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Solid phase synthesis of the proteinase of bovine leukemia virus Comparison of its specificity to that of HIV-2 proteinase lye Bl4ha, JOz,sef T0zs~r, Young Kim, Terry D. Cop¢land and Stephen Otoszlan Laboratory ~f Molecular Virology aud Cardnogcnesis, dBL.Ba,'ic R¢~'¢arc/~ Program, NCt.Frederlck Cancer Research attd Development Center, Frederick, MD 21703. USA
R~c..~ived 17 July 1992 'rh¢ 126-residue proi¢ina~= (PR) of bovine leukemia virus (BLV} was synthesized by ~olid-ph~,~ p~ptid¢ s~th~is and its activity was shown usin$ various oliltol:~ptld¢ substrat©sreprcr=:ntinll cl=tvalla rdt~ in BLV, human T-call leukemia virus tyl~ I (HTLV- 1), murinc leukemia virus (MuLV) and human immunod¢fieieney virus type 1 (H IV-I}. The specificity of the BLV PR was also temporal to that of chemically synthesized human
immunodefieienc3,virus tylX 2 (HIV.2) Pg, Many of the l~ptides werecleaved at the exp¢=tedsite. however,6 out of IS were hydroly'z~donly by one of the Pgs, Furthermore. one BLV peptide was proc¢:ss~ddiffer=ally by the two cnaym~, Them results, tOll=that with the relative activiti~ and the lack of inhibition of BLV PR by two HIV.I PR inhibitor~, sUlll~t that the flLY PR specificity is substantially different from that of HIV
PRs. Solid phar~ pcptidc synthe=igBLV proteinas¢: Substratc specificity
I. I N T R O D U C T I O N
We have used solid phase peptide synth~is to produce the prot¢olytic enzyme of BLV. Previously, BLV
Human {mmunodeficicncy viruses (HIV) and human T.cell leukemia viruses (HTLV) arc intcnsivcb, studied, since they are the causative agents of a~uir©d immu.
PR has b ~ n obtained from purified virus, and partially characterized [14]. It is ¢ompo ',~xl of 126 residue, and it is the larger known retroviral proteinase. In this study the substrate specificity of the BLV PR was compared to that o f also chemically synth~ized HIV-2 proteinas¢.
nodeficiency syndrome (AIDS) and adult T-~II leukemia (ATL). resp~tively [1]. The retroviral proteinas¢ (PR), especially that of HIV has also received a great deal o f attention as a potential target for chemotherapy [2], since the PR activity is r ~ u i r e d for the maturation and infectivityof the viruses [3-5]. The lentiviralP g is also suggested to be involved in the early phase of viral life-cycle [6--8], However. far less is known about the HTLV P g , the proteolytic enzyme of th= first bona fide human retrovirus, which has oniy b ~ n recently cloned
[9].
Bovine leukemia virus (BLV) is the etiologic agent for enzootic bovine leukosis [10], which is an economically
important disease of cattle. BLV shows the closest relationship to H T L V [I I-I 3]. therefore, as the virus itself, B L V P g can also be considered as a model of HTLV-I PR.
2, MATERIALS A N D METHODS
2, I. S)'.thezis of pcptid)'l.rexin, h,)'deogenfluoPide e/eat,age and refoldo tnfl Solid phar,¢pcptid¢syathclis was performedusinlla VegaCoupler 250C peptide synthesizer(DuPont Co.), ztartinli with 330 roll (0,25 stool) Boc.Gly.phenylac~tamidommhylre.in (Appli~ niosyztems, lno,), with a prolPam which has be~n dc~ribcd curlier [15], Eight equivalentsof protectedaminoacids(Baehcm,Inc.)wereused in each cycle,The sidechain-protectingIlroupswereI~nzyl5~rAsp, Glu. ,~r. Thr; 2.chloro~ru~loxycar~nyl for Lys; 2-bromo-~azyloxycarbonyl for Tyr; formyl for Trp and tosyl for Arll, At the end of the 12S-cycle synthesis, the pcptidyl-resin weighed 2,06 II. The peptide was cleaved from the r~in by the following different ways (using 270--270 mS aliquots): 6) stirring for 1 h at 0'C in the mixture of liquid HF (8 ml) and p.cresolc (0,3 rnl); (iS)stirrinlt for I h at 0*C in the mixture of liquid HF (12 ml), anisol¢ (1 ml). and ethanedithiole (l ml); (lid by low-hillh
Correspondenceaddrexx: S, Oroszlan, Laboratory of Mol=ular Virol. ogY and Carcinog=n~is,Net.Frederick Cancer Res~zrch and Dev¢l. opment C.cnter, P.O, Box B, Frederick, MD 21702, USA.
cleave@ac¢ordinllto Tam et al. [16],The crude peptides(25-25 mid weredizmlw.din 2 ml8 M 8uanidinc-HCl,purifiedby RP.HPLC, and
Permanent addresses: lye Blttha. Cz=chodovak Academy of Sciences, Institute of Organic Chemistry and Biuchemistry, Fleminllovo namesti 2 16610 Prague, Cz~hoslovakia, Jd~z~fT~z..~r, Department of Biochemistry, Medical University ofDebrcc=n, P,O. Box 6, I-I-t012 D=brceen, Hunsary,
P-40, a,~dczcrib~ forH1V-2 PR [I 7],Amino acid composition of the obtained protein was determined after hydrolysis in 6 M HCi (110°C. 20 h)on a D urrum D.500 anallfz~r. Peptide obtained with HF ¢leava•
Published by EIs¢~;~rScience Pubtisher,~ B, V,
refoldcd with 20 mM PIPF...S.HCI,pH 7.0, containing I mM EDTA,
100 m M NaCI. 10% ll;lyc=rol,5% ethyl=he~lycoland 0.5% Nonidet
protocol(it) was further purifiedby ~i n-qtrationon a SuperdexG.?5 HR-IU60 column (Pharmacia LKB Biot¢chnology) and purified
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again on RP-HPLC at described for HIV.2 PR [l'/I, The rcfoldcd protcinas¢ ~tution was concentrated using Amicon-3 concentrator, SDS-PAGE of the purified FR was performed according to Laemmli
[~al, ..2.2, Oligopeplides Olijol~=ptides were synthesb'xd by solid.pha~ peptidc synthesis on a Model 430A aatomated p~ptidc syathasizer (Applied Biosystems. Inc,) or on the Vei|a Coupler 2S0C using Boc chemistry, and ~=rc purifi¢d by RP.HPLC. Stock solutions and dilutions were made in distilled water (or in ~ mM dithiothr¢itol for the Cys.contalninl pep tide), and the prefer peptid¢ concenmLtion was determined by amino acid analysis on either a Dun'urn D-$O~ or a Waters Pieo-Tall arm. ly'aer. 2.3. Prulei.as¢ ass.y Prot¢inas¢ asmys ~¢rc pwformed at 3"PC usinit chemically synthe. sized BLV and HIV-2 PRs [17.19-21| in 0,2S M potassium phosphate buffer, pH 5.6. containing 7.$% ~lycerol. S mM dithiothreitol. I mM EDTA, 0.2~ Nonidet P.40 a=~ 2 M NaCI, followed by the separation of the substrates and cleavage pr~lucts by RP-HPLC as it was d¢. ~ r i ~ d [ I 5,17,20,21 ], Amino acid anal>sis of the colh'¢ted i~aks was u~d to confirm the eleavaile site in the sul~trates.
3, R E S U L T S AND DISCUSSION
3. I. Chemical s)'nthesis of the BL V protehuts¢ While earlier techniques limited chemical peptidc syn, thesis to produce only relatively small biologically active oligopcptides and enzymes like ribonucleases [22.23]. tee,cat developments and automatization of the solid-phase peptide syathesi~ have made it possible to 1.0--
60
~1 In'In':. / i l l '/ l"
/
}
2'1. I
0.5
ta " ~
/ •
kI
__.1 . . . . .
=o
T
,0
0
6'0
Elation Time (rain) FiB. I. Rev=rsc,d.phas~ HPLC purification of chemically synth~tzed [tLV prozeinas=. The polypeptide was cleaved from the resin by HF in the prcrcnce of anisol¢ and ethanedithiole, purified by reversed, phas¢ HPLC, then purified by 8¢1 filtration in 6 M Guanidine.HCl. 100 mM Tris-HCl, The peak fraction of the ~=l filtration was a¢idifi=d to pH 2 by the addition of TFA and applied to a/~llondapak Cil¢ column (19 x 150 ram), The proteinase was elated with an acetonitril= gradient into water in the presence of 0,05% TFA, Fraction= wer= collected manually, lyophilized and rcfolded as described In sc.ction 2. Activity was measured urine the oligopeptide KTKVL.VVQPK (repre~nting tlm CgJNC cleavage slt~ in HT-L;V-I}. insert shows th= purity of the peak fraction (fractina 5) of RF-HPLC on SDS.polyaerylamid= gel, The protein was stained with Coomassic brillant blue R-2~0.
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produce larger biologically active polypeptid¢ molecules [24-26], including the 9g-residue HIVol and HIV2 PRs [19,27.28]. However, the routine synthesis of large peptides (over 50 residues) has not yet been completely resolved. Although BLV PR has already been expressed in a bacterial expression vector [29], chemical synthesis may have some advantall~: it is less labourious, taster, and contamination/degradation by cellular proteinas¢~ is not a concern. Using the solid phase peptide synthesis, w= were able to prepare the BLV PR, a 126 residue polypeptid¢, so far the largest proteinase which has been chemically synthesized, Peptidu obtained with any of the HF cleavage method showed a=tivity after refoldinB, however, the peptide subjected to HF cleavage protocol (iS) had the highest specific activity (data not shown). The amino acid composition of the peptide obtained with cleavage method (iS) and RP-HPLC purification was determined to be as follows (the number= in parentheses represent the expected values): A,~x9,63 (10): Thr 5.08 (5): Set 7.39 (9); (}Ix 10.60 (11); Pro 13.88 (14); Gly 9.61 (9): Ala 8.96 (9): Val 10.85 (11); Met 2.01 C2); I1¢ 7.36 (8); Lea 16.91 (17): Tyr 3.11 43); Phe 2.41 (2)i Lys 3.74 (3); Arg 8.45 (9): Trp was not determined. N.terminal sequence analysis performed up to 22 rcnidues with a model 470A gas-phase .sequencer (Applied Biosystcm=, Inc.) ~ v e the proper sequence. The pcptid¢ was further purified by gel filtration and RP, HPLC. The last purification step is shown in Fig. 1. The peak fraction of the HPLC separation was active after refolding and it was found to be homogenous using SDS-PAGE (Fig. I) having the expected molecular weight (15 kDa). The active fraction elated on the RP-HPLC at 50% aeetonitrile which is in good agreement to that found with the viral enzyme (52-55%) [30]. This preparation was able to cleave p=ptides in a time-dependent manner. The cleavage of the peptid¢ KTKVL*VVQPK tbllowcd the Micha=lis-Menten kinetics with an apparent K,. of 0.05 mM and apparent K,~, of 0.4 nmol.s'Umg"~ (using 1 h incubation time). 3.2. Sg¢cificity uf BL V grot¢inas¢, comparison to that of chemically syadaeM:ed HfV-2 prateinase Oligopeptides representing determined cleavage siren in the polyproteins of BLVo HTLV, I, MuLV and HIV, 1 were investigated as sabstraten of BLV PR. To corn. par= the substrate specificity of BLV PR with HIV-2 PR, the peptidcs were also investigated as substrates of chemically synthesized HIV-2 PR. Oligopeptides representing known Gag and Gag-Pro cleavage sites in BLV were all hydrolyzed by the BLV PR (Table I). Similar results w~¢ found with BLV PR expressed in E. call [29], This suggests, that the ratterS. ral PR may be able to process the respective polyproteins, as it was suggested for HIV-I and HIV-2 PR, using the same approach [20,31]. However, one BLV cleavage site peptide (peptide 2) was not hydrolyzed by
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Table l Comparison of the tub~trate Ipccificity o£ ehomimlly ~,nthe~ized BLV and HIV-2 pro~in~,~ Peptid~
Repre~nted ¢le~va~ site
Relative aciivit,,P BLV PR
HIV-2 PR
1. 2. 3. 4.
PPA[L®PliSE KQPAILeVHTPG ELECL, LSIPL PP~VG.VLDAP
BLV BLV BLV BLV
MAJCA ~' CMNC TFli~R PR/p3
1.00 0.~0 0.32 0.20
1.00 not hydrolyzt,d' 0, I P 0.23
$. 6. ?.
APQVL*PVMHP KTKVLeVVQPK DPASIL.PVIP
HTLV.I HTLV-i HTLV-I
MA/CA CMNC TF/PR
1.2 i.9 0.~2
1.5 3. ! not hydrolyt.~d~
8. 9. 10. Ih
RSSLYoPALTP' TSQAFoPLRAG' MSKLLoATV'VS' POTSLL~TLDDQ'
MuLV MuLV MuLV MuLV
MAJpi2 pl 2,'CA CMNC NCJPR
0.02 0.21 0.07 0.14
12,4 11.~ not hydroiyr,ed' not hydrolized~
12. 13. 14. 15.
VSQNY,,PWQ KARVL~AEAMS TATIM,MQ~GN VSFNF, PQITL
H|V.I HiV.I HIV.I H IV.i
MAJCA CA/X YdNC TF/PR
not hydrolyzed' 0,1~ 0.16 not h ~ r o l : ~ d '
28.8 0,1 26.9 0.25
"ActivitLes were m~sured at 0.2 mM substrate concentration, and m relative activity w u exp_r~___~'~_~ the ~tivity on a l~v~n pgptid~ divided by the aedvi[y obtained for each enwrne with p~otide I. ~'MA, matrix protein; CA, cap~id protein; X, peptide between MA and CA in HiV-I Oql; NC, nuclcocap~id prolein; TF, tran,~fr.~n¢ protein; Pit,, retrovirai prot¢iner¢ (rctrovinsl protein nomenclature from Lets rt el, [37]). CleavaBe sites am indicated by ¢sterJsks, and Liken from [9.36]. ' Hot hydrolx'~d d-ring 24 h incubation. '~Thi~ pcptid¢ was hydroiyaed by the HiV.2 PR at the -Cys*L.~u. bond, in~tead of the -Lcu*Lcu. bond found by the BLV PR, ' The p~ptid~ representinil MuLV clcava~ sites were also hydrolyzed by dLtrup|ed MuLV, at the prr,dictcd site.
10
2O
30
40
BL¥ 11111111
~TLV-1 HIV-2
A
A ~ I
10
J~
20
50
~
BLV
L PQtIHLVROY
P- . R I PAA
HTLV-1
L P I A L F 55HT
P
H[V-2
VhG-.
30
60
70
GG
SRNRYHHLQGPLTLALKP
L KH TSJV L GA]GGJQJT QDH F K L T S L P V L
[ ~ - - LG~I~IY5
PK I [ V G G ]JGGLFJX N T K E ¥ .
40
50
g0
~NVE
| RLPF
] EYL...
60
100
110
120
BLV HTL¥-!
RTTPIYLTSC
HIV-2
--GH-~VRAT! 70
L
0 TK
'
I GRDALQQCqGYLYLPEAKGPP¥
| L
TGOLTPZ[N~FGRNZITALG,SLNL ~0
90
PR [12,|4]; HTLV-I PR [9]; HIV.2 PR [19]. Compnrison is basedon multiple alignment [3S]. Tho,aeamino aci~ which are prcdiccd to b¢ in':olvcd in the enzyme.s,bstrate interaction,, (S,,.S/region) ba~.d on the strucLure~ of HIV.I PR.inhibiter ¢omplc~ [39-4l] end moi~ular mnd~iin~ of HIV.PR ',ub~trat¢ and inhibiter interactions [17,42] are boxed.
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the HIV-2 PR. while another one (peptid¢ 3) was hydrolyzed differently, at the .Cys*I.¢u- bond instead of the .l.¢u*lJu- bond. The BLV PR was also able to hydrolyzc the HTLV-I cleavage site peptides at the expected site (Table l). In fact. the highest relative activity was obtained with the oligopeptide representing the CA/NC cleavage ~ite in HTLV-I (peptide 6), which was also sufficiently hydrolyzed by the HIV-2 Pg, Since it has been noted earlier, that the HTLV/BLV cleavage site.s are homologous to the MuLV cleavage sites [32], w= also inv==tisated the cleavage of oligopeptides representing four MuLV Gag and Gag-Pol cleavage sites. In accordance with the cleavage site homolo&% these peptides were also hydroly'zed by the BLV P g at the expected site (Table I). Although two of the peptides representing MuLV cleavage sites were also substratesfor HIV-2 PR, two of them were hydrolyzed only by the BLV PR. Two peptides of HIV-I cleavage site sequen~s were not hydrolyzable by the BLV PR. while one of them (peptide 12) was the most efficient substrate of HIV-2 P g from the ~lected peptides, These results suggest, that the HIV proteinase has a substrate specificity distinct from that of the BLV (and possiblythe HTLV) PR.
ProS'-lle'~ as part of the S,-S,' binding pockets may be r~ponsible for the substantial ditTcren¢¢s in Sl~Cificity of the HIV and BLV proteinases. HIV proteinases are. potential targets for chemother. spy [2]. By analog, it ma~. b~ possible to treat ATL and enzootic bovine leukosis by administration of inhibitors of HTLV and BLV PRs. However, our initial r~ults indicate, that the BLV Pg (and po~ibly the HTLV PR) has a substantially different substrate specificity from that of HIV Pgs, as also sugg~ted by others [29]. Therefore it is likely, that inhibitors designed against the HIV Pgs will not be suitable in therapy of" enzootic bovine leukosis and ATL, The pretense of HTLV-1 has r~.=ntly been expressed in E. coil [9]. The availability of both BLV and HTLV-I PRs will allow comparative studies of their substrate specificity,
3.3, Lack of inhibttion of tile BLV PR b), inhibitors of H I V proteinaz'e= Two specific inhibitor= of HIV prot¢inases, SP-346 [33] and a phosphinicomethyl isoster¢ (Compound 3 [34]) were assayed as inhibitors of BLV Pg. None of these inhibitors were able to inhibit the BLV PR up to the highest concentration assayed (40,/zM and I / t M , r~pectively), while both were able to inhibit completely HIV-2 PR under identical conditions (ICso values were 8/~M and 0.02/JM, respectively). This result further emphasizes the differen~s between the HIV and BLV proteinases. The lack of inhibition with SP-346 is not surprising, since it is a pipecolic acid contaitfing analog of SP-2l I, an HIV Pg substrat¢ (peptid¢ 12 in Table I). which is not hydrolyzed by the BLV PR, This peptide contains a typical Class I cleavage site of the immunodeficiency viruses having Asn at P=, aromatic amino acid at Pt and Pro at P{ position [35]. Since another peptide representing Class 1 cleavage site was also not hydrolyzed by BLV PR (peptide 15), the typical Class 1cleavage sites of HIV are not efficient substrates of BLV PR. In this regard, it is important to point out, that -Tyr/ Phe*Pro- cleavage site does not occur in BLV and H T L V Gag polyproteins[36], Although the HIV-2 PR substantially differs in size and amino acid composition from BLV and HTLV-I PRs, many of th= amino acids expected to be involved in enzyme-substrate interaction are conserved (Fig. 2). However, certain amino acid changes at the crucial parts of the substrate binding pocket like the change of Iles° to Ala at the tip of the flap or the change o f T h P °.
[1] Guile, R,C. and Monlallnicr. L. (19118) S¢i. Am. 259, 4l-.4tL [2l Krauulich. H.-G. Oro•lan, S, and Wimmer. E, (Eds.).in:Cur. rent Communi~tions in Molecular Ilio]oiw: Viral Prot¢inascsas Tar~tl for Chcmothcr.,p:t, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, leaP. [3l Katoh. |,. Yoshinaka. Y., Rein, A.. Shibuya, M.. Odaka, T, and Oroszlan. S, (legS} VirololW 145, ~0-292. [4] Crawford, S, and Goff. S,P, (l~tlS) J, ViroL 53. 899-907. IS] Kohl, N,E,, Emini. E.A,, $chl¢if. W,A., Davis, L.J,. Heimbach, J,C., Dixon, R.A.F., Scolniek, E,M, and Steal, [.S, (19118) Pro¢, Natl, Acad. Sci. USA 55, 4686.-4690, [6] Roberts.M,M, and Oroszlan. S. (191~9)Diothem, Biophys, R¢~, Commu.. |60. 486-494, [7] Roberts. M.M., Copeland, T,D. and Oronlan. S, (Ig91) Pro|, Eng, 4. 695-?00. [1~] Daboonian. C., Dalgleish, A.. Bountiff, L.. Gross, J,, Orot~lan, S,, Rieketk G,, Smith-Burehndl, C,, Trok¢, P, and M¢rson, J. ([991) Biochem, Biophys, Res, Commun, 179, 17-24, [9] Kobayashi. M,, Ohi, Y,, Asano, T,, Hayakawa, T., Kate, K,, Kakinurna. A. and Hatanaka, M. (1991) FEBS l.=tt. 293, 106il0, [I0] Burn)'. A., Bruek, C,. Chantrenne. H., Cleutcr, Y,, Dek¢~l, D,, Ghysdael, J,. Kettmann, g,, l..¢ilereXl. M,, I..¢unen,J, and Mare. merickx, M.. in: Viral Oncolo~y (G. Klein, Ed.), Raven Press, New York. 1980, pp. 231-289. [11] Oroszlan. S,, Sarnliadharan. M,G., Copcland, T.D,, Kalyana. raman. V,S,. Gildcn, R,V. and Guile, R.C, (1982) Prec. Natl, Acad. Sci, USA 79. 129l-i294, [12] Rice, N,R., Steph=ns. R,M,. Burny, A, and Gilden. R.V, (1985) Virology, 142. 357-377. [13] Ri¢¢, N.R.. Stephcns. R,M, and Gilden, R.V, (1987)in: Enzooti¢ Bovine l~ukosis and Bovine Leukemia (A. Burny and M. Mum. merick~, F..ds.), Martinus Nijhoff Publishing, Boston, 19B?, pp. i 15-144. [,.4| Yo;hin=k.'z, Y., Katoh..t.. Co~_~:.~, T.J~., SmyZ.~r~, G.W, and Orosglan, 5. (1986) 3, Viroi. $7, ~26-832. ItSI Bhiha, I,, Nem¢c, J., T 0 ~ r . J. and Oroszlan, S. (1991) Int,1, Peptide Protein Res, 38, 453--458,
392
ddenmrled&r.~¢ntx: We wish to thank Cathy Hixson and Suzanne $p¢cht for their ~lp with the amino acid analysb, and Patrick W~dock foe help in ~ptidc synth~ts. We are also grateful to Ch=ri Rhod=riek for preparation of the man uscript, R=¢arch sponsor~,l by the National Cano=r Institute, DI4HS under Contract N01.CO-74t01 with ABL.
REFERENCES
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[16] Tam, J,P., Henth, W.F, and Mgrrilleld, P..D.(1983)J, Am, ehcm, Soc. 105. 6442-645:~. {I 7] T~TJ~r, J.. Weber. ~,T,, Gu~tchinn, A,, Blithe, 1,. Copcland, T,D,, Louis, J,M, and Oror~lnn, S, (1992) I]iochemistw 31,4793.-4800. [18] l.a,cmmli° U,K, (1970) Nnturc, 227. 680,-~85, [i9] Copcland. T,D, and Oroszlnn, S. (1988) Gent Anal. Tcchn. 5, 109-115. [20] T~z~r. J.. Bllhn. I.. Copclnnd. T.D.. Wondrnk. E.M. and Oroszlnn. S. (1991) FE~S Lctt. 281, 77-80. {2l ] TOm,r, J., Guitchi.n, A,, Weber. I,T,, i]14hn,l,, Wondrnk, E.M, und Oro~lnn, S. (1991) FEBS Left. 279. 355-360, [22] Guile, B. and Merrificld, R,B. (1969) J, Am. Cllcm, Soc. 91, 50!-S02, [23] H~rschmnnn, R,, Hurt, R,F,. Vnl~r, D,F,. Vitali. R,A,. Versa, S,L,, Jacob. T,A,, Holi>,, F,W, ~ntl Dcnkewaltc~', P.,G, (1969) J, Am, Chem. Soc, 91,507-508. [24] Clark-l.cwis. I,, Ae~rsold. R.. Ziltener, H.. Schrader0 J,W,, Hood. L.E, and Kent, S.B.H, (1986)Science 231,134-139, [25] Kent, S,I~,H, ,snd¢lark.Lc-,vis. ~,, in: $)'nthctic Pcptidcs in Biol. o ~ nnd Medicine (K, Alitnlo ct at., Eds.), El~vier, Amsterdam, 19fJS°pp. 29-57, [26] Kent. S.B.H. (1988) Annu. Roy. Biochem. 57. 957-9~9. (27] Schneider, .I, nnd Kent, S,B.H, (1988) Cell ~. 30~1-3~8. [28] Null, R.F,, Brad),, S,F,, Dnrke, P,L,, Ciccaron¢0 T.M., Colton, C,D,, Null, E,M., Rodkey, J,A., Bennett°C,D,, Waxntzll. L.H,, Siilal. I.S.. Anderson, P.S. nnd V~ber. D,F, (19~8) Pron. Nnll, A~d, Sol. USA 115,7129-7133. [29] Andrc~nsky. M,, Hrull~ovzl-H¢idinisf'cldovd, 0,, Scdl/t~cko J,, Konvnlinku, J,, Bl~hu, 1,, JcP.men,P,, Hoh:j|i, M,, Strop. P. and F~bry° M. (1991) FEI3S l..clt.287. 129-132.
Scptcml~r 1992
[30] Kaloh, I., Y~una~, T., Ikawn, Y. and Yosh|nnka. Y. (1987) Nature 329. 6.q4.-656. [31] Dsrkc. P.L., Null. R.F., Brady. S.F..Garsky, V.M.. CieczrGnc, T.M.. Lcu, C.-T., Lumn~, P.K, Freldingcr,R.M., Vcbcr, D.F. and Sianl, l.S. (1988) Biochem. Bioph~,s. Rcs. Commun. 156, 297-303. [32] Ormzlan. S. and Copc¼nd. T.D. (1985) Curt. Topics Microbiol. Immunol. 115. 221-233. [33] ¢opcland, T.D., Wondrak0 E.M., Toucr, J.,Robcm, M.M. ,,nd Oro-,zlan,S. (1990) Biochem. 13ioph~ts.Rcs. Commun. 109. 310314. [~] Grobcln~,, D., Wondntk, F-M,. Galardy, R.E, and Orosr.lzn, S. (1990) Biochem. Biophys. P,.cl.Commun. |69, II! t-I !16. [35] Hcodcrson. L.E., l~nvcnistc,R.E.. Sowdcr, R.. Capcland, T.D., Schultz, A.M. and Oroszlan. S. (1988) J. Virol, 62, 2587-2595. [36] OrmzJan, S. and Luftis, R,B. (lgqO) Cu~. Top, Microbiol. lmn~unol. 157. 153-|8~. [~7J I.cis, J,. Baltimore, D., i~ishop, J,h4., Coffin, L, Flcir~mer. F., GoR, S.P., Oros,~Jan, S,o RoPier,on. H.. Skalka, A.M. Tcrnin, H,M. ~nd Vost, V. (1988) J. Virol. 62, 1808-1809. [38] Weber. I.T. (1989) G©nc 85. 5f~=566. [39] Miller. M., Schneider, J., Snthyansruyazla, B.K., Toth, M.V., Marshall, G,R,, Clawson, L., SClk, L., Kent, S.B..~I. and Wlodawer, A. (1989) Science 2A6:1149-1152, [40] Swain° A,L., Miller. M,M,, Green, J.. Rich, D.H.. Schneider, J., Kent, S.B,H. and Wiodawer0 A. (1990) Proc, Nail, Acad. Sci, USA 87, 8805-8809, [41] Jaskolski, M., Tomnssclli, A,G,. Sawyer. T.K., Staples, D.G., H¢inrickson, R,L.. Schneider,J., KcnI.S.B.H. and Wlodawcr. A. (lUg1) Riochemiitry 30, 1(~0-1009. [42] Gustchina, A. nnd Weber, I.T. (1991) Proteins 10, 32~3]9.
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Solid phase synthesis of the proteinase of bovine leukemia virus Comparison of its specificity to that of HIV-2 proteinase lye Bl4ha, JOz,sef T0zs~r, Young Kim, Terry D. Cop¢land and Stephen Otoszlan Laboratory ~f Molecular Virology aud Cardnogcnesis, dBL.Ba,'ic R¢~'¢arc/~ Program, NCt.Frederlck Cancer Research attd Development Center, Frederick, MD 21703. USA
R~c..~ived 17 July 1992 'rh¢ 126-residue proi¢ina~= (PR) of bovine leukemia virus (BLV} was synthesized by ~olid-ph~,~ p~ptid¢ s~th~is and its activity was shown usin$ various oliltol:~ptld¢ substrat©sreprcr=:ntinll cl=tvalla rdt~ in BLV, human T-call leukemia virus tyl~ I (HTLV- 1), murinc leukemia virus (MuLV) and human immunod¢fieieney virus type 1 (H IV-I}. The specificity of the BLV PR was also temporal to that of chemically synthesized human
immunodefieienc3,virus tylX 2 (HIV.2) Pg, Many of the l~ptides werecleaved at the exp¢=tedsite. however,6 out of IS were hydroly'z~donly by one of the Pgs, Furthermore. one BLV peptide was proc¢:ss~ddiffer=ally by the two cnaym~, Them results, tOll=that with the relative activiti~ and the lack of inhibition of BLV PR by two HIV.I PR inhibitor~, sUlll~t that the flLY PR specificity is substantially different from that of HIV
PRs. Solid phar~ pcptidc synthe=igBLV proteinas¢: Substratc specificity
I. I N T R O D U C T I O N
We have used solid phase peptide synth~is to produce the prot¢olytic enzyme of BLV. Previously, BLV
Human {mmunodeficicncy viruses (HIV) and human T.cell leukemia viruses (HTLV) arc intcnsivcb, studied, since they are the causative agents of a~uir©d immu.
PR has b ~ n obtained from purified virus, and partially characterized [14]. It is ¢ompo ',~xl of 126 residue, and it is the larger known retroviral proteinase. In this study the substrate specificity of the BLV PR was compared to that o f also chemically synth~ized HIV-2 proteinas¢.
nodeficiency syndrome (AIDS) and adult T-~II leukemia (ATL). resp~tively [1]. The retroviral proteinas¢ (PR), especially that of HIV has also received a great deal o f attention as a potential target for chemotherapy [2], since the PR activity is r ~ u i r e d for the maturation and infectivityof the viruses [3-5]. The lentiviralP g is also suggested to be involved in the early phase of viral life-cycle [6--8], However. far less is known about the HTLV P g , the proteolytic enzyme of th= first bona fide human retrovirus, which has oniy b ~ n recently cloned
[9].
Bovine leukemia virus (BLV) is the etiologic agent for enzootic bovine leukosis [10], which is an economically
important disease of cattle. BLV shows the closest relationship to H T L V [I I-I 3]. therefore, as the virus itself, B L V P g can also be considered as a model of HTLV-I PR.
2, MATERIALS A N D METHODS
2, I. S)'.thezis of pcptid)'l.rexin, h,)'deogenfluoPide e/eat,age and refoldo tnfl Solid phar,¢pcptid¢syathclis was performedusinlla VegaCoupler 250C peptide synthesizer(DuPont Co.), ztartinli with 330 roll (0,25 stool) Boc.Gly.phenylac~tamidommhylre.in (Appli~ niosyztems, lno,), with a prolPam which has be~n dc~ribcd curlier [15], Eight equivalentsof protectedaminoacids(Baehcm,Inc.)wereused in each cycle,The sidechain-protectingIlroupswereI~nzyl5~rAsp, Glu. ,~r. Thr; 2.chloro~ru~loxycar~nyl for Lys; 2-bromo-~azyloxycarbonyl for Tyr; formyl for Trp and tosyl for Arll, At the end of the 12S-cycle synthesis, the pcptidyl-resin weighed 2,06 II. The peptide was cleaved from the r~in by the following different ways (using 270--270 mS aliquots): 6) stirring for 1 h at 0'C in the mixture of liquid HF (8 ml) and p.cresolc (0,3 rnl); (iS)stirrinlt for I h at 0*C in the mixture of liquid HF (12 ml), anisol¢ (1 ml). and ethanedithiole (l ml); (lid by low-hillh
Correspondenceaddrexx: S, Oroszlan, Laboratory of Mol=ular Virol. ogY and Carcinog=n~is,Net.Frederick Cancer Res~zrch and Dev¢l. opment C.cnter, P.O, Box B, Frederick, MD 21702, USA.
cleave@ac¢ordinllto Tam et al. [16],The crude peptides(25-25 mid weredizmlw.din 2 ml8 M 8uanidinc-HCl,purifiedby RP.HPLC, and
Permanent addresses: lye Blttha. Cz=chodovak Academy of Sciences, Institute of Organic Chemistry and Biuchemistry, Fleminllovo namesti 2 16610 Prague, Cz~hoslovakia, Jd~z~fT~z..~r, Department of Biochemistry, Medical University ofDebrcc=n, P,O. Box 6, I-I-t012 D=brceen, Hunsary,
P-40, a,~dczcrib~ forH1V-2 PR [I 7],Amino acid composition of the obtained protein was determined after hydrolysis in 6 M HCi (110°C. 20 h)on a D urrum D.500 anallfz~r. Peptide obtained with HF ¢leava•
Published by EIs¢~;~rScience Pubtisher,~ B, V,
refoldcd with 20 mM PIPF...S.HCI,pH 7.0, containing I mM EDTA,
100 m M NaCI. 10% ll;lyc=rol,5% ethyl=he~lycoland 0.5% Nonidet
protocol(it) was further purifiedby ~i n-qtrationon a SuperdexG.?5 HR-IU60 column (Pharmacia LKB Biot¢chnology) and purified
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again on RP-HPLC at described for HIV.2 PR [l'/I, The rcfoldcd protcinas¢ ~tution was concentrated using Amicon-3 concentrator, SDS-PAGE of the purified FR was performed according to Laemmli
[~al, ..2.2, Oligopeplides Olijol~=ptides were synthesb'xd by solid.pha~ peptidc synthesis on a Model 430A aatomated p~ptidc syathasizer (Applied Biosystems. Inc,) or on the Vei|a Coupler 2S0C using Boc chemistry, and ~=rc purifi¢d by RP.HPLC. Stock solutions and dilutions were made in distilled water (or in ~ mM dithiothr¢itol for the Cys.contalninl pep tide), and the prefer peptid¢ concenmLtion was determined by amino acid analysis on either a Dun'urn D-$O~ or a Waters Pieo-Tall arm. ly'aer. 2.3. Prulei.as¢ ass.y Prot¢inas¢ asmys ~¢rc pwformed at 3"PC usinit chemically synthe. sized BLV and HIV-2 PRs [17.19-21| in 0,2S M potassium phosphate buffer, pH 5.6. containing 7.$% ~lycerol. S mM dithiothreitol. I mM EDTA, 0.2~ Nonidet P.40 a=~ 2 M NaCI, followed by the separation of the substrates and cleavage pr~lucts by RP-HPLC as it was d¢. ~ r i ~ d [ I 5,17,20,21 ], Amino acid anal>sis of the colh'¢ted i~aks was u~d to confirm the eleavaile site in the sul~trates.
3, R E S U L T S AND DISCUSSION
3. I. Chemical s)'nthesis of the BL V protehuts¢ While earlier techniques limited chemical peptidc syn, thesis to produce only relatively small biologically active oligopcptides and enzymes like ribonucleases [22.23]. tee,cat developments and automatization of the solid-phase peptide syathesi~ have made it possible to 1.0--
60
~1 In'In':. / i l l '/ l"
/
}
2'1. I
0.5
ta " ~
/ •
kI
__.1 . . . . .
=o
T
,0
0
6'0
Elation Time (rain) FiB. I. Rev=rsc,d.phas~ HPLC purification of chemically synth~tzed [tLV prozeinas=. The polypeptide was cleaved from the resin by HF in the prcrcnce of anisol¢ and ethanedithiole, purified by reversed, phas¢ HPLC, then purified by 8¢1 filtration in 6 M Guanidine.HCl. 100 mM Tris-HCl, The peak fraction of the ~=l filtration was a¢idifi=d to pH 2 by the addition of TFA and applied to a/~llondapak Cil¢ column (19 x 150 ram), The proteinase was elated with an acetonitril= gradient into water in the presence of 0,05% TFA, Fraction= wer= collected manually, lyophilized and rcfolded as described In sc.ction 2. Activity was measured urine the oligopeptide KTKVL.VVQPK (repre~nting tlm CgJNC cleavage slt~ in HT-L;V-I}. insert shows th= purity of the peak fraction (fractina 5) of RF-HPLC on SDS.polyaerylamid= gel, The protein was stained with Coomassic brillant blue R-2~0.
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produce larger biologically active polypeptid¢ molecules [24-26], including the 9g-residue HIVol and HIV2 PRs [19,27.28]. However, the routine synthesis of large peptides (over 50 residues) has not yet been completely resolved. Although BLV PR has already been expressed in a bacterial expression vector [29], chemical synthesis may have some advantall~: it is less labourious, taster, and contamination/degradation by cellular proteinas¢~ is not a concern. Using the solid phase peptide synthesis, w= were able to prepare the BLV PR, a 126 residue polypeptid¢, so far the largest proteinase which has been chemically synthesized, Peptidu obtained with any of the HF cleavage method showed a=tivity after refoldinB, however, the peptide subjected to HF cleavage protocol (iS) had the highest specific activity (data not shown). The amino acid composition of the peptide obtained with cleavage method (iS) and RP-HPLC purification was determined to be as follows (the number= in parentheses represent the expected values): A,~x9,63 (10): Thr 5.08 (5): Set 7.39 (9); (}Ix 10.60 (11); Pro 13.88 (14); Gly 9.61 (9): Ala 8.96 (9): Val 10.85 (11); Met 2.01 C2); I1¢ 7.36 (8); Lea 16.91 (17): Tyr 3.11 43); Phe 2.41 (2)i Lys 3.74 (3); Arg 8.45 (9): Trp was not determined. N.terminal sequence analysis performed up to 22 rcnidues with a model 470A gas-phase .sequencer (Applied Biosystcm=, Inc.) ~ v e the proper sequence. The pcptid¢ was further purified by gel filtration and RP, HPLC. The last purification step is shown in Fig. 1. The peak fraction of the HPLC separation was active after refolding and it was found to be homogenous using SDS-PAGE (Fig. I) having the expected molecular weight (15 kDa). The active fraction elated on the RP-HPLC at 50% aeetonitrile which is in good agreement to that found with the viral enzyme (52-55%) [30]. This preparation was able to cleave p=ptides in a time-dependent manner. The cleavage of the peptid¢ KTKVL*VVQPK tbllowcd the Micha=lis-Menten kinetics with an apparent K,. of 0.05 mM and apparent K,~, of 0.4 nmol.s'Umg"~ (using 1 h incubation time). 3.2. Sg¢cificity uf BL V grot¢inas¢, comparison to that of chemically syadaeM:ed HfV-2 prateinase Oligopeptides representing determined cleavage siren in the polyproteins of BLVo HTLV, I, MuLV and HIV, 1 were investigated as sabstraten of BLV PR. To corn. par= the substrate specificity of BLV PR with HIV-2 PR, the peptidcs were also investigated as substrates of chemically synthesized HIV-2 PR. Oligopeptides representing known Gag and Gag-Pro cleavage sites in BLV were all hydrolyzed by the BLV PR (Table I). Similar results w~¢ found with BLV PR expressed in E. call [29], This suggests, that the ratterS. ral PR may be able to process the respective polyproteins, as it was suggested for HIV-I and HIV-2 PR, using the same approach [20,31]. However, one BLV cleavage site peptide (peptide 2) was not hydrolyzed by
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Table l Comparison of the tub~trate Ipccificity o£ ehomimlly ~,nthe~ized BLV and HIV-2 pro~in~,~ Peptid~
Repre~nted ¢le~va~ site
Relative aciivit,,P BLV PR
HIV-2 PR
1. 2. 3. 4.
PPA[L®PliSE KQPAILeVHTPG ELECL, LSIPL PP~VG.VLDAP
BLV BLV BLV BLV
MAJCA ~' CMNC TFli~R PR/p3
1.00 0.~0 0.32 0.20
1.00 not hydrolyzt,d' 0, I P 0.23
$. 6. ?.
APQVL*PVMHP KTKVLeVVQPK DPASIL.PVIP
HTLV.I HTLV-i HTLV-I
MA/CA CMNC TF/PR
1.2 i.9 0.~2
1.5 3. ! not hydrolyt.~d~
8. 9. 10. Ih
RSSLYoPALTP' TSQAFoPLRAG' MSKLLoATV'VS' POTSLL~TLDDQ'
MuLV MuLV MuLV MuLV
MAJpi2 pl 2,'CA CMNC NCJPR
0.02 0.21 0.07 0.14
12,4 11.~ not hydroiyr,ed' not hydrolized~
12. 13. 14. 15.
VSQNY,,PWQ KARVL~AEAMS TATIM,MQ~GN VSFNF, PQITL
H|V.I HiV.I HIV.I H IV.i
MAJCA CA/X YdNC TF/PR
not hydrolyzed' 0,1~ 0.16 not h ~ r o l : ~ d '
28.8 0,1 26.9 0.25
"ActivitLes were m~sured at 0.2 mM substrate concentration, and m relative activity w u exp_r~___~'~_~ the ~tivity on a l~v~n pgptid~ divided by the aedvi[y obtained for each enwrne with p~otide I. ~'MA, matrix protein; CA, cap~id protein; X, peptide between MA and CA in HiV-I Oql; NC, nuclcocap~id prolein; TF, tran,~fr.~n¢ protein; Pit,, retrovirai prot¢iner¢ (rctrovinsl protein nomenclature from Lets rt el, [37]). CleavaBe sites am indicated by ¢sterJsks, and Liken from [9.36]. ' Hot hydrolx'~d d-ring 24 h incubation. '~Thi~ pcptid¢ was hydroiyaed by the HiV.2 PR at the -Cys*L.~u. bond, in~tead of the -Lcu*Lcu. bond found by the BLV PR, ' The p~ptid~ representinil MuLV clcava~ sites were also hydrolyzed by dLtrup|ed MuLV, at the prr,dictcd site.
10
2O
30
40
BL¥ 11111111
~TLV-1 HIV-2
A
A ~ I
10
J~
20
50
~
BLV
L PQtIHLVROY
P- . R I PAA
HTLV-1
L P I A L F 55HT
P
H[V-2
VhG-.
30
60
70
GG
SRNRYHHLQGPLTLALKP
L KH TSJV L GA]GGJQJT QDH F K L T S L P V L
[ ~ - - LG~I~IY5
PK I [ V G G ]JGGLFJX N T K E ¥ .
40
50
g0
~NVE
| RLPF
] EYL...
60
100
110
120
BLV HTL¥-!
RTTPIYLTSC
HIV-2
--GH-~VRAT! 70
L
0 TK
'
I GRDALQQCqGYLYLPEAKGPP¥
| L
TGOLTPZ[N~FGRNZITALG,SLNL ~0
90
PR [12,|4]; HTLV-I PR [9]; HIV.2 PR [19]. Compnrison is basedon multiple alignment [3S]. Tho,aeamino aci~ which are prcdiccd to b¢ in':olvcd in the enzyme.s,bstrate interaction,, (S,,.S/region) ba~.d on the strucLure~ of HIV.I PR.inhibiter ¢omplc~ [39-4l] end moi~ular mnd~iin~ of HIV.PR ',ub~trat¢ and inhibiter interactions [17,42] are boxed.
391
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the HIV-2 PR. while another one (peptid¢ 3) was hydrolyzed differently, at the .Cys*I.¢u- bond instead of the .l.¢u*lJu- bond. The BLV PR was also able to hydrolyzc the HTLV-I cleavage site peptides at the expected site (Table l). In fact. the highest relative activity was obtained with the oligopeptide representing the CA/NC cleavage ~ite in HTLV-I (peptide 6), which was also sufficiently hydrolyzed by the HIV-2 Pg, Since it has been noted earlier, that the HTLV/BLV cleavage site.s are homologous to the MuLV cleavage sites [32], w= also inv==tisated the cleavage of oligopeptides representing four MuLV Gag and Gag-Pol cleavage sites. In accordance with the cleavage site homolo&% these peptides were also hydroly'zed by the BLV P g at the expected site (Table I). Although two of the peptides representing MuLV cleavage sites were also substratesfor HIV-2 PR, two of them were hydrolyzed only by the BLV PR. Two peptides of HIV-I cleavage site sequen~s were not hydrolyzable by the BLV PR. while one of them (peptide 12) was the most efficient substrate of HIV-2 P g from the ~lected peptides, These results suggest, that the HIV proteinase has a substrate specificity distinct from that of the BLV (and possiblythe HTLV) PR.
ProS'-lle'~ as part of the S,-S,' binding pockets may be r~ponsible for the substantial ditTcren¢¢s in Sl~Cificity of the HIV and BLV proteinases. HIV proteinases are. potential targets for chemother. spy [2]. By analog, it ma~. b~ possible to treat ATL and enzootic bovine leukosis by administration of inhibitors of HTLV and BLV PRs. However, our initial r~ults indicate, that the BLV Pg (and po~ibly the HTLV PR) has a substantially different substrate specificity from that of HIV Pgs, as also sugg~ted by others [29]. Therefore it is likely, that inhibitors designed against the HIV Pgs will not be suitable in therapy of" enzootic bovine leukosis and ATL, The pretense of HTLV-1 has r~.=ntly been expressed in E. coil [9]. The availability of both BLV and HTLV-I PRs will allow comparative studies of their substrate specificity,
3.3, Lack of inhibttion of tile BLV PR b), inhibitors of H I V proteinaz'e= Two specific inhibitor= of HIV prot¢inases, SP-346 [33] and a phosphinicomethyl isoster¢ (Compound 3 [34]) were assayed as inhibitors of BLV Pg. None of these inhibitors were able to inhibit the BLV PR up to the highest concentration assayed (40,/zM and I / t M , r~pectively), while both were able to inhibit completely HIV-2 PR under identical conditions (ICso values were 8/~M and 0.02/JM, respectively). This result further emphasizes the differen~s between the HIV and BLV proteinases. The lack of inhibition with SP-346 is not surprising, since it is a pipecolic acid contaitfing analog of SP-2l I, an HIV Pg substrat¢ (peptid¢ 12 in Table I). which is not hydrolyzed by the BLV PR, This peptide contains a typical Class I cleavage site of the immunodeficiency viruses having Asn at P=, aromatic amino acid at Pt and Pro at P{ position [35]. Since another peptide representing Class 1 cleavage site was also not hydrolyzed by BLV PR (peptide 15), the typical Class 1cleavage sites of HIV are not efficient substrates of BLV PR. In this regard, it is important to point out, that -Tyr/ Phe*Pro- cleavage site does not occur in BLV and H T L V Gag polyproteins[36], Although the HIV-2 PR substantially differs in size and amino acid composition from BLV and HTLV-I PRs, many of th= amino acids expected to be involved in enzyme-substrate interaction are conserved (Fig. 2). However, certain amino acid changes at the crucial parts of the substrate binding pocket like the change of Iles° to Ala at the tip of the flap or the change o f T h P °.
[1] Guile, R,C. and Monlallnicr. L. (19118) S¢i. Am. 259, 4l-.4tL [2l Krauulich. H.-G. Oro•lan, S, and Wimmer. E, (Eds.).in:Cur. rent Communi~tions in Molecular Ilio]oiw: Viral Prot¢inascsas Tar~tl for Chcmothcr.,p:t, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, leaP. [3l Katoh. |,. Yoshinaka. Y., Rein, A.. Shibuya, M.. Odaka, T, and Oroszlan. S, (legS} VirololW 145, ~0-292. [4] Crawford, S, and Goff. S,P, (l~tlS) J, ViroL 53. 899-907. IS] Kohl, N,E,, Emini. E.A,, $chl¢if. W,A., Davis, L.J,. Heimbach, J,C., Dixon, R.A.F., Scolniek, E,M, and Steal, [.S, (19118) Pro¢, Natl, Acad. Sci. USA 55, 4686.-4690, [6] Roberts.M,M, and Oroszlan. S. (191~9)Diothem, Biophys, R¢~, Commu.. |60. 486-494, [7] Roberts. M.M., Copeland, T,D. and Oronlan. S, (Ig91) Pro|, Eng, 4. 695-?00. [1~] Daboonian. C., Dalgleish, A.. Bountiff, L.. Gross, J,, Orot~lan, S,, Rieketk G,, Smith-Burehndl, C,, Trok¢, P, and M¢rson, J. ([991) Biochem, Biophys, Res, Commun, 179, 17-24, [9] Kobayashi. M,, Ohi, Y,, Asano, T,, Hayakawa, T., Kate, K,, Kakinurna. A. and Hatanaka, M. (1991) FEBS l.=tt. 293, 106il0, [I0] Burn)'. A., Bruek, C,. Chantrenne. H., Cleutcr, Y,, Dek¢~l, D,, Ghysdael, J,. Kettmann, g,, l..¢ilereXl. M,, I..¢unen,J, and Mare. merickx, M.. in: Viral Oncolo~y (G. Klein, Ed.), Raven Press, New York. 1980, pp. 231-289. [11] Oroszlan. S,, Sarnliadharan. M,G., Copcland, T.D,, Kalyana. raman. V,S,. Gildcn, R,V. and Guile, R.C, (1982) Prec. Natl, Acad. Sci, USA 79. 129l-i294, [12] Rice, N,R., Steph=ns. R,M,. Burny, A, and Gilden. R.V, (1985) Virology, 142. 357-377. [13] Ri¢¢, N.R.. Stephcns. R,M, and Gilden, R.V, (1987)in: Enzooti¢ Bovine l~ukosis and Bovine Leukemia (A. Burny and M. Mum. merick~, F..ds.), Martinus Nijhoff Publishing, Boston, 19B?, pp. i 15-144. [,.4| Yo;hin=k.'z, Y., Katoh..t.. Co~_~:.~, T.J~., SmyZ.~r~, G.W, and Orosglan, 5. (1986) 3, Viroi. $7, ~26-832. ItSI Bhiha, I,, Nem¢c, J., T 0 ~ r . J. and Oroszlan, S. (1991) Int,1, Peptide Protein Res, 38, 453--458,
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ddenmrled&r.~¢ntx: We wish to thank Cathy Hixson and Suzanne $p¢cht for their ~lp with the amino acid analysb, and Patrick W~dock foe help in ~ptidc synth~ts. We are also grateful to Ch=ri Rhod=riek for preparation of the man uscript, R=¢arch sponsor~,l by the National Cano=r Institute, DI4HS under Contract N01.CO-74t01 with ABL.
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FEB$ LETTERS
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