 94/29456 PCTIUS94/05920
CmUt. The 5' primer for paTt Cmut was:
~. It
co~ inc an XbaI site for cl~tning~ the Aflm site, the Ala-Gly mutation and 33
nucleotides of hologous se~lucnce. The 3' primer was idçntir~l to the 3'
s primer used forpart C des.;lil~d in EA~11)1C 7. PCR was conAI)cted as
described in Fy~mrle S to give a 223 1 .Icl~4li~1es PCR pl~lu~
Parts A, B and CmUt were cloned in pJO201 to give the 5.0 kilok~ces
pl~cmid pAlB6CmUt which was t~ Ç~ ,ed in XLl-Blue to give strain
SSHP3. The strain was grown and evaluated for cA~ssion as described in
o Example 5. A band coll~ Ain~ to the predicted mole _ul~r weight for the
fusion protein (61 kDa) was observed on SDS-PAGE as shown in Fgure 3. A
55 kDa band which would co~ ond to self-procecce~ CKS/HSV-2 pr~t~ase
was not obsGl ~cd.
FY~nu?le 9
Con~t~ u~:lion of pA2B6 ~nA P~. ~s~ion of HSV-2 Prot~c~.(1-247)
Tr~ncl~tionally Co~led to CKS
HSV-2 protease(l-247) was eAI.l.,ss~d tr~nCl~tinn~lly coupled to CKS.
A rihosc,.~e binding site was il~SG t~ ~h.~n a 40 nl~c1eotiAes 5' fragment of
20 k~sB (described in Example 5) and the gene for HSV-2 protease in order to
l,r~luce non-fused HSV-2 pnJt~ase. The cqC~tte system was as des~ihed in
F.Y~nple S; part A2 was a new PCR product, part B was iA~nti~l to PY~nP1e
5. The 5' primer for part A2 was:
S-TAGAI~I~ACIAAGGA~AI~AI~Al~AGCAGAAAlWG
25 l~AAaGTl'rAGAGG~~ iCmGAaC~3'. It cont~ins a Sall site for
clc-ning, a cO~ s rihosome binding site and a TAA stop codon for CKS
which overlaps with the ATG start site for HSV-2 plotease. The codons for the
first 10 amino acids of HSV-2 protease were optimized for E. coli in the third
position; the following 21 nucleotides were coll-?letely homologollc for PCR
30 priming. The 3' primer was id~ntir~l to the 3' primer for part A in FY~ C 5.
PCR was conduct~d as descrihe~ in F.Y~rr, 1~ 5 to give a 216 nucleotides PCR
product.
The c~ ssion vector was pJO201. A unique SalI site at 40
nucleotides downs~ of the kdsB ATG start codon and the PstI site in the
35 multi-cloning site were used to clone the PCR pl.Jdu~ls. Tran~ ion~l
216~601
iVO 94/29456 pcTlus94los92o
-23-
coupling yielded a 19 aa pioducl from kdsB and HSV-2 pl~tease (1-247). Parts
- A2 and B were cloned in pJO201 to give P1~Cm;~ pA2B6 of 4.1 kilobases. The
plasmid was llansÇwll.cd in E. coli strain XL1-Blue to give strain SSHP4.
The strain was grown and evaluated for eA~,ession as described in Example 5.
5 A band ccll~;,~nding to the pl~ ,t~l n~le~ wdght of HSV-2 protease
(27 kDa) was ob~ined on SDS-PAGE (Figure 4) and Western Blot (Figure 5).
FYQm~l?le 10
Coh~ll uclion of ~A2B6C3 ~nr1 F ~ CC ~ of HSV-2 E!~ut~ ~Cf (1-307)
o Tr~nclatiQnally Cou~led to CK.~
HSV-2 ~lotease(1-307) was eA~ ssel tr~nc~ on~lly cou~led to CKS.
The cAc~e~e system ~es~r~ in Fy~nrle S and Figure 2 was used again in
this example; part A2 was ~esc~ in F~ 1C 9, parts B and C were
described in FY~ l,les 5 and 7. Parts A2, B and C were cloned in pJO201to
lS give the 4.3 kilobases plasrnid pA2B6C3. The pl~ l was ll~n;,rullll~ in E.
coli strain XL1-Blue to give strain SSHP5. The strain was grown and
evaluated for ~A~ ion as ~es~rik~d in PYr . 'c 5. As shown in Figure 4, a
band cull~,s~on-ling to the lJl~ictud mole~ r weight for HSV-2 ~lot~(1-
307) was observed (33 kDa). In r 'rlitir)n~ a band c~ to the
~o mnle~ r weight for self-l,noGesce~l HSV-2 protease (27 kDa) was also
observed.
FY~n~
Consll uclion of pA2B6Cmut and EA~ ssion of HSV-2 P~otease with Amino
2s Acid 247 ~ n
The m~ ion at the amino acid 247 ~,lot~ase self-pl~ces~ site described
in Fy~mrle 8 was hlccll~l..te;l into the non-fused tr~nCl~tion~lly co~led
protease. The cqccette system in FY~ , l S was again used: part A2 was as
des~ibe~l in FY~mrle 9 part B described in FY~mrle 9 and part Cmut
30 desc~ihed in Py~mp!e 8 were cloned in pJO201 to give the 4.3kb plqcmy
pA2B6Cmut. The plqcmitl was ~ r.,-. .~d in E. coli strain XL1-Blue to give
strain SSHP6. The strain was grown and evaluated for eAl,l.,~sion as des( ~
in FY~mplc 5. As shown in Figure 3, a band c~ ~ ndil~g to the predicted
mole nlslr weight for HSV-2 plut~c(1-307) was observed (33 lcDa). There
216460 l
WO 94/294s6 pcTlus94los92o
- 24 -
was no band co~ ~n~l;ng to the mole~vl~r weight for self-processed HSV-2
plot~,ace (27 ld~a).
F~Y~mDle 12
s Construction of pHPB-l
In this e,~ !e a suhcl~ ne was collsll uct~,d of pH2ProB (pl~cd as
described in Example 3) which cont~ined only the 0.7 k~ ses BamHI
fr~gmP,nt e~co~ g the ~ i..g sequen~e of the ~cse~bly protein ICP35.
The 0.7 kilobases Ban~II L~ cnl from pH2ProB was subcloned in pUC18.
o This construct was needed for subsequent con;,lluulion of full length UL26
clones conti~;ning HSV-2 plot~ase and ICP35.
F.x~ 13
Consl, uclion of ~SSPI1-11 and F~ ssion of HSV-2 F~ c.~/ICp35
lS A CKS fusion was col~shu~t~d to the entire UL26 e-.co~ -g HSV-2
plo~ase and ICP35 as O~ 1 in Figure 6. Plasll,ld pAlB6C, describe~l in
~Y~rnple 7, was digest~ with NcoVPsd and. in a triple lig~tion~ was ligated to
a 557 bp NcoVSall fragment of pH2ProA and a 81bp SalVPsd fragment of
pHPB-1, des~ in Example 12. The resvlting h~t~ e clone plTL~6
20 was then ~iges~ed with RsrIVHir~m and ligated with a 610 bp RsrIVHindm
fr~ nt of pHPB-1 to give the final pl~cmid pSSPI1-11 of 5.9 lrilob~ces The
p1~crnid was lla,l ,r~,l.lled in E. coli strain XL1-Blue to give strain SSHP7. The
strain was grown and evaluated for ~ ,,sion as describe~ in Fy~nlrle 5. A
band COll1S~OI ding to the predicted -'ec~ r weight for CKS/HSV-2
2s protease/ICP-35 fusion was obs~ cd (95 IcDa).
FY~rn~l?le 14
Constluction of pALTHM-4 Co.~la;~ His-148 Ml1t~tion
The histidine residue at posil;ol- 148 of HSV-2 protease was mllt~geni
30 to an alanine residue. The Altered Sites Mvt~g~nesis Kit (supplied by
~ollleg~) was used following the m~nllf;~tllrerls protoc~lc A KpnVSphI
fr~gm~nt of pSSPI1-11 was snbclQned in the pALTER-l mut~gçnesic vector
provided in the kit. Mutagen~c;c at l~ucl~l;des 442-443 of HSV-2 lJlUt~ ase
was ~xlÇwll~d: CAC to GCC converting His-148 to Ala and creating an EagI
2~46~
WO 94/29456 PCT/US94/05920
-25 -
site for analysis of ~ Anl~ pALTHM-4 conlA;nr~ the desired m~ltq-tion
which was col,LIl~ by DNA s~u~ n~ g~
Fsqmnle 15
s Con~l u ;lion of pSSPI2 and T~ ,ssion of HSV-2 Protease AnA ICP35
Tr.qnslqtionally Cou~led to ~K~
In this . rlc the UL26 gene e-~ç~i~g HSV-2 Protease and ICP35 was
trAn~lqti~nqlly cour ~sd to CKS. The plasmid pA2B6C was the parent vector
for this Col sllu~;~ A 1441bp NcovHimim fragment from pSSPIl-l l
o co~A;n;ng the 3' portion of HSV-2 Protease and the ICP35 gene was cloned in
pA2B6C to give the 5.2 kilobqces plqcmiA pSSPI2. The plAcmiA was
l-~-s~l,ed in E. coli strain XLl-Blue to give strain SSHP8. The strain was
grown and evaluated for e,.~l.,s;,;on as ~esr~ in FY- . '^ 5. As shown in
Figure 7, a band coll~"~nd;ng to the predicted mole ulq~ weight for HSV-2
15 ~ ,t~ase/ICP-35 fusion was obs~ (67 kDa) along with the 27 kDa self
ss~d protein cc~ ol~l;ng to the m~le ~ weight for HSV-2 Protease
(1-247).
F~.."~lf 16
20 C~ ll uclion of pSSPI2M qnA F~ s~ion of Mutant HSV-2 Protease ~qntl
ICP35 Tr~qnclqtionqlly Cmtl?led to C~
The UL26 gene çnf~o~ g HSV-2 protease with the arnino acid 247
APlehon~ described in FY~ 8, and ICP35 was trn~lq-tin~qlly cc upled to
CKS. The plqcmiA pA2B6Cmut was the parent vector for this consllu.;L A
25 1441 bp NcovHi~ m fragm~.nt from pSSPIl-l l conlq~ g the 3' portion of
HSV-2 pr~t:ase and the ICP35 gene was cloned in pA2B6Cmut to give
plq~miA pSSPI2M. The plqcrniA was llAn~r~"...ed in E. coli strain XLl-Blue to
give strain SSHP9. The strain was grown and evaluated for eA~l~,s~ion as
described in FYr , '- 5. A band coll.,~llding to the predicted mo~e .llq.
30 weight for HSV-2 plu~seJICP-35 was observed (67 kDa) as shown in Figure
7. There was no evidence of the 27 kDa self pl~ces~,d protein cc,ll~s~,on~ g
to the mqle ~ Illqr weight for HSV-2 plu~ase (1-247) on SDS-PAGE or Western
blots.
216~0i
WO 94/29456 PCT/US94/05920
FY~m~l?le 17
Expression of U~ 26 in S. cerevi~in~
The pl~cmi-l pSSPIl~ e~ ed as ~esçribe~ in Example 13, was
s ~1i~st~PIi with EcoR1 and the sticky ends were blunt ended by large fragment of
DNA poly,ll~,.ase accolLng to manufacturer's i~.s~ cl;ollC. The DNA was then
digPstP.~ with Xbal and el~llu~hul~,sed ûn 1% low melt agarose gel. A 2.3
ob~ces fragment co~ g the UL26 gene was excised from the gel and
then ligated to pVT100-U at the Pvull/Xbal sites. The lig~qtic!n mix was
o tran~Ç,lllled into E. coli DH5a~ The plqc~ pVT100-U UL26, co..l~in;~g the
UL26 gene was idP-ntifip~ by ~ esl;on with Hindll 1/ Xbal which released a
2.3 lril~a~Ps fr~gmPnt
Yeast strain YJO was ll~ Çolllled with pVT100 U or pVTlO~U UL26
employing selec~ion for uracil ~l~,tc)tl~h~. Tl.ulsr~ were grown at
lS 30 C in liquid media lacking uracil and col~ ining 2% glucose and eY~mined
for e.~ssion as d~Ps~ihed h Example 5. An ;~ no, ~ , band of 27 kDa
was ~,h~te~ (Figure 8, lanes 5, 6) ;ndi~ g that the UL26 gene pluduCI
(HSV-2 p,ulease) is active in S. cerev-siae.
FY~T1U71P 18
Expression of Wild-t~e and Mut~nt HSV-2 P~o ~cP in the Baculovirus
System and Demonstration of In Vivo Activity
In order to obtain high levds of HSV-2 protease e,A~ ion, the gene
was cloned into the ~ r~, vector pVL1392, placing it under the control of the
2s strong Polyhedrin ~iu~llut~r of the Baculovirus, Autographa californica
Nuclear Polyl,e~usis Virus. The gene was then inte~ed into the linealiLcd
R~Cn1QgO1(1 virus in the insect cell host by homologous Iccc,~bino~;Qn
l?eco...b;n~ viruses were then used to infect insect cells in tissue culture to
produce the protease. A DNA fra~grnent coding for the HSV-2 p~tease UL26
gene was excised from pl~m;d pSSPI1-11 (desçrihe~l in Flc .. l)le 13) in the
following ,~ n~. the ~ ll.id was first ligest~ with Xba I, then treated with
the Klenow r,~,ll~,l,t of DNA Polylll~,.ase to gen~al~ a blunt end, followed by
~i~sti~n with Eco ~. A res~llting DNA fragment of applu~ ly 2
kilobases was purified through an agaTose gel and ligated to the ll~Sr~ vector
3s pVL1392 which was previously ~igested with Eco RI and SmaI, to create the
NO 94/294s6 2 ~. 6 4 S 9 L PCTIUS94/05920
-27-
plqcmi~l pAcUL26. Similarly, the ~lq~miA pALTHM-4 (~lesçribe~l in Example
13) con~ g the HSV-2 UL26 gene with a mvt~qtion at the active site His-148
was iigested with Hindm, made blunt with Klenow polylllc.ase, cut with Eco
RI, and the 2 kilobases DNA fragment g~- ~c-~ted was gel p~ fied and ligated
s to Eco RI and Sma I cut pVL1392, to ~n~,~atc the pl~c~irl pAcH148.
~ e~o...bi~-q~-~ viruses were derived by co-ll~llsÇ~(ion of S ~g of either
pAcUL26 or pAcH148 with 0.5 llg of lineq-~i7~d Baculogold DNA using the
reagentsforcqlr,illm~ho~l~hq~ n ~ pliedinthekit. The
Baculogold DNA co~ ;n~ a lethal deleti~n of ORF1629 and is not viable
10 unless rescued by r~ n with the l,~lsr. l vector co~ .g ORF1629
and the gene to be eA~I~,s~d, thus providing a sele~ion for re~o...bi~ virus.
After ll~u~sr~lion, the Sf9 cells were incllbqted in 4 ml TMN-FH media for 5
days and ...~ d for signs of i~lr~iOI~, ie, enl&g~nlenl of cells, loss of cell
viability, or lysis. S~ were harvested, cells were removed by low
lS speed cenllir~,g~q-tion~ and 1 ml of this low titre viral stock was used to infect a
new flask con~;ni~g 2 x 106 Sf9 cdls. Once more, cells were inc~lbqt~ for 3
to 5 days until signs of i..l~-;ol- were a~. t, thus intlir-q-tin~ cl;on of a
high titre virus stock.
To assay for C.~ssiol of either UL26 or the His-148 mutant gene,
20 fresh Sf9 cells were inÇ~:t with the high dtrc stocks of vAcUL26 or
vAcH148 g_ne,~cd as ~les~ihe~ above and soluble extracts were l,.~zlcd.
Figure 9 shows the results of SDS-PAGE analysis of 20 111 aliqoutC of soluble
eA~ i of ullinr~t~ cells (lane 3), wild-type Baculovirus inrcclcd lysate (lane
4), vAcUL26 inr~;te~ lysate (lane5), and vAcH148 i.~ lysate (lane 6),
2s after st~ining with Cool~ic blue. As e-pcct~, the polyprotein from the
UL26 gene made in vAcUL26-infçcte~ cells und~ ;~s auluylvtcolysis to
produce the 27 kDa protease and the 40 kDa ICP-35 protein, in~ ting that the
HSV-2 pl~t~ ~ce is active in these cells. In vAcH148 in~ect~d cells, the
mllt~tion in the active site of the ~loleasc greatly ~luces aulo~ t~olysis and
30 the 67 kd poly~,lulein is the pl~ form observed. This shows that
procescing of the polyplotc~l is nût due to an endogenous protease of viral or
cellular origin but an ;~ r fun.;lion of the active HSV-2 ~lvt~ase, itself.
The Western blot, shown in Figure 10 cour~ I"c that the ~lulci,ls l~luduce~ in
virus infected cells are the pro and mature forms of HSV-2 I,lut~,ase.
3s
216~601
WO 94/2g456 PCT/US94/05920
FYqm~ e 19
,~lion of antibody to the 236-247 r~ .n of HSV-2 protease
A peptide of the se~u~,nce Gln-Ala-Gly-Ile-Ala-Gly-His-Thr-Tyr-Leu-
Gln-Ala was 5~ .Ps;7~d co.. -, ~;ially using standard l~,ch.~ es. This
s se~u~ e CC~ (lc to the residues 236-247 of the UL26 gene pr~lu~;l and
the c.ul,u~y terminal 12 amino acids of the mature form of HSV-2 I,lutease.
The pepdde was conjugated to keyhole limpet h~ o ~ h~ using a
cc.. - ~ially available kit (Pierce). The conjugated pepdde was then
emlllsiflçd with Freund's adjuvant and inJ~t~ into several su~;u~ eo~c
o dorsal sites of New 7Pql-qn~l white rabbits (approx. 0.5 mg peptide per
h~ u~ At;on). T.. ~ l;on boosts were also ulminict~red two and six
weeks later. The qnimqlc were bled prior to ~Jl;ll~ i.. -;,~;on and also at
four, eight and ten weeks after l,lill~ ;.. ~.;,At;ol- The serum was
c~llect~ from clotted blood by cel,l.ifvg,qtion, heated to 56C for 30 ~ JI~S,
and then stored at -20 C.
F.Yqm~ e 20
E,Y~ression of the U~ 26 ~nP in insect l-q-rvae ll~i~ a ,e~o-.lb;~ baculovirus
vector.
Cq,bbq~gç looper larvae were grown on the semi~rnlhetic diet at 27 C
and 50% reladve hllmi~lity undl the fourth instar stage. Shortly after molting,
the insects were l'~,~lW-Cd from their diets, housed individually in plastic Petri
dishes, and starved for 18 hours. The insects were then placed into contact
with 10 ~1 of insect cell culture fluid (see FYqmrle 18) which was inÇt~t~,d
25 with either 1) wild-type nuclear poly},ed-~sis virus (Autographa californica ),
2) recombinqnt baculovirus with the UL26 gene for HSV-2 ( vAcUL26), 3)
leco.nh;---q-n~ baculovirus and wild type nuclear polyl,e~l,os;s virus in a 10: 1
rado, or 4) no baculovirus. Each of the cell culture fluids cont~ined 10%
sucrose to stim~ te ~1rin~in~. The larvae were observed undl they drank part
30 of their culture fluid, and then they were left in contact with their ~s~ e
culture fluid for an ;~,dd;l;ol-~l three hours. Afta this period they were placed
onto dle su~face of fresh æmi~"ll,.,lic diet (< 6 larvae/cup) and ,~ u;~ at
28 C and 70% reladve hllm; lity. On days 3-5 posli~r~lion, dead and
m~ bllml larvae wae cQll~cted and frozen at -70 C.
WO 94/29456 216 4 6 01 PCT/US94/05920
-29-
Frozen larvae were thawed and placed into extraction buffer (50 mM
Kphosphqte, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.4 mM ~ a~oe~ nol,
0.5% Triton X-100, and 10% glycerol) in a 10 ~11 burr~ g larvae ~ ~l~ion.
The larval ~V~ipe~ ollc were ho---og.~ r~ using a mortar and pestle. Aliquots
5 were removed . nd cenlrifilg~A in a llli~,lofuge for 3 .~;n-lt~,S at high speed.
Aliquots from the ~ t fractions were d~l~alul~l and cle~ ~hol~s~ on
12.5% SDS-polyacrylamide gels. The gels were either stained for protein
using Coomqc;e R ~ ^nt Blue R-250 or elec1.~ho~lically ll~ulsr~ d to
PVDF mc.l~ ules for Western Blot analysis. The pll~ antibody sollltion~
o were ~ d in TBST buffer CQ~ g 0.75% bovine senum qlbl~m;rl and an
antibody derived either from the 236-247 HSV-2 pl~tease peptide (F
19) or the CKS-HSV-2 pl~tease fusion protein (from FY~qmrle 6).
The Coomasie stained gel, shown in Figure 11, shows the plesence of a
27 kDa band not present in larvae ;~-r~A with only wild-type baculovirus or
lS with no virus . These data suggest that the .~....~ vAcUL26 baculovirus
ihlr~t~d larvae eA~ ssed a protein of about 27 kDa The Western blot analysis
(Figure 12) co~.r;~ this int4,~ iol~, showing that the pl;~ antibody to
the 236-247 peptide fragment of HSV-2 protease reacts positively only with
the 27 kDa band in vAcUL26 treated larvae and some minor de~ lqtion
20 products. These results show that the UL26 protein plUllUCed in Trichoplusia
ni is active and capable of self-~ ce~h~ to the mature 27 kDa fo,rm.
It will be appreciated by those sldlled in the art that the spec-ific
eml~l;--~ of the present invention can be m~ifie~ or chqnged willloul
d~al~ing from the scope or spirit of the in~clllion. For e-~qmr!e, due to codon
2S ~eg~ Cy, çhqn~es can be rnade in the und~"lying DNA ~luence wi~ u
affecting the protein s~u~"~ce. Moreover, due to ~ ~?ogirql filnrtionql
equivalency ccncid~ations, çhsngÇs can be rnade in the protein ~llu~;luie and
still achieve a useful plutease. All such m~ifirqtion~ are intended to be
l~lded within the scope of the ~1~ nd~d claims.
WO 94/29456 PCT/US94105920
21~460 1
-30-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: STEFFY, K. et al
(ii) TITLE OF lNv~h,lON: HERPES SIMPLEX VIRUS TYPE 2 PROTEASE
(iii) NUMBER OF SEQUENCES: 12
tiv) CORRESPON~NC~ ADDRESS:
~A) ADDRESSEE: ABBOTT LABORATORIES
(B) STREET: ONE ABBOTT PARK ROAD
(C) CITY: ABBOTT PARK
(D) STATE: IL
(E) CO~h- KY: USA
~F) ZIP: 60064
(v) COMPUTER R~AnPRTT FORM:
~A) MEDIUM TYPE: Floppy disk
(B) CG.Lu~: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) ~u~R~hl APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/073,819
(B) FILING DATE: 06-JUN-1993
(viii) Al ORN~Y/AGENT INFORMATION:
(A) NAME: crowley, 8.
(B) REGISTRATION NUMBER: 31604
(C) R~K~N~/DOCKET NUMBER: 5363.US.Pl
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 708-938-7742
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2151 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
2 ~ 0 ~.
WO 94/29456 PCT~US94/05920
(vi) ORIGINAL SOURCE
(A) ORGANISM HSV 2
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: 211 951
(D) OTHER INFORMATION /product= nHSV2 PROTEASE"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1126 2151
(D) OTHER INFORMATION: /producte nICP35 PROTEIN"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1
G~....~.~. .~..~,..~. G~ CCGC~-G GTCACTAAAA GGCACGCCCC TTACATACTG 60
CGGGC---AG CCCGGlCCC GGAC-~-CGG CTGr~Ar-A~-AC AACAACGGCT GGGCCCGTGG 120
G.GGG.AAGT GGi,CGGGG G Q ~-G~,~ TA..CC~G CCCG~-~C Q CCCCCCCCCC 180
TTCCC~l... ~...~l..~l GCG~,GCCC ATG GCG TCG GCG GAA ATG CGC GAG 234
Met Ala Ser Ala Glu Met Arg Glu
1 5
CGG TTG GAG GCG CCT CTG CCC GAC CGG GCG GTG CCC ATC TAC GTG GCC 282Arg Leu Glu Ala Pro Leu Pro A-qp Arg Ala Val Pro Ile Tyr Val Ala
GGG TTT TTG GCC CTG TAC GAC AGC GGG GAC TCC GGC GAG CTG GCC CTG 330Gly Phe Leu Ala Leu Tyr A~p Ser Gly A~p Ser Gly Glu Leu Ala Leu
GAC CCA GAC ACG GTG CGT GCG GCC CTG CCT CCG GAG AAC CCC CTG CCG 378A~p Pro Asp Thr Val Arg Ala Ala Leu Pro Pro Glu Asn Pro Leu Pro
ATC AAC GTA GAC CAC CGC GCT CGG TGC GAG GTG GGC CGG GTG CTC GCC 426Ile Asn Val Asp His Arg Ala Arg Cys Glu Val Gly Arg Val Leu Ala
GTG GTC AAC GAC CCT CGG GGG CCG TTT TTT GTG GGG CTG ATC GCG TGC 474Val Val Asn Asp Pro Arg Gly Pro Phe Phe Val Gly Leu Ile Ala Cy~
GTG CAG CTG GAG CGC GTC CTC GAG ACG GCC GCC AGC GCC GCT ATT TTT 522Val Gln Leu Glu Arg Val Leu Glu Thr Ala Ala Ser Ala Ala Ile Phe
100
GAG CGC CGC GGA CCC GCG CTC TCC CGG GAG GAG CGT CTG CTG TAC CTG 570Glu Arg Arg Gly Pro Ala Leu Ser Arg Glu Glu Arg Leu Leu Tyr Leu
105 110 115 120
216460i
W 0 94/29456 ~ `~ PCTAJS94/05920
ATC ACC AAC TAC CTG C Q TCG GTC TCG CTG TCC ACA AAA CGC CGG GGG 618
Ile Thr Aqn Tyr Leu Pro Ser Val Ser Leu Ser Thr Lys Arg Arg Gly
125 130 135
GAC GAG GTT CCG CCC GAC CCC ACC CTG TTT GCG CAC GTG GCC CTG TGC 666
AQP Glu Val Pro Pro Asp Pro Thr Leu Phe Ala Hi~ Val Ala Leu Cy~
140 145 150
GCC ATC GGG CGT CGC CTT GGA ACC ATC GTC ACC TAC GAC ACC AGC CTA 714
Ala Ile Gly Arg Arg Leu Gly Thr Ile Val Thr Tyr Asp Thr Ser Leu
155 160 165
GAC GCG GCC ATC GCT CCG TTT CGC CAC CTG GAC CCG GCG ACG CGC GAG 762
Asp Ala Ala Ile Ala Pro Phe Arg Hi~ Leu A-qp Pro Ala Thr Arg Glu
170 175 180
GGG GTG CGA CGC GAG GCC GCC GAG GCC GAG CTC GCG CTG GCC GGG CGC 810
Gly Val Arg Arg Glu Ala Ala Glu Ala Glu Leu Ala Leu Ala Gly Arg
185 190 195 200
ACC TGG GCC CCC GGC GTG GAG GCG CTC ACA CAC ACG CTG CTC TCC ACC 858
Thr Trp Ala Pro Gly Val Glu Ala Leu Thr Hiq Thr Leu Leu Ser Thr
205 210 215
GCC GTC AAC AAC ATG ATG CTG CGT GAC CGC TGG AGC CTC GTG GCC GAG 906
Ala Val Asn Aqn Met Met Leu Arg Aqp Arg Trp Ser Leu Val Ala Glu
220 225 230
CGG CGG CGG CAG GCC GGG ATC GCC GGA CAC ACG TAC CTT CAG GCG 951
Arg Arg Arg Gln Ala Gly Ile Ala Gly Hiq Thr Tyr Leu Gln Ala
235 240 245
AGCGAAAAAT TTAAAATATG GGGGGCGGAG .~.GCCC~,G CGCCG~AGCG CGGGTATAAA 1011
ACCGGCGCCC CGG~-.GCCAT Gr.A~ArATCC CCCGCCGC~A GC~.,CCCGC GCCGCAGGTC 1071
GCC~.CCGlG CGCG~AAGT CGC6~CG-CG .CG ~--~-- C----CCGGC ACCG GCC 1128
Ala
GAT ATG AAC CCC GTT TCG GCA TCG GGC GCC CCG GCC CCT CCG CCG CCC 1176Aqp Met Aqn Pro Val Ser Ala Ser Gly Ala Pro Ala Pro Pro Pro Pro
GGC GAC GGG AGT TAT TTG TGG ATC CCC GCC TCT CAT TAC AAT CAG CTC 1224Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser Hiq Tyr Asn Gln Leu
GTC ACC GGG CAA TCC GCG CCC CGC CAC CCG CCG CTG ACC GCG TGC GGC 1272Val Thr Gly Gln Ser Ala Pro Arg Hi~ Pro Pro Leu Thr Ala Cys Gly
CTG CCG GCC GCG GGG ACG GTG GCC TAC GGA CAC CCC GGC GCC GGC CCG 1320Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly Hi~ Pro Gly Ala Gly Pro
wo g4,29456 2 1 6 Ll 6 0 1 PCT/US94/05920
TCC CCG CAC TAC CCG CCT CCT CCC GCC CAC CCG TAC CCG GGT ATG CTG 1368
Ser Pro His Tyr Pro Pro Pro Pro Ala His Pro Tyr Pro Gly Met Leu
70 75 80
TTC GCG GGC CCC AGT CCC CTG GAG GCC CAG ATC GCC GCG CTG GTG GGG 1416
Phe Ala Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala Ala Leu Val Gly
85 90 95
GCC ATC GCC GCC GAC CGC CAG GCG GGT GGG CTT CCG GCG GCC GCC GGA 1464
Ala Ile Ala Ala AYp Arg Gln Ala Gly Gly Leu Pro Ala Ala Ala Gly
100 105 110
GAC CAC GGG ATC CGG GGG TCG GCG AAG CGC CGC CGA CAC GAG GTG GAG 1512
A~p Hi~ Gly Ile Arg Gly Ser Ala Ly~ Arg Arg Arg His Glu Val Glu
115 120 125
QG CCG GAG TAC GAC TGC GGC CGT GAC GAG CCG GAC CGG GAC TTC CCG 1560
Gln Pro Glu Tyr Asp Cys Gly Arg Aqp Glu Pro A~p Arg Asp Phe Pro
130 135 140 145
TAT TAC CCG GGC GAG GCC CGC CCC GAG CCG CGC CCG GTC GAC TCC CGG 1608
Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro Val A~p Ser Arg
150 155 160
CGC GCC GCG CGC CAG GCT TCC GGG CCC CAC GAA ACC ATC ACG GCG CTG 1656
Arg Ala Ala Arg Gln Ala Ser Gly Pro Hi:~ Glu Thr Ile Thr Ala Leu
165 170 175
GTG GGG GCG GTG ACG TCC CTG CAG QG GAA CTG GCG CAC ATG CGC GCG 1704
Val Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala Hi~ Met Arg Ala
180 185 190
CGT ACC CAC GCC CCC TAC GGG CCG TAT CCG CCG GTG GGG CCC TAC CAC 1752
Arg Thr Hi~ Ala Pro Tyr Gly Pro Tyr Pro Pro Val Gly Pro Tyr Hi-~'
195 200 205
CAC CCC CAC GCA GAC ACG GAG ACC CCC GCC CAA CCA CCC CGC TAC CCC 1800
HiS Pro Hi~ Ala Aqp Thr Glu Thr Pro Ala Gln Pro Pro Arg Tyr Pro
210 215 220 225
GCC GAG GCC GTC TAT CTG CCG CCG CCG CAC ATC GCC CCC CCG GGG CCT 1848
Ala Glu Ala Val Tyr Leu Pro Pro Pro HiQ Ile Ala Pro Pro Gly Pro
230 235 240
CCT CTA TCC GGG GCG GTC CCC CCA CCC TCG TAT CCC CCC GTT GCG GTT 1896
Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro Pro Val Ala Val
245 250 255
ACC CCC GGT CCC GCT CCC CCG CTA CAT CAG CCC TCC CCC GCA CAC GCC 1944
Thr Pro Gly Pro Ala Pro Pro Leu His Gln Pro Ser Pro Ala His Ala
260 265 270
CAC CCC CCT CCG CCG CCG CCG GGA CCC ACG CCT CCC CCC GCC GCG AGC 1992
His Pro Pro Pro Pro Pro Pro Gly Pro Thr Pro Pro Pro Ala Ala Ser
275 280 285
216~601
WO 94/29456 ` : ~ PCT/US94/05920
TTA CCC CAA CCC GAG GCG CCC GGC GCG GAG GCC GGC GCC TTA GTT AAC 2040
Leu Pro Gln Pro Glu Ala Pro Gly Ala Glu Ala Gly Ala Leu Val A~n
290 295 300 305
GCC AGC AGC GCG GCC CAC GTG AAC GTG GAC ACG GCC CGG GCC GCC GAT 2088
Ala Ser Ser Ala Ala HiQ Val Asn Val Asp Thr Ala Arg Ala Ala Asp
310 315 320
CTG TTT GTG TCA CAG ATG ATG GGG TCC CGC TGA TGG GGT CCC GCT AAC 2136
Leu Phe Val Ser Gln Met Met Gly Ser Arg * Trp Gly Pro Ala Aqn
325 330 335
TCG CCT CCA GGA TCC 2151
Ser Pro Pro Gly Ser
340
~2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 247 amino acid~
(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 A~p
1 5 10 15
Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr A~p Ser
Gly A~p Ser Gly Glu Leu Ala Leu AQp Pro Asp Thr Val Arg Ala Ala
Leu Pro Pro Glu Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Arg
Cy~ Glu Val Gly Arg Val Leu Ala Val Val A~n Asp Pro Arg Gly Pro
Phe Phe Val Gly Leu Ile Ala Cy~ Val Gln Leu Glu Arg Val Leu Glu
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 Lyc Arg Arg Gly A~p Glu Val Pro Pro A~p Pro Thr
130 135 140
Leu Phe Ala Hi~ Val Ala Leu Cy~ Ala Ile Gly Arg Arg Leu Gly Thr
145 150 155 160
WO 94/29456 2 1 6 4 fi O 1
PCT/US94/05920
Ile Val Thr Tyr Asp Thr Ser Leu A~p Ala Ala Ile Ala Pro Phe Arg
165 170 175
His Leu A~p 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 A3n A~n 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 Hi~ Thr Tyr Leu Gln Ala
245
(2~ INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 amino acid~
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala A~p Met Asn Pro Val Ser Ala Ser Gly Ala Pro Ala Pro Pro Pro
1 5 10 15
Pro Gly A~p Gly Ser Tyr Leu Trp Ile Pro Ala Ser Hi~ Tyr A~n Gln
Leu Val Thr Gly Gln Ser Ala Pro Arg Hi~ Pro Pro Leu Thr Ala Cys
Gly Leu Pro Ala Ala Gly Thr Val Ala Tyr Gly His Pro Gly Ala Gly
Pro Ser Pro His Tyr Pro Pro Pro Pro Ala Hi~ Pro Tyr Pro Gly Met
Leu Phe Ala Gly Pro Ser Pro Leu Glu Ala Gln Ile Ala Ala Leu Val
Gly Ala Ile Ala Ala Acp Arg Gln Ala Gly Gly Leu Pro Ala Ala Ala
100 105 110
Gly Asp His Gly Ile Arg Gly Ser Ala Lyq Arg Arg Arg Hi~ Glu Val
115 120 125
Glu Gln Pro Glu Tyr A~p Cy3 Gly Arg A~p Glu Pro A~p Arg Asp Phe
130 135 140
216~601
WO 94/29456 PCT/US94/05920
- 36-
Pro Tyr Tyr Pro Gly Glu Ala Arg Pro Glu Pro Arg Pro Val Asp Ser
145 150 155 160
Arg Arg Ala Ala Arg Gln Ala Ser Gly Pro His Glu Thr Ile Thr Ala
165 170 175
Leu Val Gly Ala Val Thr Ser Leu Gln Gln Glu Leu Ala His Met Arg
180 185 190
Ala Arg Thr His Ala Pro Tyr Gly Pro Tyr Pro Pro Val Gly Pro Tyr
195 ~ 200 205
His His Pro His Ala Asp Thr Glu Thr Pro Ala Gln Pro Pro Arg Tyr
210 215 220
Pro Ala Glu Ala Val Tyr Leu Pro Pro Pro His Ile Ala Pro Pro Gly
225 230 235 240
Pro Pro Leu Ser Gly Ala Val Pro Pro Pro Ser Tyr Pro Pro Val Ala
245 ~ 250 255
Val Thr Pro Gly Pro Ala Pro Pro Leu His Gln Pro Ser Pro Ala His
260 265 270
Ala His Pro Pro Pro Pro Pro Pro Gly Pro Thr Pro Pro Pro Ala Ala
275 280 285
Ser Leu Pro Gln Pro Glu Ala Pro Gly Ala Glu Ala Gly Ala Leu Val
290 295 300
Asn Ala Ser Ser Ala Ala His Val Asn Val Asp Thr Ala Arg Ala Ala
305 310 315 320
Asp Leu Phe Val Ser Gln Met Met Gly Ser Arg * Trp Gly Pro Ala
325 330 335
Asn Ser Pro Pro Gly Ser
340
(2) INFORMATION FOR SEQ ID NO:4:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: ~ C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
vO 94,29456 21 fi ~ 6 n 1 PCTrUS94/05920
TAGATGAATT ~TA~AGGT CGTATGGCAT CAGCAGAAAT GCGTGAACGT TTAGAGGCGC 60
.~.GCCCGA CCGG 74
(2) INFORMATION FOR SEQ ID NO:5:
~i) SEQOE NCE CHARACTERISTICS:
(A) LENGTH: 41 ba~e Pair~
(B) TYPE: nUC1eiC aCid
(C) STRANDEDNESS: Qing1e
(D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL NO
(Vi) ORIGINAL SOURCE:
(B) STRAIN 5~W.~.1C
(Xi ) S'~U~N~ DESCRIPTION: SEQ ID NO:S:
AGTAGTCTAG AGTCTACGTT GATCGG~,AGG GG~ -~-CCG G 41
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQ OE NCE CHARACTERISTICS:
(A) LENGTH: 38 ba3e Pair~
(B) TYPE nUC1eiC aCid
(C) STRANDEDNESS: ~ing1e
(D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL NO
(Vi) ORIGINAL SOURCE:
(B) STRAIN SYN~h~1C
(Xi) SEQOE NCE DESCRIPTION SEQ ID NO:6:
AGTAGTCTAG ~TA~AC~ACC GCGC.GG6.G CGAGGTGG 38
(2) INFORMATION FOR SEQ ID NO:7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 41 baQe Pair~
(B) TYPE: nUC1eiC aCid
(C) STR~NDEDNESS Sing1e
(D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL NO
2l6~6nl
WO 94/29456 PCT/US94/05920
~vi) ORIGINAL SOURCE:
~B) STRAIN: SY~.~h-lC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
AGA G~,GCA GTTACGCCTG AAGGTACGTG .G~CCGGCGA T 41
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 ba~e pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 3ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) BYPOTHETI Q L: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: SYhL~-lC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~:
TAGATCTAGA A.CGCCGti~C A QCGTACCT T Q GGC 36
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 36 ba~e pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: ~ingle
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETI Q L: NO
~vi) ORIGINAL SOURCE:
~B) STRAIN: ~Yh~ C
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TAGATCTAGA ATCGCCGr~C A QCGTACCT T Q GGC 36
~2) INFORMATION FOR SEQ ID NO:10:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 34 ba~e pairs
~B) TYPE: nucleic acid
~C) STR~NDEDNESS: single
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
2164601
94ng456 PCT/US94/05920
-39-
(iii) HYPOTHETI Q L: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: SYh~ C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAACTG QGT TAA,CGGCCG ~j.GCCGGAAA AGAA 34
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 ba~e pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: -~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETI Q L: NO
(vi) ORIGINAL SOURCE:
~B) STRAIN: ~N.~.lC
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
TAGATCTAGA A.CGCCG~C ACACGTACCT TrAGGjGr,Ar,C GAAA 44
~2) INFORMATION FOR SEQ ID NO:12:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 78 ba-Qe pair~
~B) TYPE: nucleic acid
~C) STRA~DEDNESS: ~ingle
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
~vi) ORIGINAL SOURCE:
~B) STRAIN: SYh-~h-lC
~xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:12:
TAGAGTTCGA CTTAAG~AAG GTGATCTAAT GG QTCAGCA GAATGCGTGA ACGTTTAGAG 60
GCGCC-~GC CCGACCGG 78
