VIROLOGY

185,140-150(1991)

Purification, Properties, and Mutagenesis of Poliovirus 3C Protease ELLEN Z. BAUM, GERALDINE A. BEBERNITZ, OLGA PALANT, THOMAS MUELLER, AND STEPHEN .I. PLOTCH Molecular Biology Section, Lederle Laboratories,

Medical Research Division, American Cyanamid Company, Pearl River, New York 10965

Received February 12, 199 1; accepted July 22, 199 1 Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia co/i under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies. The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate. Proteolytic activity was assayed using as substrate either [%]methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B. Poliovirus protein 3CD (protease-polymerase) was also expressed in bacteria. About 25% of this protein apparently autodigested in viva, releasing immunoprecipitable protein 3D (polymerase). No further autodigestion of 3CD could be detected in vitro, nor could addition of purified protein 3C effect digestion in trans. Both the serine protease inhibitors PMSF, TPCK, and 3,4-dichloroisocoumarin, and the cysteine protease inhibitors cystatin and zinc, were effective inhibitors of the 3C protease. Six new mutants of the protease, with altered or no enzymatic activity, were identified based on the observation that low level expression of wild type enzyme severely retards growth of bacterial colonies harboring the expression plasmid. c 1991 Academic

Press, Inc.

structural proteins (Ypma-Wong et al., 1988; YpmaWong and Semler, 1987; Jore et al., 1988). In this paper, we report the cloning and expression of 3C protease in fscherichia co/i. We present a simple and rapid purification scheme which resulted in higher yields of biologically active protease than previously published reports (Nicklin et al., 1988; Takahara et al., 1989). The activity of 3C protease and its sensitivity to protease inhibitors was examined using natural substrates and oligopeptides. In addition, we have cloned and expressed 3CD and examined its autodigestion and susceptibility to digestion in trans by purified 3C. We also present a collection of six novel mutants of 3C protease, including one which exhibits temperature sensitivity, which were isolated based on our observation that wild type 3C protease is detrimental to the growth of E. coli.

INTRODUCTION Poliovirus provides an excellent example of the strategy of protein processing which is employed by many RNA viruses and retroviruses. The 7.5-kb positive strand RNA genome of poliovirus encodes a 247-kd primary polyprotein, which is cotranslationally processed initially into three smaller polyproteins, Pl , P2, and P3, by two distinct proteases encoded within the polyprotein (reviewed in Krausslich and Wimmer, 1988; Kay and Dunn, 1990; Palmenberg, 1990). Pl contains the capsid proteins 1A, 1 B, 1C, and 1 D; P2 is comprised of proteins 2A, 2B, and 2C; and P3 is comprised of proteins 3A, 3B, 3C, and 3D. Protein 2A is the protease responsible for the primary cleavage at the Tyr-Gly junction which separates the Pl capsid precursor from the nonstructural protein precursor P2-P3. Protein 3C is the core of a protease that carries out all subsequent cleavages at Gln-Gly junctions, recognizing 9 of 13 such pairs found within the poliovirus polyprotein. The 3C protease is a cysteine protease (Ivanoff eta/., 1986) which is required for viral infectivity (Dewalt and Semmler, 1987). In poliovirus-infected cells, autocatalytic cleavage of the P3 precursor polyprotein results in the release of two different protease forms: mature protease (3C) and protease-polymerase (3CD). Both 3C and 3CD possess proteolytic activity, but with different specificities. 3CD is responsible for the release of the capsid proteins from Pl , whereas 3C and 3CD cleave the P2 and P3 polyproteins to generate the non0042-6822191

$3.00

Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

MATERIALS

AND METHODS

Plasmid constructions Plasmid pT7-3C (Fig. la), encoding poliovirus protease 3C, was constructed as follows: Plasmid pT7PVl-5 (Van der Wet-f et a/., 1986) which contains a full-length cDNA of the poliovirus genome (Type 1, Mahoney strain) was digested with &$I and Asull, releasing a 687 nucleotide fragment (poliovirus nucleotides 5325-6011). After agarose gel purification, the DNA was digested with Asul, releasing a 515 nucleotide Asul-Asul fragment (nucleotides 5439-5953). To in140

POLIOVIRUS

sert this fragment into the bacterial expression vector pT7K (Plotch et al., 1989) and to rebuild the complete coding sequence of the 3C protease (poliovirus nucleotides 5438-5986) preceded by a methionine codon for the initiation of translation, a pair of ,flanking oligonudeotide duplexes were synthesized. The 5’ duplex recreates vector sequences from the Xbal site downstream of the 17 promoter (Rosenberg et al., 1987) to the Ndel site, followed by an additional G residue (which reconstructs poliovirus nucleotide 5438). The 3’ duplex includes the sequence from the 3’Asul site of the 3C protease (nucleotide 5954) to the end of the gene (nucleotide 5986) followed by 2 stop codons and a BarnHI site. The 515 nt poliovirus DNA fragment and synthetic oligonucleotides were ligated to pT7K which had been digested with Xbal and BarnHI, followed by transformation into E. co/i strain DH5a. The resultant plasmid, pT7-3C, was transferred into the expression strain BL21 (DE3)pLysS (Studier and Moffatt, 1986; Moffat and Studier, 1987). Mutagenesis of 3C protease was accomplished by passing pT7-3C through the E. co/i mutator strain LE30 mutD, as described previously (Baum et al., 1990). Plasmid pT7-3CD (Fig. 1b) was constructed by subcloning a 2.4-kb AvalllEcoRI fragment, encoding poliovirus protease/polymerase polyprotein (poliovirus nucleotides 5439-7441 followed by an 84 nt poly dA-dT tract) from pT7PVl-5 (Van der Wet-f et al., 1986) and the EcoRI-BamHl region from pBR322, into the XbalBarnHI sites of pT7K. The 5’ end of this construct was rebuilt using the same oligonucleotide duplex as for pT7-3C. The 3’end of this construct is identical to pT7POL (Plotch et al., 1989). Plasmid pTi”-2C3AB (Fig. 1 c) contains poliovirus nucleotides 4124-5437 and was constructed as follows: plasmid pT7PVl-5 was digested with Sphl and Hindlll, releasing a 1.9-kb fragment (nucleotides 4159-6056). After purification by agarose gel electrophoresis, this fragment was digested with Bspl2861 at nucleotide 5412 to release a 1.25-kb Sphl-Bsp12861 fragment containing most of 2C, all of 3A, and most of the 3B genes. Upstream synthetic oligonucleotide duplexes rebuilt the 2C gene, adding a 5’ terminal Ndel sequence that includes an initiating methionine codon. Downstream oligonucleotide duplexes rebuilt the 38 gene, followed by two stop codons and a BamHl site. The 1.25-kb DNA fragment and oligonucleotides were ligated to IVdel-BarnHI-digested pT7K, creating plasmid pT7-2C3AB. Plasmid pT7-A3ABC (Fig. 1d) encodes a 13-kd polyprotein and was constructed as follows. The Bglll EcoRl region of pT7PVl-5 (Van der Werf et a/., 1986) encoding poliovirus nucleotides 5325-7441 followed by an 84 nt poly dA-dT tract and the 375 nt EcoRI-

3C

141

PROTEASE

BamHl region from pBR322 was subcioned into the Ndel-BarnHI sites of pT7K, using an oligonucleotide pair encoding the first nine amino acids of f7 gene 10 flanked by Ndel and Bg/l cohesive ends. The resultant plasmid was digested with Bgfll (at poliovirus nucleotide 5601) followed by fill-in with Klenow enzyme and ligation of the blunt ends, resulting in a frameshift followed by translation termination 15 amino acids downstream from the f3g/ll site. The final construct, pT7A3ABC,thus encodes part of 3A, all of 3B, and part of 3c. Purification

of 3C protease

Two liters BL2 1(DE3)pLysS harboring pT7-3C were grown at 37” with shaking to an absorbance of 1 .O (600 nm) in Luria broth containing 25 ~g/mt kanamycin and 10 pg/ml chloramphenicol. lPTG was added to 1 mM and the incubation was continued for 3 hr. All subsequent steps (Fig. 2) were done at 4’. Ceils were pelleted by centrifugation and lysed by sonication in buffer A(50 mlMTris-HCI, pH 7.6, 1 mMEDTA, 50 mMNaCI, 1 mNI DTT) (Plotch et al., 1989). After centrifugation (Beckman SS34 rotor, 12k rpm, 15 min) and removal of the supernatant, the pellet was washed ohce with buffer A. The pellet was then dissolved in 5 mi of buffer B (50 mMTris-HCI, pH 7.6, 1 mM EDTA, 1 .O Iw NaCI, 0.5% NP-40, 1 mM DTT, 10% glycerol, 8 M urea) and dialyzed against the same buffer lacking urea (buffer C) followed by dialysis against buffer D (50 m&I Tris-HCI, pH 7.6, 1 mM EDTA, 0.5% NP-40, 1 mAA DIT, 10% glycerol). After removal of insoluble materia) by centrifugation, the supernatant was loaded on a DEAE-Sephaccl column (1.5 x 20 cm) (Pharmacia) eq&ibrated in buffer D. After washing with 1 column volume of buffer D, 3C protease was eluted in buffer D containing 100 ml\ll NaCI. The protease was again dialyzed against buffer D and loaded onto a phosphoce#&se column (1.5 X 15 cm) (Whatman) equilibrated with buffer D. After washing with buffer D, 3C protease was ejuted in buffer D + 200 mll/l NaCI. The purified protease (about 15 mg) was stored at -80”. For experiments on mutant protease (Fig. 7), 50-ml cultures of mutant and wild type 3C protease plasmids in BL21(DE3)pLysS cells were induced with IPTG as described above. After sonication in buffer A and centrifugation, protease was prepared either from the supernatant (clones 6-1, 2-3, and 2-2) or from the pellet (clones 8-1, 2-4, and 8-2). The supernatants were applied directly to a DEAE-Sephacet column and eluted as described above. The petlets were resuspended in 2 ml buffer A, and an equal volume of settied Q Sepharose was added. Protease in the pellet was sotubiiized and eluted from the Q Sepharose as described (Hoess

142

BAUM ET AL.

eta/., 1988). Wild type protease was purified from both the supernatant and the pellet fractions for use as controls. The amount of purified protease was quantitated by Coomassie blue staining of each preparation following electrophoresis on SDS-polyacrylamide gels. Protease

assays

For use as a substrate, 2C3AB polyprotein was labeled with [36S]methionine as follows: 5 ml BL21(DE3)pLysS harboring pT7-2C3AB was grown at 37” with shaking to an absorbance of 0.5 (600 nm) in Luria broth containing 25 pg/ml kanamycin and 10 pg/ml chloramphenicol. IPTG was added to 1 mA# and the incubation was continued for 30 min. Cells were pelleted and resuspended in M9 medium + 0.2% glucose (Maniatis et a/., 1982) containing 1 ml\/l IPTG. After 20 min, rifampicin (20 fig/ml) was added to suppress transcription by endogenous E. co/i (but not T7) RNA polymerase, and the incubation was continued for 20 min. [35S]methionine was then added to a final concentration of 500 rCi/ml. After 45 min, cells were pelleted and sonicated in buffer A as described above. Most of the 2C3AB was found in the insoluble pellet fraction following centrifugation. To solubilize 2C3AB, the pellet was resuspended in 1 .O ml buffer B and dialyzed against buffers C and D as described above for 3C protease. After removal of insoluble material by centrifugation, the supernatant was adjusted to 50 mlM NaCl and passed through a l-ml DEAE-Sephacel column. The flowthrough contained 2C3AB. For protease assays, radioactive 2C3AB was typically incubated with l-5 pg of 3C protease for 30 min at 30” in buffer A, unless otherwise noted. Since 2C3AB was present in insufficient quantity to be detected by Coomassie blue staining, its molar quantity could not be estimated. Therefore, the ratio of protease to substrate was 91. Reaction mixtures were subjected to SDS-polyacrylamide gel electrophoresis (12% acrylamide/0.3% bis) followed by autoradiography. Substrate 63ABCwas labeled with [35S]methionine and extracted from the pellet fraction by solubilization with urea followed by dialysis, as described for 2C3AB. To prepare 3CD protein for autodigestion studies and for digestion by protease 3C, BL21(DE3)pLysS cells harboring pT7-3CD were labeled with [36S]methionine as described above for pT7-2C3AB. Cells were lysed by sonication in buffer A. After centrifugation, the pellet was extracted with buffer C and centrifuged again. The two supernatants containing 3CD were pooled and dialyzed against buffer A and used for the experiment described in Fig. 5b.

Peptide substrates Synthetic 16-mer peptides composed of amino acids flanking poliovirus cleavage junctions were prepared using an Applied Biosystems 430A Peptide Synthesizer. Peptide 3A/3B is TyrLysLeuPheAlaGlyHisGlnGlyAlaTyrThrGlyLeuProAsn; peptide 2C/3A is AsnCysMetGluAlaLeuPheGlnGlyProLeuGlnTyrLysAsnLeu. Secretin, growth hormone reducing factor, and serum thymic factor were purchased from Sigma. Peptides (5 pg) were incubated with 3C protease (5 pg) in buffer A for 16 .hr at 30”. The digestion mixtures were separated by reverse phase HPLC (Brownlee C4 cartridge, 5 pm particle size, 30*2.1 mm) using O-70% acetonitrile in 0.1% trifluoroacetic acid for 2 hr at room temperature (flow rate of 0.25 ml/min). Sequence analysis of peptides was performed on an Applied Biosysterns 477A liquid-phase microsequenator and phenylthiohydantoin (PTH) derivatives of amino acids were identified by reverse phase HPLC using a Model 120A on-line PTH analyzer (Applied Biosystems), with experimental protocols for both procedures as supplied by the manufacturer. RESULTS AND DISCUSSION Expression

and purification

of 3C protease

The Mahoney poliovirus 3C protease was expressed under the control of the phage T7 promoter in E. co/i strain BL21 (DE3)pLysS (Fig. 1a). This strain contains a chromosomal copy of T7 RNA polymerase under lac promoter control. The synthesis of T7 RNA polymerase, and, in turn, 3C protease, is therefore inducible with IPTG. This strain also contains plasmid-encoded lysozyme, which inactivates T7 polymerase (Studier and Moffatt, 1986; Moffat and Studier, 1987) and lowers the basal level of 3C protease in the absence of induction. Upon induction with IPTG, these cells produce 3C protease, as demonstrated by SDS gel analysis (Fig. 2, lanes 2 and 3). Most of the protease partitions into insoluble inclusion bodies (Fig. 2, lane 3). We chose to purify the enzyme from this insoluble fraction, using DEAE-Sephacel followed by phosphocellulose chromatography to remove trace impurities (Fig. 2, and Materials and Methods). The yield (“8 mg/liter) of our highly purified Mahoney strain 3C protease appears to be greater than other 3C protease preparations from Sabin strains described in previous reports (Nicklin et a/., 1988; Takahara et a/., 1989). Takahara et a/. (1989) used a different expression vector, and although our vector is essentially identical to that of Nicklin et a/. (1988) those authors purified 3C protease from the soluble fraction.

POLIOVIRUS 3C PROTEASE Ndel

f

3c a

(-2OKD)

3CD b

5985

EEoRJ BarnHI

Nd8l 3c

3D

5438

’

EarnHI

3c I 5438

(-73KD)

2C3AB

143

Ndel E

t

7441

-

3A

138 . Barn HI

2c

(-!iOKD) 4124 Ndel

(38KD)

t (IOKD)

5437 (2KD)

Bgll

(5.4KD)

’ (7.8KD)

FIG. 1. Poliovirus cDNA constructs under T7 promoter control in pT7K vector. Numbers refer to poliovirus nucleotides; aatenska are stop codons. (a) 3C, encoding protease. (b) 3CD, encoding protease-polymerase. The gray box denotes the 84 nt PO@ dRdT tract; the bfack box denotes pflR322 sequences. (c) 2C3AB, encoding a polyprotein substrate for 3C protease. (d) ABABC. encoding a polypmtein substrate for 3C protease. This construct lacks the amino terminal portion of 3A and the carboxy terminal portion of 3C. The gray box denotea T7 gene 10 sequences. The black box denotes a frameshift starting at the &/II site of 3C protease, resulting from fill-in with Klenow enzyme as described under Materials and Methods. The observed cleavage sites are indicated by arrows.

To provide a functional assay for 3C protease, the 2C3AB region of the poliovirus genome was subcloned into the T7 expression vector (Fig. 1c). This construct encodes a 50 kDa substrate for the 3C protease, providing cleavage sites at the 2C/3A and 3A/3B junctions. The 2C3AB substrate was labeled with [35S]methionine. Complete cleavage of 2C3AB by 3C

3C

FIG. 2. Purification of 3C protease. Supernatant fractions were prepared from E. co/i BL2 1(DE3)pLysS cells harboring (lane 2) or lacking (lane 1) pT7-3C as described under Materials and Methods. The pellet fraction containing 3C protease (lane 3) was resolubilized, applied to a DEAE-Sephacel column, and eluted (lane 4). After dialysis, the eluant was applied to a phosphocellulose column which was washed with buffer D (lane 5). 3C protease was eluted with buffer D containing 200 mM NaCl (lane 6). The figure is a Coomassie bluestained SDS gel of equal proportions of each purification step. Molecular weight markers are shown at left, and the position of 3C protease is indicated.

protease should liberate products of 38, 10, and 2.5 kDa, the last of which lacks methionine n&dues and is therefore undetectable in this assay. 3C pWWaae was incubated with 2C3AB substrate, and ctaavage was monitored by SDS get electrophoreeis and autmadiography. As shown in Fig. 3a, lane 2, purified JC protease is enzymatically active, as judged by the &savage of the 2C3AB substrate. Two deavage products are observed: a 38 kDa product whose size ia coWetent with 2C, and a smaller cleavage product of -12 kDa. The latter is probably uncleaved 3AB, since it corn&rates with recombinant 3AB and reacts with antibody against 3B (data not shown). The lack of cleavage of 3AB is consistent with studies on synthetir: peptides which showed that the 3AB linkage is a ~~~~i~ poor substrate for 3C protease (Pattai et& I%%?), and with our own studies with recombinant @&I, in which no cleavage by 3C protease was detected (data not shown). In contrast, Hammerle et a/. (19gl) have recently shown autocatalytic cleavage of the 3A8 junction following expression of recombinant JABC. Thus, the 3AB junction appears susceptible to cleavage in cis but not in trans. The activity of 3C protease appears relatively unaffected by the denaturation/renaturation protocoJ. The activity of the renatured, purified 3C was compared to that of the crude enzyme present in the soMe fraction of the bacterial sonicate, using CoomasGe blue staining of SDS-acrylamide gels to estimate the amount of

BAUM ET AL.

144

a M

1

23456

7

b

C

1234567

123456

-2C3AB-

-2CJ

FIG. 3. (a) Cleavage of substrate 2C3AB by 3C protease and sensitivity to protease inhibitors. [35S]methionine-labeled 2C3AB was incubated without 3C protease (lane 1) or with 3C protease (lanes 2-7) in the presence of TPCK (lane 3), TLCK (lane 4), PMSF (lane 5), 3,4-dichloroisocoumarin (lane 6), or ZnCI, (lane 7), followed by gel electrophoresis and autoradiography as described in Table 1 and under Materials and Methods. The position of substrate 2C3AB and cleavage product 2C are indicated. Molecular weight markers (lane M) are shown at left. (b) Cleavage of 2C3AB by purified vs crude 3C protease. Purified 3C protease resolubilized from the pellet fraction (0.2, 1, and 2 pg, lanes 1, 2, and 3. respectively) and crude protease from the supernatant fraction of BL21(DE3)pLysS/pT7-3C cells sonicated in l/l 00 culture volume of Buffer A (0.5,2, and 10 ~1,lanes 4, 5, and 6, respectively), was incubated with [36SImethionine-labeled 2C3AB for 30’, followed by gel electrophoresis and autoradiography. The position of uncleaved 2C3AB and 2C cleavage product are indicated. Lane 7, 2C3AB without added protease. (c) Coomassie blue staining of the same gel shown in (b). The migration of molecular weight markers and of 3C is shown,

3C protease present in this fraction (Fig. 3~). We found that equivalent amounts of renatured 3C protease and crude protease (Fig. 3c, lanes 3 and 5) had similar enzymatic activities (Fig. 3b, lanes 3 and 5). Inhibition

of 3C protease

activity

Previous reports have suggested that 3C is a member of the cysteine protease class. The cysteinespecific reagents iodoacetamide, N-ethyl maleimide, para-chloromercuribenzoate, and 1,3-dibromoacetone block polyprotein processing; also, site-directed mutagenesis of Cys 147, a residue which is conserved in picornaviruses, inactivates 3C protease (Ivanoff et al., 1986 and references therein; Nicklin et a/., 1988). However, computer analysis demonstrates significant homology between 3C protease and the serine proteases (Gorbalenya et al., 1986; Bazan and Fletterick, 1988), and TPCK, an inhibitor of the serine protease chymotrypsin, also inhibits cleavage of polyprotein in poliovirus-infected HeLa cells (Summers et a/., 1972). Since we can assay 3C activity using purified protease with 2C3AB as a substrate in vitro, we can directly determine the sensitivity of 3C protease to a variety of inhibitors whose mechanism of inhibition is known. A collection of commercially available protease inhibitors and other compounds (Table 1) was tested for the ability to inhibit 3C protease. We found that the serine protease inhibitors TPCK, PMSF, and 3,4-dichloroisocoumarin were effective inhibitors of the protease (Fig. 3a, lanes 3, 5, and 6, and Table 1). Since the mecha-

nism of these inhibitors is extremely specific for the active site of serine proteases, our results suggest that 3C protease has an active site similar to the serine proteases. In the case of TPCK, we cannot exclude the possibility that inhibition is due to alkylation of 2C3AB which renders it uncleavable, as noted by Summers et al. (1972). Cystatin and zinc are known to inhibit cysteine proteases, and both have been previously reported to inhibit poliovirus polyprotein processing in infected cells TABLE 1 TEST OF INHIBITORS

Compound Antipain Aprotinin CaCI, Calpain inhibitor I Calpain inhibitor II Cystatin 3,4-Dichloroisocoumarin E-64 Leupeptin a2-Macroglobulin &-Cl, Pepstatin A PMSF TLCK TPCK Trypsin inhibitor ZnCI,

OF

3C PROTEASE

Cont. used

Inhibition

50 as/ml

No No No No No Weak Yes No No No No No Yes Weak Yes No Yes

2 b&ml 2.5 mM 20 pg/ml 10 &ml 20 &ml 20 aglml 10 rglml 5 pglml 2 units/ml 2.5 mM 3.5 pglml 20 pg/ml 50 ,ug/ml 120 fig/ml 20 ag/ml 2.5 mM

POLIOVIRUS

(Korant et al., 1985; Takegami et al., 1983). We found that ZnCI, and cystatin also inhibit 3C protease (Table 1). Taken together, these results provide experimental support for computer modeling which suggested that cysteine is in the active site of 3C protease, but that the active site is structurally similar to that of serine proteases (Gorbalenya et al., 1986, 1989; Bazan and Fletterick, 1988). Recent site-directed mutagenesis of poliovirus and rhinovirus 3C proteases (Hammerle et al., 1991; Cheah et a/., 1990) also supports this notion.

Thermal inactivation of substrate 2C3AB A significant proportion (-20%) of 2C3AB remains undigested after 2 hr at 30” (Fig. 3a, lane 2) and even more undigested 2C3AB is observed at 37” (Fig. 4a), despite a large excess of 3C protease over substrate (molar ratio & 1; see Materials and Methods). The residual undigested 2C3AB remains uncleaved even after extended incubation times (16 hr, data not shown). Initially, we could not exclude the possibility that a portion of the 2C3AB substrate is irreversibly denatured during purification and is therefore resistant to cleavage. However, incubation at lower temperature (22”) resulted in essentially complete cleavage of 2C3AB (Fig. 4a). The basis for the lack of complete cleavage of 2C3AB observed at 30” and 37” was investigated further. Supplementation with additional 3C protease midway through 2 hr incubation at 37” did not increase the amount of cleavage of 2C3AB (Fig. 4b, lane 3) suggesting that the lesion is with the substrate and not with the protease. Temperature shift experiments were performed to determine if incomplete cleavage of 2C3AB at 37” is reversible. Substrate 2C3AB and protease were incubated together for 1 hr at 37’, followed by 1 hr at 22” (Fig. 4b, lane 7). No additional cleavage of 2C3AB is observed upon the temperature downshift, even if more protease is added (lane 6) again suggesting that the lesion is with 2C3AB and that it is irreversible. Next, substrate 2C3AB was preincubated for 1 hr at either 22” or 37”, followed by addition of protease with continued incubation for 1 hr at each temperature (Fig. 4~). Preincubation of 2C3AB at 37” resulted in incomplete cleavage, regardless of the temperature of incubation with protease (Fig. 4, lanes 2 and 3). In the reciprocal experiment, preincubation of 3C protease at 37” (Fig. 4d, lane 6) had no effect on subsequent incubation at 22”. Thus, we conclude that 2C3AB becomes resistant to cleavage upon incubation at 37”. Protein 3A contains a hydrophobic region and is presumably membrane bound during poliovirus infection (e.g., Takegami era/., 1983). In our in vitro system, it is possible that some unfolding and exposure of 3A occurs at 30-

3C

PROTEASE

145

37O, resulting in aggregation of 2C3AB and resistance to digestion. Preliminary experiments in which recombinant protein 3Af3 exhibits higher than expected molecular weight (as determined by gel Wf?ration chromatography) support this notion (data not shown). It is worth noting that cleavage of 2C3AB by 3C protease is reproducibly observed even if the incubation is carried out at 0” (Fig. 4d, lanes 1,4, and 7) suggesting that the protease is extremely active in cleaving this substrate. An additional substrate, A3ABC, was also used to monitor the activity of 3C protease. Cleavage of this 13 kDa substrate should generate A3AB (5.4 kDa) and A3C (7.8 kDa). Following incubation with 3C protease, a single cleavage product migrating at -6.5 kDa is observed (Fig. 4e). Immunoprecipi&tion experiments suggest that this band is A3C (data not shown). An additional, lower molecular weight band which is probably A3AB is visible upon longer axposure of the autoradiogram. As shown in Fig. 4e, 3C protease cleaves this substrate equally well at 22” and 37”. We observe at most only -50% cleavage of the 3W3C junction, consistent with peptide studies whioh showed that 3B/ 3C cleavage is less efficient than 2C/3A (Pallai et al., 1989).

We also examined the cleavage of synthetic 16-mer peptides which correspond to the 2C/3A and the 3# 3B junctions (Fig. 5). After incubation with 3C protease, the cleavage products were resolved by HPLC and sequenced to confirm that cleavageoccurred at the expected Gln/Gly junction. The 2C/3A 16-mer is digested to completion after an overnight incubetion with 3C protease (Fig. 5b). In contrast, the 3A/38 16-mer is not as good a substrate, since after overnight digestion, about 20% of the starting materiai remains uncut (Fig. 5d). The efficient cleavage of 2C13A compared to 3# 38 is in agreement with previous peptide studies by Pallai et al. (1989) and is consistent with the situation in poliovirus infected HeLa cells, in which protein containing the 3AI3B linkage accumulates, but protein containing the 2U3Alinkage is not present in appreciable amounts (Krausslich and Wimmer, 1988). As noted above, recombinant protein 3AB was not cleaved at all by 3C protease, indicating that the protein is even more resistant to cleavage than the peptide substrate. Although 3C protease cleaves at Gtn-Gly junctions, it is well established that this sequence atone is insufficient for cleavage. Four out of 13 Gln-Gly bonds in the poliovirus precursor are resistant to cleavage by 3C protease (Krausslich and Wimmer, 1988). Nevertheless, we examined some commercially available synthetic peptides which contain Gin-Gly junctions for susceptibility to cleavage by 3C protease. We found that secretin, growth hormone reducing factor, and

146

BAUM

'4

2

1 6

-2.5 -12 O"4

2

1 .5 a25.12 0'

jq3C

ET AL.

serum thymic factor were all resistant to cleavage by 3C protease (data not shown). Autodigestion

370 '4'0

4

220

2 1 6'10

4

2

1

-5 ' pg3c

21.5 8

-A.WC

14.3 -

+cw

0.5 *'

:,:,

FIG. 4. (a) Temperature dependence of 2C3AB cleavage by 3C protease. The indicated amount of 3C protease was incubated with substrate 2C3AB at either 22” or 37” for 1 hr, followed by gel electrophoresis and autoradiography. (b) Irreversibility of incomplete cleavage of 2C3AB. 2C3AB was incubated at 22” for 2 hr with (lane 1) or without 2 pg 3C protease (lane 2) or at 37” for 2 hr with (lane 4) or without 3C protease (lane 5). Lane 3 is exactly as lane 4, except that an additional aliquot of protease was added after 1 hr. Lane 7, incubation of 2C3AB and 3C protease for 1 hr at 37” followed by 1 hr at 22”. Lane 6 is exactly as lane 7, except that an additional aliquot of protease was added upon shifting temperature. (c) Effect of preincubation of 2C3AB substrate. 2C3AB was preincubated at 37’ for 1 hr, followed by addition of 3C protease and continued incubation at either 37” (lane 2) or 22” (lane 3). 2C3AB was preincubated at 22” for 1 hr, followed by addition of 3C protease and continued incubation at either 37” (lane 5) or 22” (lane 6). Lane 1, incubation of 2C3AB without protease for 2 hr at 37”, or at 22” (lane 4). (d) Effect of preincubation of 3C protease. 3C protease (2 ag) was preincubated for 1 hr on ice (lanes l-3), or at 37” (lanes 4-6) or at 22” (lanes 7-9). 2C3AB substrate was then added, and the incubation was continued on ice (lanes 1,4, and 7) at 37” (lanes 2.5, and 8) or at 22” (lanes 3,6, and 9). Lane 10,2C3AB without protease. (e) Lack of temperature dependence of A3ABC cleavage by 3C protease. The indicated amount of 3C protease was incubated with substrate A3ABCat either 22” or 37” for 16 hr, followed by gel electrophoresis and autoradiography. The positions of uncleaved 2C3AB or ABABC

of 3CD

The 3CD polyprotein, composed of the 3C protease and the 3D polymerase regions of the poliovirus genome, has been found to accumulate during poliovirus infection of HeLa cells (Hanecak et a/., 1982; Krausslich and Wimmer, 1988). In fact, most of the 3CD in infected cells remains unprocessed. Thus, in vivo, a pool of 3CD appears resistant to autodigestion and to digestion by free 3C protease in trans. To directly examine the autodigestion of 3CD polyprotein, E. co/i cells harboring pT7-3CD were labeled with [35S]methionine. Cell lysates were immunoprecipitated with anti-3D antiserum (Plotch et a/., 1989) and the amount of cleavage of 3CD was determined by electrophoresis and autoradiography. As shown in Fig. 6a, approximately 25% of 3CD polyprotein autodigested, releasing 3D protein (lane 2). Identical results are obtained when the E. co/i are labeled at 22, 30, or 37” (data not shown). These data suggest that autodigestion of 3CD expressed in E. co/i is also inefficient, consistent with results in poliovirus-infected cells (Hanecak et al., 1982; Krausslich and Wimmer, 1988), with expression of various 3C precursors in E. co/i (Hanecak et al., 1984; lvanoff et a/., 1986; Richards et al,, 1987) and with translation of 3C precursors in vitro (Ypma-Wong and Semler, 1987; Ypma-Wong et a/., 1988). A synthetic peptide which spans the 3CD cleavage junction is also a relatively poor substrate for 3C protease in vitro (Pallai et a/., 1989). The residual 3CD which is not autodigested in vivo does not further autodigest in vitro (Fig. 6b, lane 2). In addition, the residual 3CD is not cleaved in tram by purified 3C protease (Fig. 6b, lane 4) whereas control 2C3AB is cleaved (Fig. 6, lane 3). This experiment was performed at 30”; identical results are obtained at 22” and 37” (data not shown). The residual 3CD may fold into a conformation which is resistant to autodigestion and to digestion in trans. Because 3CD is itself a protease required for the proper processing of the Pl region of the poliovirus polyprotein (Ypma-Wong and Semler, 1987; Ypma-Wong et a/., 1988; Jore eta/., 1988) this resistance to complete digestion may be a regulatory feature necessary to ensure proper virion assembly. Mutagenesis

of 3C protease

Several point mutants of polio 3C protease have been reported, and most were constructed by site-diand their respective lar weight markers

cleavage products are shown at left.

are indicated

at right;

molecu-

POLIOVIRUS 3C PROTEASE

a

b

-L---A tt

d

tt

FIG. 5. Cleavage of synthetic peptides by 3C protease. 16-mer oligopeptides 2C13A (a) and 3N3B (c)were incubated overnight with 3C protease (b and d), followed by HPLC as described in the text. The positions of intact oligopeptide and cleavage products (f) are indicated. They axis is absorbance at 2 10 nm; the x axis is retention time.

rected mutagenesis of 3C in regions which were known to be highly conserved among picornaviral proteases. For example, substitution at Gly 51 results in noninfectious virus (Dewalt and Semmler, 1987). Similarly, substitution at His 161 or Cys 147 (Ivanoff et a/., 1986) inactivated the 3C protease. Hammerle et al. (199 1) recently constructed mutants of the putative catalytic triad (His 40, Glu 7 1, Cys 147) which also inactivated the enzyme. Mutations at or near Lys 60 attenuate the enzymatic function of 3C protease and are manifested as a small plaque phenotype (Dewalt et al., 1990). We have employed a different strategy in isolating mutants of 3C. Previously, we observed that a plasmid bearing human immunodeficiency virus (HIV) protease under T7 promoter control cannot be transformed into E. co/i strain BL21(DE3) which lacks the pLysS plasmid. Lysozyme inhibits T7 RNA polymerase, and when plasmid pLysS encoding lysozyme was also present in the BL21(DE3) host, these cells could be successfully transformed with the HIV protease plasmid (Baum er a/., 1990). In strain BL21(DE3) lacking lysozyme, the low level of expression of HIV protease that occurs even in the absence of IPTG induction is sufficient to kill these cells, presumably due to cleavage of an unidentified essential E. co/i protein(s). An extensive collection of mutants of HIV protease was obtained by selecting for growth in BL2 1(DE3) after mutagenesis of the HIV protease plasmid (Baum et a/., 1990). Poliovirus protease plasmid pT7-3C was examined for its ability to transform E. co/i host strains BL21 (DE3) and 6L21(DE3)pLysS. We found that although both strains were transformed by pT7-3C with approximately equal efficiency, the colony size of BL2 1(DE3)

147

transformants was much smaller than BL2? (DE3)pLysS transformants (data not shown). PLasmid pT7POL, which encodes the poliovirus RNA pclymerase (Plotch et a/., 1989), was used a$ a control, and the colony sizes of these transform@?& w%%?similar in the two strains. We hypothesize that 3C protease digests an unidentified E. co/i protein(s), and that this digestion leads to a slower growth rate. We attempted to isolate mutants of poliovirus 3C protease, based.on the hypothesis that mutant protease transformed into BL21(DE3) would not inhibit cell growth, resulting in large size colonies. Plasmid pT7-3C was mutagenized by passage through the mutator strain E. co/i LE30 mutD, as described previously (Baum ef a/., 1990). Independent pools of mutagenized pT7-3C were then used to transform strain BL21(DE3) (at 37”), and colonies which were significantly larger than those obtained with witd type pT7-3C were selected for further investigation. Plasmid was isolated from these clones and used to retransform BL21(DE3), to confirm the IaFge colony phenotype. These putative mutants were then examined for 3C protease production by SDS-PAGE, and each produced protein consistent with the molecular

a 1

b 2

Ml

2

3456

FIG. 6. (a) Autodigestion of 3CD in E. co/i. BL21(DEd)pLysS cells harboring pT7-POL (lane 1) or pT7-3CD (lane 2) were grown to midlogarithmic phase, followed by IPTG induction and Iab&rtg of protein with [%]methionine. Ceil lysates were irnrnun~~~~~~ with antiserum against 3D polymerase (Plotch et al., lslir@, followed by gel electrophoresis and autoradiography. Molacufar w@ght markers are shown at left, and the positions of 3CD and 30 are indicated. (b) Lack of further autodigestion of 3CD in vitro or cleavage in tram by 3C. 3CD or 2C3AB was incubated in the presence or absence of purified 3C protease (3 pg) for 1 hr at 30” and ioadad directly on an SDS-polyacrylamide gel (without immunoprecip&jtion) followed by autoradiography. Lane 1, 2C3AB without pro%-; lane 2, 3CD without protease; lane 3, 2C3AB + proteas@; lane 4, 3CD + protease; lane 5,2C3A5 unincubated; lane 6,3CD unincubated. Molecular weight markers are shown at left, and the positions of 3CD, 3C, 3D, 2C3AB, and 2C are indicated. A di#use bend migt-aling at -34 kDa in lanes 1, 3. and 5 is an impurity in the 2C3AB preparation.

148

BAUM

a 3Cd

-2c3AB -2C

370

FIG. 7. Expression and enzymatic activity of mutant and wild type 3C protease. (a) f. co/i BL21(DEB)pLysS cells harboring the indicated pT7-3C mutants (Table 2) were grown to midlogarithmic phase, and protease synthesis was induced with IPTG. Cells (0.2 ml) were boiled in SDS sample buffer and subjected to polyacrylamide gel electrophoresis, followed by Coomassie blue staining. Molecular weight markers are shown at left; 3C protease is indicated. (b) Cleavage of 2C3AB by mutants of 3C protease at 22,30, and 37”. Purified wild type (WTs from supernatant, WT” from pellet, see Materials and Methods) or mutant 3C protease (1 or 0.2 fig) was incubated with [35S]methionine-labeled 2C3AB, followed by gel electrophoresis and autoradiography. The positions of molecular weight markers are shown at left; uncleaved 2C3AB and the 2C cleavage product are indicated.

weight of 3C (Fig. 7a). The 3C protease region of seven putative mutants was subjected to DNA sequence analysis. In each case, a single base change, leading to a single amino acid substitution, was identified (Table 2). Thus, this selection procedure for isolating mutants of 3C protease based on colony size appears to be valid, analogous to our previous studies using the toxicity of HIV protease in E. co/i to isolate protease mutants. One mutation of 3C protease, Ala 172 + Pro, was identified in two independent clones (8-2 and 5-3). Two other mutations, Ala 144 + Val and Gln 146 + Leu (clones 6-l and 2-3, respectively) are near Cys 147, which may be the active site residue (Ivanoff eta/., 1986; Gorbalenya eta/., 1989; Hammerle et al., 1991). Mutation Pro 38 + Thr (clone 8-l) is likewise near putative active site residue His 40 (Hammerle et a/., 1991). Since the X-ray structure of 3C protease has not

ET AL.

yet been determined, it is unclear how the other mutations we describe would alter the activity of 3C protease. To determine if any of the mutants displayed temperature sensitivity, transformations of BL21 (DE3) with the wild type or mutant protease plasmids were plated at both 30” and 37”. Wild type protease plasmid produced small colonies at both temperatures, compared to the control pT7-POL. Of the mutants, only 2-2 (His 168 --) Leu) displayed temperature sensitivity, in that colonies were small at 30°, but large at 37”. We hypothesize that this mutant protease is able to cleave its putative E. co/i substrate at 30”, but not at 37” (the temperature at which all mutants were initially identified). To determine the effect of these mutations on the cleavage of poliovirus polyprotein substrates, it was necessary to purify the mutant proteases. Supernatant and pellet fractions were prepared for wild type protease and for each mutant. For clones 6-1, 2-3, and 2-2, -75% of the protease was in the pellet, and 25% was in the supernatant, exactly as for wild type protease. In contrast, all of the protease produced from clones 8-1, 2-4, and 8-2 was reproducibly found in the pellet fraction. Thus, the latter three mutations have increased the insolubility of 3C protease relative to wild type, raising the possibility that these mutant proteases fail to inhibit E. co/i growth because they are sequestered in inclusion bodies. Wild type or mutant protease was purified as described (Materials and Methods) and incubated with [35S]methionine-labeled 2C3AB at various temperatures, followed by gel electrophoresis and autoradiography (Fig. 7b and Table 2). Whether isolated from the supernatant or solubilized from inclusion bodies, wild type 3C protease digested most of the 2C3AB substrate at 22” (Fig. 7b). The three mutants which partitioned exclusively into inclusion bodies (2-4, 8-1, and 8-2), cleave 2C3AB poorly or not at all following solubilization, suggesting that their lack of toxicity may be due to lack of protease activity. In contrast, mutant proteases 2-3 and 6-l have diminished activity in cleaving 2C3AB, compared to wild type protease, but they are still capable of cleavage. In fact, protease 2-2 appears slightly more active than wild type in cleaving 2C3AB. Essentially identical results are obtained for each protease with substrate A3ABC (data not shown). These data indicate that certain mutations of 3C protease interfere with cleavage of the putative E. co/i protein required for growth, but have only a small or no effect (mutants 2-2 and 6-l) on the ability to cleave 2C3AB or A3ABC. Variation in the efficiency of cleavage at different sites is well established for the poliovirus polyprotein

POLIOVIRUS

3C TABLE

149

PROTEASE 2

POLIO 3C PROTEASE MUTANTS Relative

Position Clone

8-2,

Nucleotide

Amino

8-l

5549

38

2-4

5849

138

6-l

5868

144

2-3

5874

146

2-2

5940

168

5951

172

5-3

Mutation

acid

CCA -+ ACA Pro Thr TAC + AAC Asn Tv GCA -, GTA Ala Val CAG + CTG Gln Leu CAC --* CTC His Leu GCG + CCG Ala Pro

Wild-type Note. Mutants were mined by densitometty a At each temperature,

isolated as described in the text and subjected of Fig. 7b. the cleavage of 2C3AB by mutant protease

(Pallai et al., 1989) and may well be a regulatory mechanism. The mutation Ile 74 + Thr apparently alters site selectivity, in that 3C protease autodigested at its amino terminus, but not at its carboxy terminus (Kean et al., 1988). That mutation also exhibited temperature sensitivity in poliovirus. Molecular modeling by Gorbalenya et al. (1989) suggests that the region Thr 142 Arg 143 may also play a role in site selection. In addition, chimeras between poliovirus and coxsackievirus 3C proteases demonstrate that at least some site selection is conferred by the carboxy terminal domain (residues 126-l 83) (Lawson et al., 1990). Our mutations Val 144, Leu 146, and Leu 168 are within that domain. Val 144 and Leu 146 are potentially in or near the presumptive active site (Cys 147, lvanoff et al., 1986; Hammerle et al., 1991), and Leu 168 is in or near the putative substrate-binding pocket (Bazan and Fletterick, 1988; Gorbalenya et al., 1989). We hypothesize that the geometry of the enzyme has been altered such that binding and/or cleavage of 2C3AB and A3ABCis only slightly altered, but that binding and/or cleavage of an E. co/i protein is significantly reduced, resulting in the normal colony growth rate. Thus, these mutations appear to change the substrate specificity of 3C protease. It is possible that cleavage at some of the other seven poliovirus polyprotein cleavage sites would be affected by these mutations. For each mutant, the extent of cleavage of 2C3AB and A3ABCis similar at each temperature tested (22, 30, 37”; Fig. 7b, and data not shown) especially if these data are normalized to wild type (Table 2). Thus, the apparent temperature sensitivity of mutant 2-2 ob-

to DNA was

sequence

normalized

analysis. to cleavage

cleavage

of 2C3ABe

22”

30”

37”

0

0

0

0

0

0

0.8

0.5

0.7

0.3

0.3

0.4

0.9

1.1

13

0

0

0

1.0

1.0

1.0

2C3AB

cleavage

by wild-type

determination

was

deter-

protease.

served in cleaving its putative f. c&i ssrbstrate is not reflected in the cleavage of the racombinartt polio substrates. In summary, the availability of large quantities of purified 3C protease, along with the methodology to rapidly select for mutants with diminished proteolyric activity, should greatly facilitate future structure-function studies of this important prototypical picornaviral enzyme. ACKNOWLEM3MENTS We thank Y. Gluzman, cussion of the manuscript.

I. Kovesdi,

1. Morin,

and

5. O’Hara

for dis-

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