To whom correspondence should be addressed. 11 Fellow DRG-848 of the Damon Runyon-Walter Winchell Cancer. Fund. Present address: Research Institute ...
Communication
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264 No. 17 Issue of June 15 p 9738-9741 1989 0 1989 by The American Societ; for Bidchemistry and MbLiular Bioloh, Inc. Printed in U.S. A.
Cleavage of Synthetic Peptides by Purified Poliovirus 3 C Proteinase* (Received for publication, December 21, 1988) Peter V. PallaiSB, Frederick BurkhardtS, Mark SkoogS, Kurt SchreinerS, Patricia BaxS, Kenneth A. CohenS, Gordon HansenS, Deborah E. H. PalladinoS, Kevin S. HarrisT, Martin J. NicklinT(1, and Eckard WimmerT From the $Research and Development Center, Boehringer Ingelheim Pharmaceuticals Inc.,Ridgefield, Connecticut 06877 and theTDepartment of Microbiology, State University of New York, Stony Brook, New York 1I794
Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain GlnGly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3 C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated halflife of the homologous precursor proteins detected in poliovirus-infected cells. The peptide mostresistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3 C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P l ’ , respectively, contributes to substrate recognition. Ac-Gln-Gly-NHz was not a substrate for 3C.
is responsible for cleavages within P2 and P3 polyprotein precursors. 3C cleaves predominantly at Gln-Gly pairs (for reviews see Refs. 1 and 2). Processing events in the P2 and P 3 region liberate polypeptides 2A, 2B, 2C, 3A, 3B, 3C, and 3D. 3CD-induced cleavages in the capsid precursor P1 yield a complex of capsid proteins (VPO,VP3, VP1) that can assemble to form an icosahedral procapsid. Structural factors that govern polyprotein processing are poorly understood. Processing of the capsid polyprotein appears to be dependent not only on primary sequence signals (1)but also on correct tertiaryfolding (3,4). Capsid precursor P1 is efficiently cleaved only by 3CD (the precursor to3C and RNA polymerase 3D) (5-7). Purified 3C in high concentration slowly hydrolyzes the bond between VP3 and VP1 in vitro (7). Although the x-ray structures of poliovirus (8) and rhinovirus (9) capsid proteins are available, they offer limited insight into the substrate structure, as the x-ray structures portray a state in which the processed termini are no longer adjacent to each other. 3C-mediatedcieavages in the P2 and P 3 region may have less stringent substrate structure requirementsthanthose in the P1 region. A strongrelationship between folding and proteolysis, however, is indicated by the occurrence of stable alternative cleavage products of a single precursor (10). Nine Gln-Gly sites have been found to be cleaved in vivo in the poliovirus polyprotein. Four additional Gln-Gly sites, which occur in VP2, 2A, 2C, and 3D, do not appear to be cleaved in vivo or in vitro (1).In order to study structural requirements for proteolytic processing by poliovirus 3C we have synthesized peptides corresponding to poliovirus polyprotein sequences and studied theirbehavior as substratesfor the purified recombinant enzyme. Peptides that correspond to the P2 and regions P3 of the genome were selected because precursors originating from these regions can be cleaved i n vivo by 3C, whereas P1 is known to be cleaved by 3CD only (5-7). EXPERIMENTAL PROCEDURES
The Picornaviridae is oneof the largest families of human pathogens andincludes the genera of enteroviruses (e.g. poliovirus) and rhinoviruses. During the replication cycle picornaviralRNAistranslatedinto asinglelarge polyprotein precursor which undergoes proteolytic processing to yield functional component proteins. The maturation process is dependent on the action of virus-encoded proteinases. Enteroand rhinoviruses encode two core proteinases, 2A and 3C. Proteinase ZA, which maps to the P2 region of the polyprotein (shown in Fig. l ) , cleaves only at itsown N terminus between Tyr-Gly residues while still being translated, possibly by an intramolecular mechanism. Proteinase 3C, thought tobe generated also by self-cleavage, is encoded in the P3 region and
Synthesis and Purificationof Peptides-Peptides were synthesized by the solid phase method (11)using either an Advanced Chemtech or an Applied Biosystems 430 automatic synthesizer. Peptides were cleaved from the resin by liquid H F (0 “C, 1 h) in the presence of anisole and dimethyl sulfide (HF:anisole:dimethyl sulfide, 1O:l:l) as scavengers. After evaporation of HF, the peptide resin mixtures were washed with diethyl ether containing a suspension of calcium carhonate and extracted with water80% andacetonitrile-water. The extracts were lyophilized, and the crude peptides were purified by high performance liquid chromatography (HPLC)’ on a reversed phase column (C18, 22 mm X 25 cm) using 0.1% trifluoroacetic acid, solvent A, and 60% acetonitrile-40% water-0.1% trifluoroacetic acid, solvent B, as mobile phase solvents. The lyophilized products were characterized by fast atom bombardment mass spectrometry, HPLC, and amino acid analysis. Peptide Analysis-Fast atom bombardment analysis of peptides was obtained with a Kratos MS80RFAQ double focusing mass spectrometer with operating conditions of 10 kV xenon atoms at 35 pA discharge current. Samples were dissolved in a glycerol matrix containing 2% acetic acid. The reported spectra were a summation of three to five 100 s/decade scans acquired in the raw data acquisition mode. Masses were assignedrelative toCsIclusterionreference
* This work was supported by Boehringer Ingelheim Pharmaceuticals Inc. and by United States Public Health Grants AI 15122 and CA 28146 (to E. W.). The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. The abbreviations used are: HPLC, high performance liquid chro11 Fellow DRG-848 of the Damon Runyon-Walter Winchell Cancer Fund. Present address: Research Institute of Molecular Pathology, matography; HEPES,4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. 9738
Peptide Cleavage by Poliovirus 3C Proteinase peaks at m/z 912.335 and 1172.145.Amino acid composition of peptides was determined by the pico-tag methodology (Waters Associates). Proteolytic Cleavage of Peptides by Poliovirus Proteinase 3CProteinase 3Cof poliovirus type 2 was expressed in bacterial cells and purified as recently described (7). The enzyme was 95-98% pure and identical in sequence to 3C produced by poliovirus type 2 infection of HeLa cells except the initiator methionine residue of the N terminus was retained on the recombinant enzyme. Peptide solutions were prepared at 1 mg/ml in HEPES buffer (10 mM HEPES, 0.1 M NaC1, 1 mM EDTA, 5 mM NaOH, pH 7.4). A stock solution of3C 0.246 was4.8 mg/ml (240 JLM) in HEPES buffer (as above) containing peptides was carried additional 1 mM dithiothreitol. Digestion of the0.226 out at 30 "C. Progression of Peptide Cleavage-Peptides l a0.550 and 3a (50 JLM) were 0.563 digested with proteinase 3C (1 JLM). Progression was monitored by 0.813 reversed phase HPLC analysis of samples taken a t 30 min and 4 h. HPLC conditions were: Vydac C18column, 4.60.876 mm X 25.0 cm; linear 2 ml/min flow rate; gradient, 5% B/95% A to 100% B in 30 min,0.884 monitored at 215 nm. Consumption of substrates and generation of two products were observed. Complete cleavage of both peptides was indicated by the disappearance of a substrate peak in the 4-h sample. Specific Cleavageof Substrate la-Peptide l a (8JLM) was incubated with 3C (1 JLM) overnight, and the digest was analyzed by HPLC (conditions as above). In addition to the fragment and substrate peaks, no other peptidic material was observed as all remaining HPLC peaks could be identified as buffer component materials. Fractions corresponding to the major peaks were collected and analyzed by mass spectrometry, which proved that the expected C- and N-terminal fragments were present in the HPLC peaks eluting at retention times identical to those of the synthesized standards. In fast atom bombardment a MH' of 932.2 was found for the C-terminal fragment and a MH' of 780.3 for the N-terminal fragment. As further confirmation of their identities, the collected C- and N-terminal fragments were also subjected to amino acid and sequence analysis. Last, HPLC quantifications of 3C digests obtained under conditions causing partial cleavage gave levels of products and substrate leading to proper mass balance. Competition Experiments for Ranking Peptide Substrates by V-1 K,-Reactions were carried out at 30 "C in pH 7.5 buffer consisting of 0.1 M HEPES, 0.3 M KCl, 1 mM dithiothreitol, and 1 mM EDTA. Proteinase 3C, final concentration 2 JLM, wasadded to a mixture of two substrates, one usually l a , each a t 50 JLM. Aliquots were removed, added to an equal volume of 2 % trifluoroacetic acid in water, and stored at -20 "C prior to HPLC. Peptides were separated on a Nucleosil C-18 column eluted at 1 ml/min with a 26-min linear gradient from 9% to 56% acetronitrile in water with 0.05% trifluoroacetic acid. Elution was monitored at 210 nm. For each substrate
9739
and itsproducts, the area under theircombined peaks was independent of the extent of conversion of the substrate.Substrateand products were, therefore, detected and recovered with equal efficiency, and comparison of areas yielded the extent of conversion. These values yield ratios of rate constants using ( V ~ ~ ~ / K ~ ) 1 / ( V m =~=/K~)z log(1 - F,)/log(l - F2) where F is the fraction of substrate that is converted to product (12, 13). For example, the value of (Vmex/K,,&~ for substrate 3 was determined from the following values of F. t (min) 6 10 10 15 30 30 60 90 90
Peptide 3 0.069 0.135 0.111 0.187 0.303 0.314 0.548 0.625 0.642
Peptide la 0.158 0.367
Demonstration of Lack of Cleavage-The following peptides containing either one or both residues (Gln, Gly) of the scissile bond were incubated with 3C (11.5 JLM) for 20 h: SQLFPIS, Ac-ITTAGPSN H z +, E T Y G F Y , S Q L L P L RV , KDLQGDGLADVA, VSFNFPQITLW,andAc-STKDLTTYGGHQNKA-NH2.Peptide:enzyme ratios ranged between 1:4 and 1:lO. HPLC analysis of digests did not indicate formation of new products. Ac-Q-G-NHz (500 p M ) was also incubated with 3C (2 JLM) for 3 h. Quantitative analysis of the digest by HPLC indicated no consumption of substrate. RESULTS ANDDISCUSSION
The amino acid sequences within the poliovirus polyprotein (14, 15) surrounding the scissile Gln-Gly sites that are recognized by proteinase 3C do not share significant primary structure similarity. Moreover, comparison of sequences surrounding the four Gln-Gly pairs of the polyprotein that are not cleaved suggests no additional determinants characteristic of all substrate sequences. An exception may bethe preference for an alanine residue in the P4 position (1).In light of the diversity of cleavage site sequences, i e . the limited identities within these sequences, a demonstration of cleavage specificity and definition of structural requirements for 3C-induced proteolysis are desirable. Accordingly, we synthesized a variety of peptides with sequences corresponding to poliovirus cleavage sites (Table I) and studied these peptides as sub-
TABLEI Sequences of cleavage sites in the poliovirus polyprotein and relative cleavage efficienciesof synthetic peptides by proteinase 3C Peptides listed correspond to sequences of poliovirus type 2 (Sabin) (16, 17). Cleavage sites are indicated in the left column. The efficiency of cleavage is shown in the right column and is expressed as ( VmaX/K,,Jml. A, peptides l a and 3a were used to establish specificity of cleavages by proteinase 3C by analysis of their products. B, 16residue peptides 1-5 correspond to five different cleavage sites and were used in competition experiments in the presence of l a to establish V,,,.,/K, ratios relative to la.C, peptide 6 and its TP4A analog, 6 (TP4A), are shown. Cleavage sites in polyprotein
Code number P1'
A
P1
Relative efficiency of cleavage
Sequence P4
la 3a
2C/3A 3B/3C
1 2 3 4 5
6 6(TP4A)
PLQYKDL-NHz PGFDYAV-NHz
1.00 0.39 + 0.02
2C/3A 2B/2C 3B/3C 2A/2B 3A/3B
Ac-NCMEALFQ'GPLQYKDL-NHz Ac-EIPYAIEQ'GDSWLKKF-NHz Ac-TIRTAKVQ*GPGFDYAV-NHz Ac-YEEEAMEQ*GISNYIES-NHZ Ac-YKLFAGHQ*GAYTGLPN-NHz
1.84 f 0.11 2.77 f 0.16 0.46 k 0.03 0.05 f 0.01 0.04 f 0.01
3C/3D
A~-RSYFTQIQ*GEIQWMRP-NH~ Ac-RSYFAQIQ*GEIQWMRP-NHz
