Download Hydrolysis of a Series of Synthetic Peptide Substrates by the Human

J. gen. Virol. (1989), 70, 2931-2942.
Printed in Great Britain
2931
Key words: H R V-14/recombinant proteinase/picornavirus
Hydrolysis of a Series of Synthetic Peptide Substrates by the Human
Rhinovirus 14 3C Proteinase, Cloned and Expressed in Esckerichia coli
By D A V I D C. O R R , 1 A U D R E Y C. L O N G , 2 J O H N K A Y , : B E N M. D U N N 3
AND J A N E T M. C A M E R O N 1.
1Department of Virology, Glaxo Group Research Limited, Greenford Road, Greenford,
Middlesex UB6 OHE, 2Department of Biochemistry, University College Cardiff, P.O. Box 78,
Cardiff" CF1 1XL, U.K. and 3Department of Biochemistry and Molecular Biology,
J. Hillis Miller Health Center, University of Florida, Gainesville, Florida 32610, U.S.A.
(Accepted 27 June 1989)
SUMMARY
The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth
disease virus (FMDV) and encephatomyocarditis virus (EMCV), have been
demonstrated to be cysteine-type proteinases, involved in the processing of the
respective polyproteins expressed by the monocistronic RNA genome. Nucleotide
sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome
encodes a homologous 3C protein. The HRV-14 3C protein was purified to
homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The
enzyme was assayed against peptides corresponding to those residues, predicted (by
nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein.
The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D
cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all
cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein
liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly
bond in each of the peptides. The ready availability of such convenient substrates
facilitated the further characterization of the 3C proteinase. By contrast, peptides
corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing
was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by
the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic
peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein.
Studies with known proteinase inhibitors substantiated the conclusion that the HRV14 3C protein appears to be a cysteine proteinase and that the Cys residue at position
146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an
important role, analogous to that implied for the poliovirus 3C proteinase, in the
replication of the virus and thus represents a potential target for antiviral
chemotherapy.
INTRODUCTION
The human rhinoviruses (HRVs), implicated as the major causative agents of upper
respiratory tract infections collectively known as the common cold (Larson et al., 1980), belong
to the largest genus of the picornavirus family. The other genera of the picornavirus family are
the enteroviruses (poliovirus, echovirus, coxsackie virus), cardioviruses [encephalomyocarditis
virus (EMCV), mengovirus] and aphthoviruses [foot-and-mouth disease virus (FMDV)]
(Rueckert & Wimmer, 1984). The picornaviruses share their genomic organization and
translational strategy. The icosahedral protein particles contain a single-stranded positive-sense
infectious RNA molecule of Mr 2-5 x 106 (approximately 7500 nucleotides). The RNA has a
small virus-specific protein, 3B (VPg) covalently linked at its 5' end (Wimmer, 1982) and a
poly(A) tail at the 3' end (Yogo & Wimmer, 1972). [When describing the picornavirus proteins,
0000-8963 © 1989 SGM
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D.C. ORR AND OTHERS
the nomenclature adopted at the 3rd Meeting of the European Study Group on Molecular
Biology of Picornaviruses (Urbino, Italy, 1983) (Rueckert & Wimmer, 1984) has been used in
this paper. Where reference is made to papers that used the earlier nomenclature, the former
names of the respective proteins are given in parentheses for ease of reference.] The genomic
RNA acts as a monocistronic message and is initially translated to a > 200K polyprotein which
is cleaved by a series of proteolytic events to yield the mature viral products, both structural and
non-structural, that are essential for viral replication and the assembly of infectious viral
particles (Decock & Billian, 1986; Kitamura et al., 1981 ; Nicklin et al., 1986; Pallansch et al.,
1984; Palmenberg, 1987; Toyoda et al., 1986a).
Extensive studies have not been carried out on the processing of the rhinovirus polyprotein.
By contrast, the proteolytic steps involved in the production of poliovirus proteins have been
analysed in considerable detail (Nicklin et al., 1987; Palmenberg, 1981 ; Semler et al., 1981). The
first cleavage within the poliovirus polyprotein is an autocatalytic process mediated by the
poliovirus 2A protein which occurs while the polypeptide is still nascent on the ribosome. The
peptide bond hydrolysed in this initial processing step is between a tyrosine and a glycine residue
which connect the P1 and P2 regions (Nicklin et al., 1987; Toyoda et al., 1986a, b) (Fig. 1)
whereas most of the further cleavages which take place within the capsid and non-structural
protein precursors are produced by proteolysis between glutamine and glycine residues.
These Gln-Gly cleavages are carried out by the viral 3C proteinase, which is a cysteine-type
enzyme mapping in the 3'-terminal region of the genome and displays a high specificity for its
own polyprotein substrate (Hanecak et al., 1982, 1984; Ivanoffet al., 1986). The final poliovirus
proteolyic processing event, the cleavage of the capsid lAB (VP0) protein between an
asparagine and serine residue to produce 1A (VP4) and 1B (VP2), is associated with maturation
of the virion (Arnold et al., 1987).
A study of the complete nucleotide sequences of two human rhinoviruses, HRV-2 (Skern et
al., 1985) and HRV-14 (Callahan et al., 1985; Stanway et al., 1984), indicated that both genomes
were typical of the Picornaviridae family, and comparison of these two nucleotide sequences
(and their predicted amino acid sequences) with those of poliovirus types 3 and 1 (Kitamura et
al., 1981 ; Stanway et al., 1983) revealed not only a high degree of homology between the two
rhinovirus serotypes but also between the two rhinoviruses and the polioviruses. Since, as
described above, the cleavage sites in the polioviruses have been accurately determined, this
extensive homology in the predicted amino acid sequences enabled most of the cleavage sites in
HRV-2 and HRV-14 to be tentatively identified and the mature virus proteins to be mapped
onto the polyprotein. In particular, comparison of the nucleotide and deduced amino acid
sequences in the presumed 3C region of rhinovirus with those of poliovirus, other picornaviruses
and cowpea mosaic virus (CPMV, a plant virus related in gene organization) reveals substantial
homology (Argos et al., 1984; Carroll et al., 1984; Cann et al., 1983; Earle et al., 1988; Hughes et
al., 1988; Jenkins et al., 1987; Lomonossoff & Shanks, 1983; Robertson et al., 1985; Skern et al.,
1984, 1985; Stanway et al., 1983, 1984).
Therefore, it appears that the picornaviruses studied to date may all code for a similar 3C
proteinase. The 3C proteinase in EMCV and poliovirus has been shown to play a crucial role in
the processing of the viral proteins (Ivanoff et al., 1986; Semler et al., 1987; Shih & Shih, 1981 ;
Shih et al., 1978, 1979; Ypma-Wong et al., 1987a, b) and may represent an ideal target enzyme
for antiviral chemotherapy. By analogy, this is almost certainly true for the 3C proteins of all
picornaviruses, including human rhinoviruses. However there have been relatively few
comprehensive studies on the rhinovirus 3C protein (Libby et al., 1988). To ascertain not only
whether this protein expressed similar proteolytic activity but also whether it is a suitable target
for antiviral chemotherapy through the rational design of specific inhibitors, it was considered
that detailed molecular characterization of this enzyme was necessary.
This information was unlikely to be obtained using conventional methods to prepare viral
proteins, because of the relatively small quantities of 3C protein produced in infected cell
cultures (Decock & Billian, 1986). Therefore, appropriate fragments of cDNA copies of the
HRV-14 RNA genome were cloned and expressed in Escherichia coli. The 3C protein expressed
by these constructs was purified and subjected to biochemical characterization (Knott et al.,
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Hydrolysis by recombinant H R V proteinase
2933
1989). Previous investigations into structure/function relationships with 3C proteinases from
other picornaviruses have been hampered by the lack of availability of convenient substrates
such that most of the studies on proteolytic activity have, of necessity, been indirect, using
expression in E. coli of plasmids containing various cloned fragments of the picornavirus
genome (Hanecak et al., 1984; Ivanoff et al., 1986; Klump et al., 1984; Semler et al., 1987;
Toyoda et al., 1986a, b) or cell-free expression systems (Parks et al., 1986; Shih & Shih, 1981 ;
Shih et al., 1978, 1982; Vos et al., 1984, 1987; Ypma-Wong & Semler, 1987a, b).
We report on the characterization of the HRV-14 3C proteinase, which was facilitated by
examination of its interaction with synthetic peptide substrates corresponding to the residues,
predicted by nucleotide sequencing data, to occur at authentic sites within the HRV-14
polyprotein (Callahan et al., 1985; Stanway et al., 1984).
METHODS
Purification of3C proteinase. The HRV-14 3C proteinase and its derivative containing the cysteine (146) to
serine mutation were produced directly in E. coli. The induction and growth of the E. coli cells expressing the
respective plasmids and the details of the purification protocol were as described previously (Knott et at., 1989).
Briefly, the 3C proteins were purified from the soluble extract of induced cells using a two-stage high pressure
liquid chromatography (HPLC) procedure on a cation-exchange column (TSK SP5PW) and a gel permeation
column (TSK G3000SW). The HRV-14 3C proteins were homogeneous, as judged by SDS-PAGE, isoelectric
focusing and amino acid analysis. Western blot analysis demonstrated that the purified proteins had crossreactivity with antibodies raised against synthetic peptides corresponding to presumed antigenic sites of the HRV14 3C protein.
Synthesis of peptides corresponding to the putative cleavage sites in the viral polyprotein. Eight peptides, each
spanning a putative (Gln-Gly; Gln-Ala; Glu-Gly) cleavage site in the rhinovirus polyprotein were synthesized
(Fig. 1). Occasional changes were made in the naturally occurring (predicted) sequence, for example by
substitution of a tyrosine residue at the C terminus of peptide IV in order to facilitate u.v. detection of the parent
peptide and its degradation products. In all cases where such a strategy was employed, the replacement residues
were distant from the predicted P1-P~ scissile peptide bond. The changes are indicated in the sequences in Fig. 1
by inclusion of the naturally occurring residues in parentheses (Lys) following the residue, Tyr, which replaced it
(e.g. in peptide IV). A derivative of peptide VII was also synthesized in which the putative Gln-Gly scissile
peptide bond was altered to Gtu-Gly. The sequer~ce of this peptide, peptide VII (Q-*E) was thus Arg-Pro-¥al-ValVal-Glu-Gly-Pro-Asn-Thr. Two further peptides were prepared. Peptide IX had the sequence Thr-Leu-Glu-SerGln-Thr-Val-Gln-Gly-Gly-Thr-Val-Glu-Arg-Leu and peptide X had the sequence Gly-Gly-Asn-Gly-Arg-GlnGly-Phe-Ser-Ala-Gln-Leu-Lys, corresponding to residues 162 to 174 of the HRV-14 3C protein except that the
native Ala at position 166 was replaced by Arg in the synthetic peptide.
Peptides were synthesized using the Merrifield solid-phase technique on phenylacetamidomethyl polystyrene
resins (Applied Biosystems). Synthesis was carried out with an Applied Biosystems Model 430A peptide
synthesizer utilizing ABI chemicals, the manufacturer's standard protected symmetric anhydride programmes
and a double coupling protocol. A portion of each synthetic peptide was removed after the addition of the glycine
(alanine in the case of peptide III) to provide a sample of the C-terminal fragment expected from enzymatic
hydrolysis at the respective Gln-Gly (or Ala) or Glu-Gly peptide bonds. After removal of the peptides from the
resins with 9 0 ~ anhydrous HF, 10~ anisole, the samples were extracted with 10 to 5 0 ~ acetic acid, diluted with
water, and lyophilized to dryness. Reversed phase HPLC was performed on each peptide [Waters C 18 #Bondapak
in Radial Pak cartridges, 10 to 9 0 ~ acetonitrile gradients with 0-1 ~ trifluoroacetic acid (TFA)-water as solvent
A]. All the resulting peaks were analysed by u.v. spectroscopy as well as by amino acid analysis on a Beckman 6300
analyser after acid hydrolysis.
The composition of each of the substrate peptides together with those for the C-terminal cleavage fragments was
ascertained. In all cases, the determined compositions was in good agreement with the theoretical values. The
desired peptide in each of the preparations was 84 to 9 0 ~ of the total material present.
Monitoring peptide bond cleavage. Samples of each of the potential peptide substrates were incubated with
recombinant HRV-14 3C proteinase under a variety of experimental conditions. Aliquots were withdrawn at
several time points and TFA was added to give a final concentration of 0.1 ~ (v/v). The samples were centrifuged
at 5000 g for 5 rain and portions of each supernatant were analysed by reverse phase chromatography using either
C18 /tBondapak (Waters) or more commonly PepRPC fast protein liquid chromatography (FPLC) columns
(Pharmacia). The flow rate was 1 ml/min with Buffer A (A) consisting of HzO containing 0-1 ~ (v/v) TFA and
Buffer B (B) consisting of 100 ~ acetonitrile, 0.1 ~ (v/v) TFA. The gradients used were 0 to 10 min with 100 ~ A; 10
to 15 min with 0 to 15 ~ B; 15 to 40 rain with 15 to 50 ~ B; 40 to 45 min with 50 to 100 ~ B; 45 to 50 min with 100
B; 50 to 55 min with 100~ B plus 100~ A. Detection was at a wavelength of 214 nm.
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D . C . ORR AND OTHERS
Identification of the cleavageproducts generated. After prolonged incubation of potential substrate peptides with
proteinase, newly generated peaks were collected in acid-cleansed test tubes and then lyophilized. Samples (50 to
100 pmol) were acid-hydrolysed on the PICO-TAG work station for 20 h at 105 °C and analysed using the
Millipore-Waters Corporation PICO-TAG column connected to Millipore-Waters Corporation HPLC
equipment, or-Amino butyric acid (ABA) was incorporated into the samples as an internal standard.
Monitoring hydrolysis of p-nitroanilide and p-nitrophenyt ester containingpeptides. Various p-nitroanilide and pnitrophenyl ester-linked peptides and amino acids were dissolved in DMSO at a concentration of 10 to 50 mM.
Aliquots of these preparations diluted with the required buffer were added to flat-bottomed microtitre plate wells
and incubated at 37 °C with 3C proteinase which had been preincubated for 10 min at 37 °C in the presence of
5 mM-L-cysteine, 1 mM-EDTA, 5 mM-2-mercaptoethanol and 10~ (v/v) glycerol. Colour development was
monitored at 405 nm using a Titre-Tek Multiskan plate reader.
Materials
Chromogenicpeptides and other peptides. Dinitrophenyl (DNP)-GIn-Gly-Ile-Ala-GIy-Gln-o-Argand DNPPro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg were from the Peptide Institute (Osaka, Japan). The dipeptides Glu-Gly and
Gln-Gly were from Serva (Heidelberg, F.R.G.). The serum thymic factor (pGlu-Ala-Lys-Ser-Gln-Gly-Gly-SerAsn) and Fibrinopeptide A (Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg)
were from
Sigma. Other chromogenic peptides obtained from either Sigma or the Peptide Institute were Ac-L-Asp-pnitroanilide (NA), N-Succ-L-Tyr-L-Leu-L-Val-p-NA,N-Ac-L-AIa-L-Ala-L-Ala-p-NA,N-Succ-L-Ala-L-Ala-L-ProL-Phe-p-NA, N-Succ-L-Ala-L-AIa-L-Pro-L-Leu-p-NA, N-Succ-L-Ala-L-Ala-L-Val-p-NA, N-carbobenzoxy (NCBZ)-L-Gly-L-Pro-L-citrulline-p-NA, N-Ac-L-AIa-p-NA, N-CBZ-L-Gly-L-Pro-L-citrulline-p-NA, N-CBZ-L-GlyL-Gly-L-Leu-p-NA, N-Succ-L-AIa-L-Ala-L-AIa-p-NA, N-Ac-DL-Phe-p-nitrophenyl ester (NPE), N-tertbutoxycarbonyl (N-t-BOC)-benzyl-L-Glu-p-NPE, N-t-BOC-L-Gln-p-NPE, N-t-BOC-L-Asp-p-NPE and N-t-BOCL-GIy-p-NPE.
Proteinase inhibitors. Amastatin, antipain, bestatin, chymostatin, E-64, elastatinal, leupeptin and
phosphoramidon were all obtained from the Peptide Institute, ~-2-macroglobulin (from bovine plasma), and trypsin
inhibitor (from chicken egg white) were all obtained from Boehringer Mannheim. Aprotinin (from bovine lung),
bromelain inhibitor (from pineapple stem), p-chloromercuriphenyl-sulphonic acid, cystatin (from egg white),
epiamastatin, iodoacetamide, N-ethylmaleimide, 4-amidophenyl-methane sulphonyl fluoride (APMSF), PMSF,
2-nitro-4-carboxyphenyl-N, N-diphenyl carbamate (NCDC), N-p-tosyl-L-lysinechloromethyl ketone (TLCK) and
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were all obtained from Sigma.
Two synthetic chymostatin analogues containing a phenylalanine aldehyde (Z-Arg-VaI-Phe-CHO) or a
semicarbazone (Z-Arg-Leu-Phe-CH=N .NH-CONH2) reactive function respectively (Place et al., 1987) were
generous gifts from Dr R. J. Beynon, University of Liverpool, U.K. Recombinant human cystatin C (Abrahamson
et al., 1988) was kindly provided by Dr M. Abrahamson, Maim6 General Hospital, Sweden.
RESULTS
Analysis o f the cleavage site in the various peptide substrates
Samples of each of the synthetic peptides I to V I I I (Fig. 1) corresponding to the putative
polyprotein cleavage sites were incubated at neutral p H with the homogeneous 3C proteinase
and the mixtures were analysed by reverse-phase H P L C or F P L C . As internal controls, the
substrate peptide or the carboxy-terminal fragment of synthetic peptides were also incubated
with buffer alone. F o r all of the peptides except I I I and V (Fig. 1), the peak corresponding to the
intact substrate diminished upon incubation with the recombinant proteinase concomitantly
with the appearance of only two novel peaks, one of which (in every case) corresponded exactly
in its elution position with the respective synthesized C-terminal fragments (data not shown).
The newly generated peaks were collected in all cases and their compositions were determined
by amino acid analysis. Peptides I, II, IV, VI, VII and V I I I were all cleaved specifically between
the G l n - G l y residues within the respective sequences. N o novel peptides were generated when
any o f the peptides were incubated with buffer alone, with proteinase pretreated at 100 °C for 10
min or with the mutant [Cys(146)-,Ser] proteinase (D. S. Montgomery, D. C. Orr, C. W. Dykes
& A. N. Hobden, unpublished results; K n o t t et al., 1989).
By contrast, peptides I I I and V were not cleaved by the 3C proteinase, even upon prolonged
incubation (26 h) under the same conditions used to achieve significant hydrolysis of the other
synthetic substrates. Furthermore, preincubation of peptides I I I or V (120 ~tg) with proteinase
(0.5 ~tg) for 2 or 16 h prior to addition of peptide VII (120 ~tg with continuation of the incubation)
showed no inhibition of the cleavage of peptide VII. Thus, it would a p p e a r that recombinant
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Hydrolysis by recombinant HR V proteinase
(a)
VPg
Genomic RNA
Poly(A)
@
Initiation
Termination
~ Translation
P1
-~;
•
P2
~-"
P3
!
!
!
Polyprotein
2a
P1
P2
P1
P3
P2 3AB
3C
(O 3C cleavage sites)
lAB
1C
1A 1B
(b)
3D
1D
3C
w
3D
2B
2 A ~ 2C
3A
3B
i
I (2C/3A junction)
Asp-Ser-Leu-Glu-Thr-Leu-Phe-Gln-Gly-Pro-Val-Tyr-Lys
II (2A/2B junction)
Glu(Cys)-Ala-Ile-Ala-Glu-Glu-Gln-Gly-Leu-Ser-Asp-Tyr-Ile-Thr
III (2B/2C junction)
Val-Pro-Tyr-Ile-Glu-Arg-Gln-Ala-Asn-Asp-Gly-Trp-Phe-Arg-Lys
IV (1B/1C junction)
Arg-Ser-Lys-Ser-Ile-Val-Pro-GIn-Gly-Leu-Pro-Thr-Thr-Thr-Tyr(Lys)
V (1C/1D junction)
Ser-Gln-Thr-Val-Ala-Leu-Thr-Glu-Giy-Leu-Gly-Asp-Glu-Leu-Glu-Glu-Tyr(Val)
VI (3A/3B junction)
Lys-Leu-Phe-Ala-Gln-Thr-GIn-Gly-Pro-Tyr-Ser-Gly-Asn-Pro
VII (3B/3C junction)
Tyr(Leu)-Arg-Pro-Val-Val-Val-Gln-Gly-Pro-Asn-Thr-Glu-Phe
VIII (3C/3D junction)
Lys-Gln-Tyr-Phe-Val-Glu-Lys-Gln-Gly-Gln-Val-Ile-Ala-Arg
Fig. 1. (a) Representation of the generation of the mature viral proteins from the potyprotein precursor
during the replication of picornaviruses. (A) Cleavage by 2A proteinase; (0) cleavage by 3C
proteinase. (b) Sequences of eight synthetic peptides (I to VIII) each spanning the putative cleavage site
(in bold) at the indicated junctions in the HRV-14 polyprotein.
rhinovirus 3C proteinase did not (to any significant extent) recognize the sequences of peptide
III (the putative 2B/2C junction) or V (the predicted 1C/1D junction, Fig. 1).
Peptide V I I I (Q--*E) in which the Gln-Gly scissile peptide bond was aRered to a Glu-Gly
linkage was also not cleaved by the viral 3C proteinase. Peptides IX and X, both containing the
Gln-Gly pair in their respective sequences (see Methods), also were not hydrolysed by the
enzyme.
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2936
D. C. ORR AND OTHERS
Table 1. Kinetic constants for the hydrolysis of eight synthetic substrates by recombinant HRV-
14 3C proteinase
Peptide*
I
I1
III
IV
V
VI
VII
VII1
Sequence
I/max
~tmol/min/mg)
Km
(mM)
D-S-L-E-T-L-F-Q-G-P-V-Y-K
E-A-I-A-E-E-Q-G-L-S-D-Y-I-T
V-P-Y-I-E-R-Q-A-N-D-G-W-F-R-K
R-S-K-S-I-V-P-Q-G-L-P-T-T-T-Y
S-Q-T-V-A-L-T-E-G-L-G-D-E-L-E-E-Y
K-L-F-A-Q-T-Q-G-P-Y-S-G-N-P
Y-R-P-V-V-V-Q-G-P-N-T-E-F
K-Q-Y-F-V-E-K-Q-G-Q-V-I-A-R
4-3
0-03
0
< 0.05
0
7-0
11.5
< 0-1
1.2
1.0
-
NMt
2.6
0.7
NM
* Each peptide was incubated at 35 °C with HRV-14 3C proteinase in 6 mM-citric acid, 187 mM-NazHPO4
buffer pH 7.6, containing 10~ (v/v) glycerol, 5 mM-DTT and 2 mM-EDTA. At appropriate times, aliquots were
removed and reaction rates (V) at each concentration of substrate were determined from these time courses using
reverse-phase FPLC. The area of the newly generated peak corresponding to the carboxy-terminal fragment was
measured in each case and converted into nmol of product, using a calibration graph. Best values for K,, and Vm,x
were obtained by computerized curve-fitting as described previously (Jupp et al., 1988).
t ~ , Not measurable.
The recombinant HRV-14 proteinase was also tested for its ability to attack a variety of
chromogenic peptides containing p-nitroanilide or p-nitrophenyl esters as the carboxy-terminal
reporter group and a number of random peptides which chanced to contain the dipeptide
sequences Gln-Gly or Glu-Gly. Hydrolysis was not observed for any of these peptides.
Quantification of peptide cleavage by 3C proteinase
Cleavage rates for each of the peptides were obtained by monitoring the appearance of the
carboxy-terminal cleavage fragment. By applying increasing amounts (nanograms) of each of
the C-terminal peptide fragments onto the reverse-phase column and integrating the peak area
from each trace, calibration graphs relating peak area to nanograms loaded were constructed.
Having previously identified the products of the proteolytic reactions, it was thus feasible to
quantify the extent of cleavage in any of the reactions through measuring the area of the
carboxy-terminal fragments generated. Recoveries were generally in excess of 9 0 ~ of the
amount of intact substrate peptide introduced initially into the incubations. The generation of
the carboxy-terminal fragment was thus a reliable indicator of the extent of substrate cleavage so
that by removal of several samples at different times of incubation, initial rates of hydrolysis for
each of the peptides I, II, IV, VI, VII and V I I I were determined. The cleavage rates of the
peptides were linear with respect to time.
Determination of kinetic parameters (Km and V,~.0
The kinetic parameters, Km and Vmax, for the cleavage of these six peptides were determined
(Table 1). From these data, it is clear that although all six peptides were cleaved by the
proteinase, peptides I, VI and VII (all containing the scissile peptide bond within the sequence
-Gln-Gly-Pro-) were much better substrates than II, IV or VIII. Consequently peptide VII was
selected as a good substrate upon which to base further biochemical characterization of the 3C
proteinase.
pH optimum and the effects of divalent cations, temperature and thiol compounds
The rate of cleavage of peptide VII at a variety of p H values was investigated. From this, it
appeared that the proteinase was stable at alkaline pH values and had a p H optimum in the
range of 7.6 to 8.0. The temperature for maximum cleavage of peptide VII was ascertained to be
in the region of 35 °C (data not shown).
At concentrations up to 50 mM, neither Ca z÷ nor Mg z÷ had any effect on the proteolytic
activity against peptide VII. The cleavage rate of peptide VII was also assayed in the presence
of various concentrations of the reducing agents, dithiothreitol (DTT) and L-cysteine. In
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Hydrolysis by recombinant HRV proteinase
2937
Table 2. Effect of proteinase inhibitors on the hydrolysis of peptide VII by HRVol4 3C enzyme
Concentration
of
compound
~g/ml)
Compound*
p-Chloromercuriphenyl
sulphonic acid
Iodoacetamide
N-Ethylmaleimide
E64
Chymostatin
Leupeptin
Antipain
Elastatinal
TPCK
TLCK
Egg white cystatin
Human cystatin C
ct-2-Macroglobulin
Inhibition of peptide VII cleavaget
(~)
25
1O0
25
20
100
80
10
100
10
100
100
100
100
178
55
178
100
100
0
100
30
100
85
<5
0
85
60
0
0
0
* Aliquots of 3C proteinase (0.5 ktg) were preincubated with the indicated concentrations of each modifier for
15 min at 35 °C before determination of the residual proteolytic activity using Peptide VII as the substrate.
1"The inhibition of cleavage was calculated as the decrease in the carboxy terminus peptide fragment generated
in the presence of each modifier expressed as a percentage of that obtained in the respective control samples
(incubated with buffer alone or with DMSO or ethanol where required).
the absence of any thiol reducing agents, no cleavage of the peptide substrate was observed.
Maximum activation of hydrolysis was achieved between approximately 5 and 20 mM-DTT
whereas, when I.-cysteine was used as the reductant, concentrations as high as 50 mM were still
unable to stimulate maximum activation of hydrolysis.
Effects of general proteinase inhibitors on the cleavage of peptide VII by the 3C proteinase
A range of known inhibitors of serine and cysteine proteinases were studied for their effect on
the activity of the 3C proteinase towards peptide VII (Table 2). If DMSO or ethanol were
required for the solubilization of the inhibitor, these solvents were added at the required level to
the respective controls. N-Ethylmaleimide, iodoacetamide and p-chloromercuriphenyl sulphonate inactivated the enzyme completely (Table 2) and since these compounds have been shown to
be inhibitors of a wide range of cysteine proteinases including ficin, papain and stem bromelain
(Liener & Friedenson, 1970; Murachi, 1970; Rich, 1986), it would thus appear that the
rhinovirus 3C proteinase does indeed belong to the cysteine proteinase family. T L C K and
T P C K , which are also known to inhibit cysteine proteinases non-specifically by alkylation of the
sulphydryl group of the enzyme (Rich, 1986), were also seen to have some inhibitory effect on the
viral proteinase activity. Of the two peptide aldehyde inhibitors known to inhibit cysteine
proteinases, antipain and leupeptin (Rich, 1986), only leupeptin was seen to have any activity
against the 3C proteinase. E64, an epoxysuccinyl peptide shown to inactivate rapidly several
cysteine proteinases, including ficin, papain, cathepsin B and stem bromelains (Barrett et al.,
1982; Elliott & Liu, 1970; Rich, 1986), did not inhibit the viral proteinase. However, another
peptide aldehyde, chymostatin, an inhibitor of chymotrypsinqike enzymes (Place et al., 1987;
Powers & Harper, 1986b), was found to be a competitive inhibitor of the H R V 3C proteinase,
albeit not a particularly good one. Two synthetic chymotrypsin analogues, Z-Arg-Val-PheH and
Z-Arg-Leu-PheSc (Place et al., 1987), were also tested, but were found to possess no inhibitory
activity. Other serine proteinase inhibitors ( N C D C , aprotinin, elastatinal, A P M S F and PMSF)
(Powers & Harper, 1986b) and metalloproteinase inhibitors (bestatin, amastatin, epiamastatin
and phosphoramidon) (Powers & Harper, 1986a) also all failed to show any activity against the
HRV-14 3C proteinase.
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D.C. ORR AND OTHERS
None of the high Mr protein inhibitors of cysteine proteinases (Abrahamson et al., 1987;
Barrett et al., 1986) that were tested at relatively high concentrations, i.e. ct2-macroglobulin
human cystatin C or egg white cystatin, showed any inhibitory activity against the HRV-14 3C
proteinase.
DISCUSSION
The difficulties of acquiring proteinase from natural sources and the lack of availability of
convenient substrates have hampered detailed investigations into structure/function relationships in the picornavirus proteinases (2A and 3C). While the present work was in progress
however, one report did appear documenting the cloning and expression of an active HRV-14
3C proteinase. The construct used was such that the authors were able to demonstrate activity in
the proteinase only when it was associated with the membrane fraction of E. coli extracts;
attempts to obtain soluble proteinase by detergent extraction and other means were unsuccessful
(Libby et al., 1988). However, in the present study, a large proportion of the 3C protein
expressed from the cloned HRV-14 genome cDNA fragments was present in the soluble fraction
(Knott et al., 1989).
Detailed characterization of the homogeneous rhinovirus 3C proteinase was accomplished by
examining its action on a series of peptides, representing putative processing sites within the
HRV-14 polyprotein, which were designed to fulfil all of the primary sequence requirements of
3C recognition and cleavage (Callahan et al., 1985; Stanway et al., 1984). All of these peptides
(Fig. 1) containing Gln-Gly as the putative cleavage site were hydrolysed specifically at this
peptide bond by the preparations of the purified 3C proteinase. However in the case of peptide
VII (Q-~ E), substitution of Gln-Gly with a Glu-Gly sequence rendered this peptide resistant to
proteolytic attack by the 3C proteinase. Furthermore, when the HRV-14 3C proteinase was
assayed against a variety of other peptides, chosen because they contained a Gln-Gly sequence
in their structure, no cleavage of any of the peptides was detected. This suggests that the HRV14 3C proteinase not only has a stringent specificity for Gln in subsite P1 and Gly in P~ [the
nomenclature of amino acid residues at the cleavage sites is according to Berger & Schechter
(1970)] in peptide substrates but also appears to require further structural features to be met as
well. In the serum thymic factor, fibrinopeptide A, and in the various chromogenic peptides
examined as potential substrates, the Gln-Gly dipeptide was flanked by four or less amino acids
as opposed to the six upstream and five downstream residues in the standard peptide (peptide
VII). The possibility cannot be excluded that these former peptides were not cleaved simply
because they were not held in the active site in the correct conformation owing to the lack of
flanking residues. However, since two further peptides (peptides IX and X; see Methods; each
containing the target Gln-Gly dipeptide flanked by seven or five residues respectively upstream
with six downstream) were not hydrolysed at all by the proteinase, this would seem unlikely.
Some form of recognition, either in linear or spatial terms, would appear to be operative.
However examination of the primary sequences flanking the putative HRV-14 cleavage sites
reveals no apparent homology or motif (Callahan et al., 1985; Stanway et al., 1984), except
perhaps a preference for an amino acid with a hydrophobic group (Ala, Ile or Val) in position
P4. The frequency of occupation of this site by a hydrophobic residue in the cleavage sites of
other picornaviruses is also apparent (Palmenberg et al., 1987; Pallansch et al., 1984; Parks &
Palmenberg, 1987; Vos et al., 1987). Three of the HRV-14 cleavage sites are flanked on one side
by a proline residue (2C/3A, 3A/3B and 3B/3C sites) and the corresponding peptides which were
synthesized (I, VI and VII) were by far the most rapidly processed of the HRV-14 3C peptide
substrates. Whether the rapid rate of cleavage of these peptides may be attributed to sequence
recognition with the proline residue specifically occupying the P~ subsite, or whether the
presence of the proline residue in this position may predispose the peptide to a conformation
suitable for recognition and cleavage, is clearly worthy of further investigation.
In the EMCV polyprotein, cleavage by the viral 3C proteinase takes place at seven Gln-Gly
(or Ser) sequences that are flanked by proline residues (Palmenberg, 1987) whereas four other
Gln-Gly (or Ser) sequences which happen to exist but which do not have an adjacent proline
residue are not processing sites. By contrast, in poliovirus, only three of the processed Gln-Gly
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pairs are flanked by prolines (Pallansch et al., 1984), and experiments on the processing of
CPMV and EMCV capsid protein precursor molecules altered by site-directed mutagenesis
have suggested that exact proximity of a proline residue to a cleavage site is not essential for
processing (Parks & Palmenberg, 1987; Vos et al., 1987). Future experiments using derivatives
of the substrate peptide and mutated and cloned polyprotein precursor molecules which have
been shown to be processed by the HRV-14 3C proteinase (D. S. Montgomery, D. C. Orr, C. W.
Dykes & A. N. Hobden, unpublished results), should provide further information on the
requirements for particular residues to occupy specific P or P' sites in order to maintain effective
cleavage.
Peptides III and V were not hydrolysed upon incubation wth 3C proteinase. Since
preincubation of either of these peptides with 3C did not affect activity towards peptide VII,
neither would appear to be able to bind to the active site of the proteinase. The potential
cleavage sites within these two peptides were Gln-Ala and Glu-Gly respectively. The Gln-Ala
(2A/2B) cleavage site was chosen as being the most likely processing point in the predicted
region of the 2A/2B boundary (Stanway et al., 1984) although there are no reports of a Gln-Ala
site being the natural cleavage junction for any picornavirus 3C proteinase. However Parks &
Palmenberg (1987) showed that when the P] Gly residue at the poliovirus 1C/1D processing
junction was replaced by Ala, efficient processing still occurred. Peptide V was taken to
represent the H RV-14 1C/1D cleavage site although in the region of the predicted boundary, the
sequence Gln-Thr occurs twice and Gln-Gly once. It is not known with certainty which (if either)
of these sequences is recognized for processing. Stanway et al. (1984) concluded that one of the
Gln-Thr sites was used, based on the fact that this is the 1C/1D cleavage point in FMDV. Other
workers favour the Glu-Gly site to be the one processed (Callahan et al., 1985) due to the number
of cleavage events occurring at this type of junction in FMDV (Klump et al., 1984; Palmenberg,
1987; Robertson et al., 1985). Thus it would be interesting to examine synthetic peptides
representing the possible Gln-Thr sites and assess these as potential substrates. Another area for
future studies would be to ascertain whether peptides III and V are cleaved if the Gln-Ala or
Glu-Gly dipeptides respectively are replaced with Gln-Gly.
It would appear that the HRV-14 3C protein is a cysteine proteinase. In order to generate
activity towards the synthetic peptide substrates, the presence of a thiol compound (DTT or Lcysteine) was required. Furthermore, N-ethylmaleimide, iodoacetic acid, TPCK, TLCK and pchloromercuriphenylsulphonic acid, all known to be inhibitors of a wide range of cysteine
proteinases (Rich, 1986), were all found to block the action of the 3C proteinase efficiently. In
addition, specific replacement of the cysteine residue at position 146 with serine by site-directed
mutagenesis (Knott et al., 1989; D. S. Montgomery, D. C. Orr, C. W. Dykes & A. N. Hobden,
unpublished results) resulted in a total loss of activity towards the peptide substrates. This
identifies Cys (146) as a residue that may participate in the catalytic mechanism of the enzyme,
as shown previously for the poliovirus 3C proteinase (Ivanoffet al., 1986). Apart from this Cys
residue (in a -Gly-X-Cys- sequence) which is conserved together with a His residue (in position
160 for HRV-14) in the picornavirus proteinases, there is no further homology detectable
between the 3C enzymes and papain or other members of the cysteine proteinase family (Berger
& Schechter, 1970; Kamphuis et al., 1985 ; Polgar & Halasz, 1982). Therefore, it may well be that
the picornavirus 3C proteinases represent a subclass of cysteine proteinases that are mechanistically similar to but homologous with papain. A further reflection of this distinction may lie in
the value of the pH optimum observed for the HRV-14 3C proteinase (between pH 7-6 and 8-0),
whereas that for papain and other archetypal cysteine proteinases is more commonly in the acid
region below pH 7-0. The different nature of the active sites is substantiated by the observations
that the HRV-14 3C proteinase was poorly susceptible to inhibition by the peptide aldehyde
inhibitors (antipain and leupeptin). The competitive inhibition observed by chymostatin was
also a somewhat unexpected finding since this peptide is considered primarily as an inhibitor of
chymotrypsin-like serine proteinases. Similarly, ~z-macroglobulin, human cystatin C and egg
white cystatin, all very effective inhibitors of archetypal cysteine proteinases, did not act against
the HRV-14 3C proteinase even at very high concentrations. Chicken cystatin and a2macroglobulin have been suggested (Korant et al., 1986a, b) to have inhibitory activity towards
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D. C. ORR AND OTHERS
the poliovirus 3C proteinase. Unfortunately however, only binding assays were performed in
these studies, not direct inhibition measurements. In a plaque reduction assay, where the uptake
of these high M, protein inhibitors might be problematic, chicken cystatin had a minimal
antiviral effect whereas the ct2-macroglobulin was ineffectual (Korant et al., 1986a, b).
Small peptides would appear to be appropriate to use as substrates since they may be able to
assume a variety of structural conformations including those required for interaction with the
active site of the proteinase. The substrate peptides utilized in this study permitted the rapid
analysis of the proteolytic activity of the cloned HRV-14 3C protein and its demonstration as a
member of the cysteine proteinase family. The peptide cleavage data coupled with observations
on the 3C proteinase-mediated processing of cloned viral precursor polypeptides (D. S.
Montgomery, D. C. Orr, C. W. Dykes & A. N. Hobden unpublished results) indicate that the 3C
region of the HRV-14 genome represents the core sequence responsible for proteolytic
processing (at Gln-Gly) of the precursor polypeptide. Using the in vitro transcription translation
system with the poliovirus proteinase, Ypma-Wong et al. (1988) demonstrated that although an
intact 3C coding region was sufficient to support cleavage of the expressed polyprotein between
2A/2B, 2B/2C and 2C/3A, the processing of the P1 region (cleavages between the structural
proteins 1B, 1C and 1D) was more efficiently carried out by the proteinase precursor, 3CD. They
suggested that this effect may have arisen from the D sequence of 3CD making additional
specific contacts with the polyprotein that stabilize the precursor-proteinase complex (YpmaWong et al., 1988). Such studies may begin to establish the nature of the active enzyme within
the infected cells as well as the order of processing of the precursor, but parallel investigations
have yet to be carried out on the rhinovirus proteinase.
The demonstration that certain synthetic peptides are cleaved under defined conditions by
the HRV-14 3C proteinase now makes it feasible to undertake further studies to assess the
significance of the primary sequence surrounding 3C proteinase cleavage sites through the
production of new generations of peptide substrates with systematic substitutions in individual
subsites. By carrying out such studies in conjunction with experiments on the processing of
rhinovirus precursor polypeptides, valuable information would be obtained on the significance
of secondary and tertiary protein determinants in the recognition and interaction of the proteolytic enzyme with its substrates. Interpretation of such data would be greatly facilitated by determination of the three-dimensional structure of the viral proteinase by X-ray crystallography.
Such an approach may facilitate the rational design of specific inhibitors of the rhinovirus
proteinase which would prevent the 3C proteinase-mediated processing of viral precursor
polyproteins essential for replication whilst having no effect on other host cell proteinases.
The assistance of Alicia Alvarez (Protein Chemistry Core Facility, University of Florida) in the synthesis and
analysis of peptides is gratefully acknowledged. Thanks are also due to Professor J. Almond of Reading University
for the provision of the original HRV-14 cDNA.
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