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Poliovirus; Pmtcexo .X

1,lNTRODUCTIQN

2. MATERIALS 4ND METHODS

The formation of stable poliovirus proteins includes processing of the single polypcptide-precursor [ 1,2], A limited proteolysis is performed by two poliovirus proteases 2A and 3C clcavirtg peptide bonds Tyr-Gly, and Gin-Gly, respectively (3,4]. Nine of 12 established cleavage sites in the polyprotein of poliovirus are specific to 3C protease [2]. Nowadays a total antino acid sequence of the enzyme (183 amino acids of M, 19.7 kDa) and a nucleotide sequence of its cdding gene have been deciphered [5]. Protease 3C, a cystcine enzyme 16-81 with a utiique specificity (cleaves onlypeptide bond Gin-Gly), is able to split off autocatalytically from the poliovirus polyprotein [$-lo]. However, still a lot of problems on 3C function expect their solution. The role of secondary interactions and the spatial structure of the substrate for the enzyme specificity seem rather obscure. The present paper deals with isolation of the active recombinant 3C: protease of poliovirus I(M), The enzyme having a fragment of 3D polymerase in the Cterminus is shown to possess a specific proteolytic activity not only relative to the fragment of a virus polyprotein (cleaves bonds Gin-Gly surrounding peptide VPg at the N- and C-termini), but also cleaves polypeptide bonds Gin-Cly in mo’lecules of ,& galactosidase and bovine catalase.

Canxrruchg of’rccombinnnt DNA, preparation afcompctirirc E. CD/i cells and their trnnsformnrion, clone selection, isolation and

Cokspondence address: E.P. Sablina, Shemyakin Institute of Dioorganic Chemistry, USSR Academy of Sciences, ul. Miklukho. Maklaya, 16/10 117871 GSP-7, Moscow, V-437, USSR

Published

by Etsevier Science Publishers

8. V.

nnalyais of plasmid DNA were performed ai’ described in [Ill, Nucleo~lde sequencing was done according IO the MaxamGilbcrt rnchxl in G modification of Clluvpilo.Kravchcnko Ill). 2. I s Isoiulio~~ of ‘itxhtsion bodies /rat?1 bucreriat mss Cells WR 101 transformed by vcctorpTTQ8-PV and cells XL-1 Blue transformed by vector pEKI-PVs wcrr grown in the YT medium [I 11 with Rp addition (SO&ml) up to L&I = 0,2, After 1PTG addition up to the final concentration of I mM Khe cells were grown for 3 h (up to Rt,aa = 1.O), then collected by centrifugation, washed by cooled buffer A (IO mM Tris, pH 7.5, 10 mM EDTA, 150 mM N&l, I mM PMSF) and ccl1 paste was suspended in buffer A (l/100 of the initial volume OF the cell culture) contoining I mg/ml of lysozyme. After 1 II incubation at p°C the suspension was sonicatcd and collected by crntrifugation, the pellet was washed by cooled buffer A and stored at -7ooc. 2,2. Isololiori and purification of polypeprides PI and FL?. The proteins of ‘inclusion bodies’ isolated from 3 liters of bacterial culture and containing polypeptides PI and P2 were dissolved in 20 ml buffer B (10 mM Tris-HCI, pH 7.5, 1 mM EDT& I mM DTT) containing 8 M urea and loaded onto a column with DEAE Sephacel (2 x 10 cm). Elution was performed by the same buffer. The fractions unbound to an ion-exchanger were combined, dialysed against buffer B and applied to a column with cystatin-Sepharose (2x 3 cm, 2 mg of cystatin/ml of eel). The proteins bound to cystatinssepharose we’re eluted by 0,Ol M NaOH solution, pH 12, containing 0.1 M NaCI, dialysed against buffer B, concentrated and stored at -70°C. 2.3. Isolarion and purification of protein p-&polio The proteins of ‘inclusion bodies’ isolated from 1 I OFbacterial culture and containing recombinant protein @gal-polio were dissolvcd in 3 ml buffer B with 8 M we:! and applied to the column with CLsepharose 4B (2 x 70 cm). Elution was perfornied by the same buffer. The fractions containing protein@gal-polio were combined, dialysed against buffer B, concentrated and stored at -7OOC.

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3. RESULT’S AND DISCUSSION Two virus-sprreific polypcptidcs binding polyclonnl antibodies to the fragtncnt of 3C wer; obtained when cloning and expressing fragment HindII-HirrdlII (bases from 5240 to 6056 [S]) of cDNA poliovirus I (Ml conraining gene for proteasc 3C (Fig. fn) in vector pTTQ8 (Fig. lb). One of rhcm (PI) was formed at autocatalytic cleavage of bond Gin-Gly, adjoined to N-terminus of 3C in the molecule of the recombinant protein. The other (P2) was a product of translation from alternative methioninc encoded by bases 5516-5518 (51 of poliovirus eDNA (Fig, la) [lo]. Sequence analysis

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on cynmtin&phnresc of rcnaturcd prorcins from frrelinna nal bound 10 DEAE-Scphnccl and caminiq palypcpridcs PI nnd P?. Elusion: I, buffer 8; 2, buffer B ccmrnininp 0.5 M NaCI; 3, 0.01 Fvl NnOM conrnining 0. I M NaCI, PI-I l2,O. (b)’ Analysi.s in 12% Nc+Do~SQ,~PAGEprercin from pcakx I (I) and II (2) marked on chromatogramx (8).

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showed that the N-terminal sequences of 3C and PI are identical and the sequence of P2 coincides with that of 3C downstream to the residue 27 f 151. Amino acid pair Gin-Gly adjoined to the C-terminus in two cases rcmaincd uncleavable, therefore the pcptides contained fragment (25a/a) of 3D polymerase besides sequence of amino acids of 3C protease. Polypeptidcs Pl and P2 were isolated and purified as described in ‘Materials and Methods’ (Fig. 2a, b). The fragment of a virus polyprotein obtained at cloning and expression of fragment HindIII-BglII (bases 5240 to 5661 [S], Fig. la) of poliovirus cDNA in vector pEK1 was used as a natural substrate (Fig. lc, [16]). The virus polypeptide encoded by fragment HindII-BgIII and translated in the recombinant protein ,8galactosidase-polio did not possess a proteolytic activity 2

1

Fig. 1. (a) Fragment kiindll-Mind111 of poliovirus cDNA and the encoded polypeptide contalning 43 a/a of 3C protein, 22 a/a of VP, peptide, 183 a/a of 3C pPotease and 25 a/a of3D polymerase. Symbol 8 designates amino acid pair ~irr~ty recognized by 3C protease of poliomyelitis virus. Bases 5516-5518 of poliovirus cDN,$ encode mcthionineinitiating translation of an alternative polypeptide. (b) Recombinant vector pTTQ&PV containing fragment HindII-HindIlI of PoliOvirUS cDNA. (c) Recombinant vector pEKI-PVS containing fragment NindlI-&/II of poliovirus CDNA.

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and conterincd two 3C proteasc-recognized sites GlnGly surrounding pcptide VPg at the NMand C-ends (Fig, la), A rccsmbinnnr protein _&gal-polio purified to homogeneity as dcscribcd in ‘Materials and Methods’. To define a praccolytic activity the samples of PI and P2 were incubated with the substrate in the presence or absence of iodacetamidc, Electrophoresis showed (Fig. 3a, b) that only polypeptide Pt possessed a protcolytic activity as regards a natural substrate. Enzymatic hydrolysis was suppressed by 10 mM iodacetamide. Polypeptide P2 did not show a protcolytic activity thar conforms to an idea about the role of the 3C N-terminal fragment to support an active conformation of the cnzymc [17]. Besides the activity of the natural substrate polypcptide PI displayed a specific protcolytic activity relative to/l-galactosidase and bovine catalase and cleaved them at site Gln-Gly. The amino acid sequence of each of the four identical subunits of E. co/i ,&galactosidase contains 4 sites GlnGly in positions 262-263, 563-564, 675-676, and 845-846 [18]. Mowever, after a long incubation of ,& galactosidase and the enzyme ,&galactosidase was partially hydrolyzed and two fragments formed (Fig. 4a). Deciphering of the N-terminal sequences of the obtained fragments showed that peptide bond Gin-Gly split in position 563-564 that yielded fragments I (50 kDa, Nterminal sequence Gly-Gly-Phe-Val) and II (66 kDa, Nterminal sequence Thr-Met-IQ After denaturation of ,&galactosidase in buffer with 8 M urea and further dialysis against buffer without urea the rate of the enzymatic hydrolysis increased, here besides fragments I and II fragment III appeared (78 kDa, N-terminal sequence Thr-Met-IIe) (Fig. 4b), that can be explained by cleavage of bond Gin-Gly in position 675-676. An advantageous cleavage of bonds GlnGly in positions 563-564 and 675-676 occurs due to

their accessibility to the enzyme. This accords well with the fact that the two Gin-Gly sites are located in the pro. line rich region of the &alaerosidaxe maleeulc [18]. Each of four identical chains of the bovine catalaae molecutc contains 3 sites Gin-Cily in positions 239-240, 351-352 and 397-398. The latter two sites arc surrounded by proline residues [19], The native catalase is not susceptible to the cleavage by 3C but after &maturation with 8 M urea the cleavage of bond Gln*Gly in pasitians 351-352 and 377-398 yields fragment of && 39 kDa (Fig. 4c) and low molecular peptides, The cleavage of bond Gin-Gly in position 239-240 is not apparently observed due to its inaccessibility to the enzyme, REFEIWNCES Kiramure,I\i.,Semter,R.L,, Rothberg, P,G., Larsen, G,K. and Adler, CJ, (1981) Nature 291, 647-653. [2] Pattansch, M., Kcw,O,M., Scmler, B.L., Omitianovski, D.R, and Anderson, C.W. (1984) J. Viral, 49, $73-880. [31 Toyodn, H,, Nicklin, M.J., Murray, M.G., Anderson, C.W., Dunn, J.J., Studier, F.W. and Wimrncr, E. (1986) Cell 45, 761-770, [41 Hanecak,R., Semter,B.L., Anderson, C.W. and Wimmer, E. (1982) Proc, Natl, Acad. Sci. USA 79, 3973-3977. [S] Racaaietto,V.R. and Baltimore, D. (1982) Proc. Nail. Acad, Sci. USA 78, 4887-4891. [61Korant,B.D.(1973) J. Viral. 12, 556-563. I71Pelham, H. (1987) Eur. J. Biochcm. 85, 457-462, I81Ivanoff,L.F., Towatari, T., Korant, B.D. and Petteway, S.R, (1986) Proc. Nett. Acad. Sci. USA 83, 5392-5396, [9] Palmenberg, A.C. and Ruechert, R.R. (1982) J. Virot, 41, 244-249, [IO]Hanecak, R., Semler, B.L., Ariga, H,, Anderson, C,W. and Wimmcr. E (t984) Cell 37, 1063-1073. [ItI Maniatis, T., Fritsch, E.E. and Sambrook, J, (1984) in: Motecutar Cloning (Bayev, A.A. and Skryabin, K.G. eds) pp, 98-185, 239-242, Mir, Moscow, [Ial Chuvpilo, S.A. and Kravchenko, V.V. (1985) FEBS Lett. 179, 34-36. [13] Laemmti, U.K. (1970) Nature 227, 680-685. [141 Swaniy, K.H.S. and Goldberg, AL. (1982) J. Bacteriot. 149, 1027-1033. [II

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Recombinant poliovirus 3C protease. The enzyme application to protein specific fragmentation.

Active 3C protease of poliovirus 1(M) was obtained when cloning and expressing fragment HindII-HindIII (bases from 5240 to 6056) of cDNA in vector pTT...
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