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Development ofin VitroPeptide Substrates for Human Rhinovirus-14 2A Protease
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 356, No. 1, August 1, pp. 12–18, 1998 Article No. BB980746
Development of in Vitro Peptide Substrates for Human Rhinovirus-14 2A Protease Q. May Wang,1 Robert B. Johnson, Wolfgang Sommergruber,* and Timothy A. Shepherd Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285; and *Boehringer-Ingelheim Research Vienna, Bender and Company, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria
Received January 20, 1998, and in revised form May 8, 1998
Purified 2A protease from human rhinovirus serotype-14 (HRV14) was unable to efficiently cleave a 16-mer peptide representing its authentic cis-cleavage site on the viral polyprotein, implying that in vivo cis cleavage by this enzyme might be very different from its in vitro trans activity. Presence of a serine at position P2 and a leucine at P2* in the 16-mer peptide was found to be responsible for the low peptide cleavage efficiency. To search for an efficient peptide substrate for HRV14 2A, small peptides derived from other rhinovirus 2A protease cleavage sites were synthesized and tested. These results suggested that the N-terminal 8 amino acids were sufficient for HRV14 2A cleavage to occur, although the P1* and P2* residue identities were important to the cleavage of peptides with amino acids occupying both sides of the scissile bond. On the basis of the 2A substrate requirements, a sensitive fluorometric assay for the viral 2A proteases was developed using peptides with anthranilide and 3-nitrotyrosine as the resonance energy transfer donor/quencher pair. Our data indicated that these fluorescent peptide substrates were suitable for 2A protease characterization and inhibitor evaluation. © 1998 Academic Press
As the major causative agents of the common cold in humans, more than a hundred different serotypes of human rhinovirus (HRV)2 have been identified to date (1, 2). HRVs are small plus-strand RNA viruses in the picornavirus family (1), which translate their genomic information into an ;220-kDa polyprotein precursor 1 To whom correspondence should be addressed. Fax: (317) 2761743. E-mail: [email protected]. 2 Abbreviations used: HRV, human rhinovirus; DTT, dithiothreitol; pNA, p-nitroanilide.
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(2, 3). Generation of functional viral proteins from the polyprotein precursor is required for viral replication and this process is performed by virally encoded 2A and 3C proteases (2, 3). The 2A protease makes the first cleavage at the junction of capsid protein VP1 and the N-terminus of the 2A itself, which separates the viral structural proteins from the nonstructural ones (2, 3). In addition, the 2A proteases from HRV serotype-2 (HRV2) and poliovirus have been shown to be involved in the cleavage of certain important cellular proteins, including protein synthesis initiation factor eIF4G (p220), which may result in host-cell protein synthesis shutoff (4 –7). HRVs have been grouped in various ways on the basis of receptor specificity, sensitivity to antiviral agents, or amino acid sequence homology (1, 8 –11). Most of the HRV serotypes, including HRV2, are very closely related to one another by sequence alignment, with the exception of HRV14 being a more divergent strain. In fact, HRV14 is more like polio-related enteroviruses than typical rhinovirus strains (10 –12). Both HRV 2A and 3C proteins have been identified as cysteine proteases due to the presence of active-site cysteine residues (13–15), although sequence alignment data have revealed that these viral proteases do not display significant structural homology to the typical papain-type cysteine proteases (12, 13). Hence, with no known cellular homologues, these proteases are ideal enzyme targets for antiviral intervention. For this purpose, development of sensitive and quantitative assays for measurement of the viral protease activity will be essential. For the rhinovirus 3C protease, several assays using chromogenic, fluorogenic, HPLC, and radioactive peptides as substrates have been reported (16 –20). In contrast, assays for the viral 2A protease have been limited to an HPLC method using small synthetic peptides and a colorimetric assay described recently for HRV2 2A (21, 22). Although tre0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
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mendous efforts have been made investigating the 2A protease from HRV2, little is known about the 2A enzyme from other HRV serotypes. In this report, we describe the peptide substrate cleavage specificity of the purified 2A protease from HRV14. Based on the substrate requirements of HRV14 2A, we have developed a sensitive assay using fluorogenic peptides as substrates. This method allows the HRV 2A protease activity to be detected in vitro quantitatively. MATERIALS AND METHODS Materials. All peptides described in this report were synthesized on an Applied Biosystems 433A peptide synthesizer using standard Fmoc chemistry as described previously (19). Crude peptides were purified by reversed-phase HPLC as described and their sequences were confirmed by mass spectrometry. Classic protease inhibitors were purchased from Sigma. Expression and purification of HRV14 2A protease. Detailed molecular cloning of HRV14 2A protease and its expression in Escherichia coli cells will be described elsewhere. Briefly, the cDNA encoding HRV14 2A protease was cloned into a heat-inducible pH10 vector for expression in E. coli cells (16). Transformed bacterial cells were collected and resuspended in buffer A containing 25 mM Hepes, pH 8.0, 5 mM DTT, and 5% glycerol. After lysis by sonication in buffer A plus 1 M NaCl, cytoplasmic granules containing HRV14 2A protein were collected and washed with 100 ml of 1 M NaCl and then 1 M urea followed by water. The washed granules were solubilized overnight with 7 M urea in buffer A and centrifuged at 10,000g for 30 min. The supernatant, containing the denatured HRV14 2A protein, was diluted with buffer A to a concentration of 0.1 mg/ml and dialyzed overnight at 4°C against 0.1 mM ZnCl2 in buffer B (25 mM Hepes, pH 8.0, 5% glycerol, and 150 mM NaCl) to refold the 2A protease. The refolded proteins were loaded onto a Mono Q 5/5 column (Pharmacia) and then eluted with a linear gradient of 0.15–1 M NaCl in buffer A. Fractions containing the 2A protease activity were identified by the colorimetric assay described below, pooled, and loaded onto a Superdex-75 Hiload 26/60 column (Pharmacia). The proteins were then eluted with buffer B. The active 2A protease was identified, pooled, and stored at 220°C. Recombinant HRV2 2A protease was purified as described (6). Protein concentration was determined by the Bradford method using bovine serum albumin as standard. Protein purity was determined by densitometry analysis of SDS–PAGE gels. Viral protease assays. A typical colorimetric assay for HRV14 2A was performed at 25°C for the time indicated in a mix containing 25 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, purified 2A protease at the indicated concentrations, and 250 mM chromogenic p-nitroanilide (pNA) peptide RKGDIKSY–pNA. Cleavage of the pNA peptide between the P1 tyrosine and P19 pNA releases the free yellowcolored pNA which can be detected at a visible wavelength. Protease reactions (200 ml) were carried out in microtiter plate wells using a temperature-controlled microplate reader (Molecular Devices). All reactions were monitored at 405 nm against a blank in which either substrate or enzyme was not included in the reaction. A standard fluorescence assay for HRV14 2A protease (400 ml) using internally quenched fluorogenic peptides was conducted at 30°C for the time specified under the conditions described above. The fluorescence donor and quencher in these peptides are an anthranilyl group and a 3-nitrotyrosine, respectively. The intrinsic fluorescence of anthranilide can be efficiently quenched by 3-nitrotyrosine due to resonance energy transfer between these two moieties (23). Since the fluorescence signal from the anthranilyl group increased once the peptide was cleaved by the protease, these reactions were continuously monitored by a fluorometer. Reactions were started by addition
FIG. 1. SDS–PAGE analysis of the purified HRV14 2A protein. An aliquot of the pooled fractions containing active HRV14 2A protease activity (;50 ng) was separated by electrophoresis on a 16% gel and then subjected to silver staining.
of substrate and detected with a Perkin–Elmer LS50B luminescence spectrometer with the emission and excitation wavelengths set at 415 and 340 nm, respectively. To determine the kinetic parameters, reactions were run with 1 mM 2A protease and peptide substrate with a concentration range of 0.1–1 mM under the conditions described above. Data were collected prior to 10% substrate depletion in order to obtain the initial cleavage velocity. The cleavage efficiency, expressed as kcat/Km, was generated from the formula v 5 (kcat/Km)[E][S], where [E] and [S] are enzyme and substrate concentrations, respectively. Kinetic parameters were determined based on the assumption that all the 2A protein present in the reactions was active. The absence of products (detection limit, ;25 pmol) after 2 h incubation of the substrate with the enzyme was defined as no cleavage. HPLC Analyses of peptide cleavage reactions. A typical HPLC assay was carried out under the conditions described above with different synthetic peptides. Reactions (100 ml) were stopped at the time specified by addition of trichloroacetic acid to a final concentration of 5% (v/v). An aliquot of the terminated mix (50 ml) was then injected onto a reverse-phase HPLC (Shimadzu SCL-10A). The reaction mix was eluted from a C18 column (4.6 3 250 mm) at 1 ml/min with a linear gradient of 10 – 60% acetonitrile in 0.1% trifluoroacetic acid in 20 min. Peaks were monitored at 214 nm. For verification of the cleavage products by electrospray-ionization mass spectrometry, data were obtained as described previously using a PESciex API III triple-stage-quadrupole mass spectrometer (19).
RESULTS
Recombinant HRV14 2A protease was purified as described under Materials and Methods. A single protein band with a molecular mass of ;16 kDa was identified on a silver-stained SDS–PAGE gel (Fig. 1). This size was consistent with the 2A calculated molecular mass and its identity as HRV14 2A was confirmed by both N-terminal amino acid sequencing and mass spectrometry analysis (not shown). We initially assayed the HRV14 2A protease activity by using a 16-mer peptide (P8/P89) derived directly from the HRV14 2A native cis-cleavage region. HRV14 2A cleaved the 16-mer P8/P89 peptide with a kcat/Km of ;45 M21 s21 (Table I). To define the minimal sequence
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WANG ET AL. TABLE I
Influence of Peptide Length on HRV14 2A Cleavage Efficiency Peptide code
Peptide sequence
kcat/Km (M21 s21)
P8/P89 P8/P59 P8/P29 P8/P19 P5/P89 P2/P89 P1/P89 P8-pNA
RKGDIKSY-GLGPRYGG RKGDIKSY-GLGPR RKGDIKSY-GL RKGDIKSY-G DIKSY-GLGPRYGG SY-GLGPRYGG Y-GLGPRYGG RKGDIKSY-pNA
45.7 6 4.5 45.0 6 5.1 30.8 6 2.2 25.4 6 2.3 20.6 6 2.1 NC NC 335 6 20
Note. Peptide sequences represent the authentic VP1/2A cleavage site of HRV14 polyprotein. Cleavage reactions were detected by HPLC methods as described in the text. At least two independent experiments were performed for each substrate and shown is the average with variation. The absence of products after 2 h incubation with the enzyme (1 mM) is defined as no cleavage (NC).
required for HRV14 2A cleavage, a series of peptides differing in size was synthesized and tested as potential 2A protease substrates. The 2A catalytic rates toward these peptides, expressed as kcat/Km, are shown in Table I. The C-terminal truncations did not significantly alter the 2A cleavage efficiency; peptides P8/P29 and P8/P19 were still substrates for the enzyme (Table I). In contrast, the N-terminal truncations of the peptides could dramatically affect the 2A catalytic rate (Table I). Cleavage of peptide P5/P89 was reduced ;2.5-fold relative to the peptide P8/P89, and either P2/P89 or P1/P89 was no longer a substrate for the protease. For the two latter peptides, no detectable cleavage product peaks or decrease of the original substrate peak was found by HPLC analysis of the 2-h reaction mix (not shown). To search for an optimal peptide substrate for this enzyme, internally quenched peptides were synthesized
using an anthranilyl group (abz) and a 3-nitrotyrosine as the resonance energy transfer donor/quencher pair (23). To make a valid comparison, all fluorogenic peptides were designed to contain 13 residues with 3-nitrotyrosine at P1 and abz-modified lysine at P59 as seen in Table II. Similar to the results previously obtained with the unmodified peptide P8/P59 (Table I), peptide 1, containing a leucine at P29, was not efficiently hydrolyzed by HRV14 2A under the conditions employed (Fig. 2A, Table I). Analysis by HPLC revealed that less than 15% of the peptide was hydrolyzed in a 4-h reaction. As a comparison, a similar approach was also applied to the HRV2 2A protease. Purified recombinant HRV2 2A was found to hydrolyze peptide 6 derived from its native cis-cleavage site with a kcat/Km value of 1424 M21 s21 (Fig. 2B). Interestingly, HRV14 2A protease could also recognize and hydrolyze this peptide, but with nine-fold reduced rate compared to HRV2 2A under the same assay conditions (Table II). On the other hand, the HRV2 2A enzyme failed to process the HRV14 2A peptides such as P8/P89 and peptide 1(Tables I and II). Considering the preference of the HRV2 2A protease for a proline residue at P29 (21, 24) as well as the ability of HRV14 2A to process peptide 6 which contained a P29 proline, we envisioned that a proline at P29 might be an important determinant for HRV14 2A trans-cleavage efficiency, although this residue is not present in its native site on the HRV14 polyprotein. To verify this hypothesis, peptide 2 was synthesized with a substitution of proline for leucine at position P29. As seen in Table II, this resulted in a roughly 12-fold better HRV14 2A substrate than peptide 1, albeit the only difference between these peptides was the amino acid identity at P29. However, such a substitution did not make peptide 2 an effective substrate for the 2A protease from HRV2 (Table II).
TABLE II
In Vitro Peptide Cleavage Specificity of HRV 2A Proteases Cleavage efficiency kcat/Km (M21 s21) Code
Peptide sourcea
1 2 3 4 5 6 7
HRV14 HRV14 HRV14 HRV14 HRV14 HRV2 eIF4G
Peptide sequence RKGDIKTY(NO2) -GLGPK RKGDIKTY(NO2) -GPGPK RKGDIKTY(NO2) -APGPK RKGDIKAY(NO2) -GPGPK RKGDIKSY(NO2) -GPGPK TRPIITTY(NO2) -GPSDK GRTTLSTY(NO2) -GPPRK
HRV2 2A (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y
NC 14.5 6 2.5 NC NC 10.0 6 3.3 1424 6 78 397 6 25
HRV14 2A ,10.0 6 9 NC NC 8.0 6 3.5 154 6 11 418 6 35 126
Note. Lysine–anthranilide and 3-nitrotyrosine within the peptides are shown as K(abz) and Y(NO2), respectively. Peptide 6 was derived from the HRV2 2A native cis-cleavage site which has an original amino acid sequence of -TRPIITTY//GPSDMYVHon the viral polyprotein. Peptide 7 was from the HRV2 2A hydrolysis site on human eIF4G p220 protein containing an original 2A recognition sequence of 478GRTTLSTR//GPPRGGPG493 (5). All other peptides were derived from the VP1/2A site (P8/P89 as shown in Table I) of the HRV14 polyprotein with the indicated substitutions (in bold). NC, no cleavage was seen. a
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
FIG. 2. Time-dependent cleavage of fluorogenic peptides by viral 2A proteases. Peptide (100 mM) hydrolysis by the purified HRV14 2A (0.5 mM, A) or HRV2 2A enzyme (0.2 mM, B) was performed at 30°C under the conditions described under Materials and Methods. Only 10% of the collected data points are shown. The peptides (with their sequences shown in the text) used in the assays are labeled as peptide 1 (D), peptide 7 (h), and peptide 6 (E). Reaction containing no HRV2 2A enzyme (F) is also shown for peptide 6.
We also examined the amino acid requirements of HRV14 2A at positions P2 and P19. Single substitution of the amino acids at these positions caused significant effects on the HRV14 2A cleavage efficiency. As summarized in Table II, peptides with alanine substitution at either P19 or P2 were no longer substrates for HRV14 2A enzyme. Interestingly, HRV14 2A protease was capable of distinguishing between serine and threonine at P2; replacement of the P2 threonine with serine (peptide 5) resulted in a more than 12-fold reduced kcat/Km value (Table II). Taken together, our data indicated that amino acids at P2, P19, and P29 were important determinants for an efficient cleavage by HRV14 2A to occur. Based on the 2A substrate specificity studies, fluorogenic peptide 7, representing the 2A cleavage site on
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human eIF4G p220 protein, was synthesized and tested as a 2A substrate (5). This peptide was cleaved by HRV14 2A with a kcat/Km of 418 M21 s21, which manifested a roughly eight-fold fluorescence intensity increase as seen in Fig. 2A. Hydrolysis of peptide 7 by HRV14 2A was dependent on enzyme concentration (Fig. 3). Incubation of this peptide with 1 mM HRV14 2A protease for 3 h resulted in complete cleavage as determined by HPLC analysis (data not shown). Analysis of the reaction products by mass spectrometry indicated that the peptide was cleaved at the expected scissile bond (results not shown). Interestingly, HRV2 2A was also found to cleave peptide 7 with a catalytic rate similar to that for HRV14 2A enzyme (Table II). Since peptide 7 was identified as the most efficient peptide substrate for HRV14 2A, a number of groupspecific protease inhibitors were screened against HRV14 2A protease using this fluorogenic peptide as a substrate. HRV14 2A was strongly inactivated by thiol-alkylating reagents such as iodoacetamide and N-ethylmaleimide, implying an essential role for cysteine residue in the 2A catalytic reaction. A series of homophthalimide compounds, previously identified as the 2A protease inhibitors (25), inhibited the HRV14 2A at low-micromolar range when tested using the fluorogenic peptide 7 as the substrate (Table III). Overall, the HRV14 2A enzyme inhibition profile shown in Table III was very similar to that generated using the chromogenic pNA peptide as a substrate (data not shown). Therefore, these results indicated that the internally quenched small peptides could be used for 2A protease inhibitor screening and evaluation.
FIG. 3. Peptide cleavage versus HRV14 2A enzyme concentration. Peptide 7 at 100 mM was incubated with the purified HRV14 2A protease at the indicated concentration for 30 min at 30°C under the conditions described under Materials and Methods. Readings were taken against the control which contained no enzyme. Shown is an average of two measurements.
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WANG ET AL. TABLE III
Evaluation of HRV14 2A Protease Inhibitors Using Internally Quenched Peptide as Substrate Inhibitor Control EDTA E-64 Iodoacetamide N-Ethylmaleimide Pepstatin Aprotinin Elastatinal TLCK LY338387 LY343813 LY343814 LY353350
Inhibitor type No addition Metalloprotease Cys protease Cys protease Cys protease Asp protease Ser protease Ser protease Ser/Cys protease HRV14 3Ca HRV14 2A HRV14 2A HRV14 2A
Concentration
25 100 1 1 0.1 15 100 1 25 25 25 25
mM mM mM mM mM mM mM mM mM mM mM mM
2A inhibition (%) 0 0 0 100 100 0 23 6 3 68 6 6 100 0 61 6 2 59 6 5 67 6 4
a
LY338387 (N-Cbz-Phe-Met-SO2) is a peptide-based inhibitor designed specifically for HRV14 3C protease (32). LY343813, LY343814, and LY353350 are homophthalimide-type inhibitors identified for HRV14 2A (25). Protease assays were performed using 100 mM fluorogenic peptide 7 as a substrate under the conditions described under Materials and Methods. Control was run under the identical conditions except only the solvent (no inhibitor) was included in the reaction. E-64, L-trans-epoxysuccinylleucylamide-4-(guanidino)-butane; TLCK, tosyllysylchloromethyl ketone.
DISCUSSION
Both 2A and 3C proteases encoded by HRVs are important for viral polyprotein maturation and replication. In the case of HRV14, previous studies have been very much focused on the 3C protease. For example, detailed expression, purification, assay development, structural analysis, and inhibitor studies have been conducted for this enzyme (14, 16–20, 25–27). As a comparison, much less attention has been paid to the 2A protease from the same serotype. Our current understanding about the viral 2A protease arises from the extensive studies with the 2A protein from either HRV2 or poliovirus (4–6, 28–30). It is generally accepted that HRV2 and HRV14 belong to different subgroups (2, 11). Amino acid sequence comparison of the 2A proteases from HRV2 and HRV14 reveals only 40% identity and 57% similarity (data not shown). Actually, HRV14 2A shares even higher similarity to the 2A protease from coxsackievirus (data not shown). Therefore, it is interesting to investigate the 2A protease from distinct HRV serotypes, and such biochemical comparison studies would generate a better understanding of these viral enzymes. As the 2A protein crystal structure is not available, substrate-specificity studies of the 2A protease will facilitate rational design and synthesis of potent 2A inhibitors. To address these issues, we decided to purify active HRV14 2A protease. With a bacterial cell expression system similar to that used for HRV 14 3C protease (16), we have successfully expressed, refolded, and purified the active HRV14 2A protease, which was used for enzyme characterization studies. It is surprising to see that small peptides representing the native HRV14 2A cis-cleavage region could not be effectively processed in vitro by the purified enzyme. Al-
though peptide cleavage efficiency of HRV14 2A could be elevated by introducing a P29 proline in these peptides, the data raised a question about how the HRV14 2A processes the polyprotein precursor in vivo. In the case of poliovirus, substrate secondary and tertiary structures are believed to be the important determinants for the enzyme–substrate interaction in vivo (31). This conclusion may extend to HRV14 polyprotein cleavage by the 2A protease in vivo. Since the 2A maturation cleavage has been thought of as a cis or an autocatalytic event (2, 3), it is possible that protease folding and its ways to access the protein substrate might be quite different from those seen with the in vitro peptides. Another explanation is that the specific activity of the 2A protease in the HRV-infected cells could be regulated by certain cellular factors. Nevertheless, this observation marks the most significant difference between HRV14 2A and its counterpart from HRV2. Since the HPLC-based methods are usually inconvenient and time consuming, we have used fluorogenic peptides to study the 2A protease substrate specificity. Considering that the fluorophore attached to the peptides might affect the interaction between the enzyme and its substrate, we put the fluorophore and the quencher at the same positions for all the peptides in order to generate valid comparison results. Overall, our data suggest that in vitro substrate requirements of HRV14 2A are similar to those described for 2A proteases from HRV2 and coxsackievirus (21, 24, 29) in terms of their preference for a glycine at P19, a proline at P29, and a threonine at P2. The two 2As demonstrated very similar cleavage efficiency toward the peptide derived from the eIF4G p220, indicating that this
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
cellular protein might also be a potential target for HRV14 2A enzyme. Although the identity of the residues at P19 and P29 is important to the cleavage of peptides with amino acids occupying both sides of the scissile bond, the N-terminal 8 amino acids seem to be sufficient for HRV14 2A cleavage to take place (Table I). Substitution of the original prime-side amino acids with a single pNA molecule generates a new chromogenic peptide P8-pNA, which was a much better HRV14 2A substrate than the native 16-mer peptide P8/P89 (Table I). We have observed a dramatic rate difference between HRV2 2A and HRV14 2A proteases on processing the pNA peptides. HRV2 2A hydrolyzed its pNA peptide TRPIITTA-pNA with a kcat/Km of 19,500 M21 s21 (22). In comparison, HRV14 2A exhibited 65-fold less efficiency toward its own P8-pNA peptide and had a kcat/Km value of 335 M21 s21. The observed low pNA peptide cleavage rate of HRV14 2A could not be explained solely by the absence of an essential threonine at P2 in its peptide because the pNA peptide (TRPIITTApNA) designed for HRV2 2A was an even worse substrate for HRV14 2A. Apparently, the other residues upstream of the scissile bond are also important for the HRV14 2A cleavage to occur. It is possible that the substrate binding pocket at the prime side (especially S19 and S29) of HRV2 2A is more suitable for the pNA moiety than for Gly-Pro residues, because HRV2 2A prefers pNA peptides to its 16-mer P8/P89 peptide as determined in a competition cleavage assay (20). Similar observations have been made on the basis of the 2A protease inhibition profiles using homophthalimides (25). Taken together, these data suggest that HRV2 2A and HRV14 2A share a similar active-site conformation, although amino acids involved in substrate binding might be very different. The 2A protease of HRVs has been viewed as an important enzyme target for antiviral intervention, therefore, development of quantitative and convenient assays for its enzymatic activity measurement would be essential for facilitating antiviral drug discovery efforts. Parallel to the chromogenic pNA peptide specifically designed for the HRV14 2A enzyme (Table I), we have also developed several fluorogenic substrates for both HRV2 2A and HRV14 2A enzymes using small internally quenched peptides. HRV2 2A was able to cleave the fluorogenic peptide derived directly from its cis-cleavage site, while HRV14 2A preferred the peptide with the sequence derived from the human eIF4G p220 protein. Compared to the HPLC method, fluorescence assays are more intrinsically sensitive, especially for those using internally quenched peptides based on resonance energy transfer (23). For peptides with anthranilyl moiety and 3-nitrotyrosine as the fluorescence donor/quencher pair, another advantage is that these peptides can be prepared directly with the solid-phase method as described here. It is hoped that these assays developed for both HRV2 2A and HRV14 2A protease will facilitate not only the 2A
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characterization studies but also the protease inhibitor evaluation. Additionally, the peptides generated in this study may provide valuable insight into the HRV 2A protease structural determinants and help the rational design of specific 2A protease inhibitors. ACKNOWLEDGMENTS We thank John Richardson for performing mass spectrometry experiments and Drs. J. Colacino and B. A. Heinz for critically reading the manuscript.
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28. Ko¨nig, H., and Rosenwirth, B. (1988) J. Virol. 62, 1243–1250. 29. Sommergruber, W., Ahorn, H., Klump, H., Seipelt, J., Zo¨phel, A., Fessl, F., Blaas, D., Kuechler, E., Liebig, H.-D., and Skern, T. (1994) Virology 198, 741–745. 30. Molla, A., Hellen, C. U. T., and Wimmer, E. (1993) J. Virol. 67, 4688 – 4695. 31. Ypma-Wong, M. F., Filman, D. J., Hogle, J. M., and Semler, B. L. (1988) J. Biol. Chem. 263, 17846 –17856. 32. Shepherd, T. A., Cox, G. A., McKinney, E., Tang, J., Wakulchik, M., Zimmerman, R. E., and Villarreal, E. C. (1996) Bioorg. Med. Chem. Lett. 6, 2893–2896.
Vol. 356, No. 1, August 1, pp. 12–18, 1998 Article No. BB980746
Development of in Vitro Peptide Substrates for Human Rhinovirus-14 2A Protease Q. May Wang,1 Robert B. Johnson, Wolfgang Sommergruber,* and Timothy A. Shepherd Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285; and *Boehringer-Ingelheim Research Vienna, Bender and Company, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria
Received January 20, 1998, and in revised form May 8, 1998
Purified 2A protease from human rhinovirus serotype-14 (HRV14) was unable to efficiently cleave a 16-mer peptide representing its authentic cis-cleavage site on the viral polyprotein, implying that in vivo cis cleavage by this enzyme might be very different from its in vitro trans activity. Presence of a serine at position P2 and a leucine at P2* in the 16-mer peptide was found to be responsible for the low peptide cleavage efficiency. To search for an efficient peptide substrate for HRV14 2A, small peptides derived from other rhinovirus 2A protease cleavage sites were synthesized and tested. These results suggested that the N-terminal 8 amino acids were sufficient for HRV14 2A cleavage to occur, although the P1* and P2* residue identities were important to the cleavage of peptides with amino acids occupying both sides of the scissile bond. On the basis of the 2A substrate requirements, a sensitive fluorometric assay for the viral 2A proteases was developed using peptides with anthranilide and 3-nitrotyrosine as the resonance energy transfer donor/quencher pair. Our data indicated that these fluorescent peptide substrates were suitable for 2A protease characterization and inhibitor evaluation. © 1998 Academic Press
As the major causative agents of the common cold in humans, more than a hundred different serotypes of human rhinovirus (HRV)2 have been identified to date (1, 2). HRVs are small plus-strand RNA viruses in the picornavirus family (1), which translate their genomic information into an ;220-kDa polyprotein precursor 1 To whom correspondence should be addressed. Fax: (317) 2761743. E-mail: [email protected]. 2 Abbreviations used: HRV, human rhinovirus; DTT, dithiothreitol; pNA, p-nitroanilide.
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(2, 3). Generation of functional viral proteins from the polyprotein precursor is required for viral replication and this process is performed by virally encoded 2A and 3C proteases (2, 3). The 2A protease makes the first cleavage at the junction of capsid protein VP1 and the N-terminus of the 2A itself, which separates the viral structural proteins from the nonstructural ones (2, 3). In addition, the 2A proteases from HRV serotype-2 (HRV2) and poliovirus have been shown to be involved in the cleavage of certain important cellular proteins, including protein synthesis initiation factor eIF4G (p220), which may result in host-cell protein synthesis shutoff (4 –7). HRVs have been grouped in various ways on the basis of receptor specificity, sensitivity to antiviral agents, or amino acid sequence homology (1, 8 –11). Most of the HRV serotypes, including HRV2, are very closely related to one another by sequence alignment, with the exception of HRV14 being a more divergent strain. In fact, HRV14 is more like polio-related enteroviruses than typical rhinovirus strains (10 –12). Both HRV 2A and 3C proteins have been identified as cysteine proteases due to the presence of active-site cysteine residues (13–15), although sequence alignment data have revealed that these viral proteases do not display significant structural homology to the typical papain-type cysteine proteases (12, 13). Hence, with no known cellular homologues, these proteases are ideal enzyme targets for antiviral intervention. For this purpose, development of sensitive and quantitative assays for measurement of the viral protease activity will be essential. For the rhinovirus 3C protease, several assays using chromogenic, fluorogenic, HPLC, and radioactive peptides as substrates have been reported (16 –20). In contrast, assays for the viral 2A protease have been limited to an HPLC method using small synthetic peptides and a colorimetric assay described recently for HRV2 2A (21, 22). Although tre0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
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mendous efforts have been made investigating the 2A protease from HRV2, little is known about the 2A enzyme from other HRV serotypes. In this report, we describe the peptide substrate cleavage specificity of the purified 2A protease from HRV14. Based on the substrate requirements of HRV14 2A, we have developed a sensitive assay using fluorogenic peptides as substrates. This method allows the HRV 2A protease activity to be detected in vitro quantitatively. MATERIALS AND METHODS Materials. All peptides described in this report were synthesized on an Applied Biosystems 433A peptide synthesizer using standard Fmoc chemistry as described previously (19). Crude peptides were purified by reversed-phase HPLC as described and their sequences were confirmed by mass spectrometry. Classic protease inhibitors were purchased from Sigma. Expression and purification of HRV14 2A protease. Detailed molecular cloning of HRV14 2A protease and its expression in Escherichia coli cells will be described elsewhere. Briefly, the cDNA encoding HRV14 2A protease was cloned into a heat-inducible pH10 vector for expression in E. coli cells (16). Transformed bacterial cells were collected and resuspended in buffer A containing 25 mM Hepes, pH 8.0, 5 mM DTT, and 5% glycerol. After lysis by sonication in buffer A plus 1 M NaCl, cytoplasmic granules containing HRV14 2A protein were collected and washed with 100 ml of 1 M NaCl and then 1 M urea followed by water. The washed granules were solubilized overnight with 7 M urea in buffer A and centrifuged at 10,000g for 30 min. The supernatant, containing the denatured HRV14 2A protein, was diluted with buffer A to a concentration of 0.1 mg/ml and dialyzed overnight at 4°C against 0.1 mM ZnCl2 in buffer B (25 mM Hepes, pH 8.0, 5% glycerol, and 150 mM NaCl) to refold the 2A protease. The refolded proteins were loaded onto a Mono Q 5/5 column (Pharmacia) and then eluted with a linear gradient of 0.15–1 M NaCl in buffer A. Fractions containing the 2A protease activity were identified by the colorimetric assay described below, pooled, and loaded onto a Superdex-75 Hiload 26/60 column (Pharmacia). The proteins were then eluted with buffer B. The active 2A protease was identified, pooled, and stored at 220°C. Recombinant HRV2 2A protease was purified as described (6). Protein concentration was determined by the Bradford method using bovine serum albumin as standard. Protein purity was determined by densitometry analysis of SDS–PAGE gels. Viral protease assays. A typical colorimetric assay for HRV14 2A was performed at 25°C for the time indicated in a mix containing 25 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, purified 2A protease at the indicated concentrations, and 250 mM chromogenic p-nitroanilide (pNA) peptide RKGDIKSY–pNA. Cleavage of the pNA peptide between the P1 tyrosine and P19 pNA releases the free yellowcolored pNA which can be detected at a visible wavelength. Protease reactions (200 ml) were carried out in microtiter plate wells using a temperature-controlled microplate reader (Molecular Devices). All reactions were monitored at 405 nm against a blank in which either substrate or enzyme was not included in the reaction. A standard fluorescence assay for HRV14 2A protease (400 ml) using internally quenched fluorogenic peptides was conducted at 30°C for the time specified under the conditions described above. The fluorescence donor and quencher in these peptides are an anthranilyl group and a 3-nitrotyrosine, respectively. The intrinsic fluorescence of anthranilide can be efficiently quenched by 3-nitrotyrosine due to resonance energy transfer between these two moieties (23). Since the fluorescence signal from the anthranilyl group increased once the peptide was cleaved by the protease, these reactions were continuously monitored by a fluorometer. Reactions were started by addition
FIG. 1. SDS–PAGE analysis of the purified HRV14 2A protein. An aliquot of the pooled fractions containing active HRV14 2A protease activity (;50 ng) was separated by electrophoresis on a 16% gel and then subjected to silver staining.
of substrate and detected with a Perkin–Elmer LS50B luminescence spectrometer with the emission and excitation wavelengths set at 415 and 340 nm, respectively. To determine the kinetic parameters, reactions were run with 1 mM 2A protease and peptide substrate with a concentration range of 0.1–1 mM under the conditions described above. Data were collected prior to 10% substrate depletion in order to obtain the initial cleavage velocity. The cleavage efficiency, expressed as kcat/Km, was generated from the formula v 5 (kcat/Km)[E][S], where [E] and [S] are enzyme and substrate concentrations, respectively. Kinetic parameters were determined based on the assumption that all the 2A protein present in the reactions was active. The absence of products (detection limit, ;25 pmol) after 2 h incubation of the substrate with the enzyme was defined as no cleavage. HPLC Analyses of peptide cleavage reactions. A typical HPLC assay was carried out under the conditions described above with different synthetic peptides. Reactions (100 ml) were stopped at the time specified by addition of trichloroacetic acid to a final concentration of 5% (v/v). An aliquot of the terminated mix (50 ml) was then injected onto a reverse-phase HPLC (Shimadzu SCL-10A). The reaction mix was eluted from a C18 column (4.6 3 250 mm) at 1 ml/min with a linear gradient of 10 – 60% acetonitrile in 0.1% trifluoroacetic acid in 20 min. Peaks were monitored at 214 nm. For verification of the cleavage products by electrospray-ionization mass spectrometry, data were obtained as described previously using a PESciex API III triple-stage-quadrupole mass spectrometer (19).
RESULTS
Recombinant HRV14 2A protease was purified as described under Materials and Methods. A single protein band with a molecular mass of ;16 kDa was identified on a silver-stained SDS–PAGE gel (Fig. 1). This size was consistent with the 2A calculated molecular mass and its identity as HRV14 2A was confirmed by both N-terminal amino acid sequencing and mass spectrometry analysis (not shown). We initially assayed the HRV14 2A protease activity by using a 16-mer peptide (P8/P89) derived directly from the HRV14 2A native cis-cleavage region. HRV14 2A cleaved the 16-mer P8/P89 peptide with a kcat/Km of ;45 M21 s21 (Table I). To define the minimal sequence
14
WANG ET AL. TABLE I
Influence of Peptide Length on HRV14 2A Cleavage Efficiency Peptide code
Peptide sequence
kcat/Km (M21 s21)
P8/P89 P8/P59 P8/P29 P8/P19 P5/P89 P2/P89 P1/P89 P8-pNA
RKGDIKSY-GLGPRYGG RKGDIKSY-GLGPR RKGDIKSY-GL RKGDIKSY-G DIKSY-GLGPRYGG SY-GLGPRYGG Y-GLGPRYGG RKGDIKSY-pNA
45.7 6 4.5 45.0 6 5.1 30.8 6 2.2 25.4 6 2.3 20.6 6 2.1 NC NC 335 6 20
Note. Peptide sequences represent the authentic VP1/2A cleavage site of HRV14 polyprotein. Cleavage reactions were detected by HPLC methods as described in the text. At least two independent experiments were performed for each substrate and shown is the average with variation. The absence of products after 2 h incubation with the enzyme (1 mM) is defined as no cleavage (NC).
required for HRV14 2A cleavage, a series of peptides differing in size was synthesized and tested as potential 2A protease substrates. The 2A catalytic rates toward these peptides, expressed as kcat/Km, are shown in Table I. The C-terminal truncations did not significantly alter the 2A cleavage efficiency; peptides P8/P29 and P8/P19 were still substrates for the enzyme (Table I). In contrast, the N-terminal truncations of the peptides could dramatically affect the 2A catalytic rate (Table I). Cleavage of peptide P5/P89 was reduced ;2.5-fold relative to the peptide P8/P89, and either P2/P89 or P1/P89 was no longer a substrate for the protease. For the two latter peptides, no detectable cleavage product peaks or decrease of the original substrate peak was found by HPLC analysis of the 2-h reaction mix (not shown). To search for an optimal peptide substrate for this enzyme, internally quenched peptides were synthesized
using an anthranilyl group (abz) and a 3-nitrotyrosine as the resonance energy transfer donor/quencher pair (23). To make a valid comparison, all fluorogenic peptides were designed to contain 13 residues with 3-nitrotyrosine at P1 and abz-modified lysine at P59 as seen in Table II. Similar to the results previously obtained with the unmodified peptide P8/P59 (Table I), peptide 1, containing a leucine at P29, was not efficiently hydrolyzed by HRV14 2A under the conditions employed (Fig. 2A, Table I). Analysis by HPLC revealed that less than 15% of the peptide was hydrolyzed in a 4-h reaction. As a comparison, a similar approach was also applied to the HRV2 2A protease. Purified recombinant HRV2 2A was found to hydrolyze peptide 6 derived from its native cis-cleavage site with a kcat/Km value of 1424 M21 s21 (Fig. 2B). Interestingly, HRV14 2A protease could also recognize and hydrolyze this peptide, but with nine-fold reduced rate compared to HRV2 2A under the same assay conditions (Table II). On the other hand, the HRV2 2A enzyme failed to process the HRV14 2A peptides such as P8/P89 and peptide 1(Tables I and II). Considering the preference of the HRV2 2A protease for a proline residue at P29 (21, 24) as well as the ability of HRV14 2A to process peptide 6 which contained a P29 proline, we envisioned that a proline at P29 might be an important determinant for HRV14 2A trans-cleavage efficiency, although this residue is not present in its native site on the HRV14 polyprotein. To verify this hypothesis, peptide 2 was synthesized with a substitution of proline for leucine at position P29. As seen in Table II, this resulted in a roughly 12-fold better HRV14 2A substrate than peptide 1, albeit the only difference between these peptides was the amino acid identity at P29. However, such a substitution did not make peptide 2 an effective substrate for the 2A protease from HRV2 (Table II).
TABLE II
In Vitro Peptide Cleavage Specificity of HRV 2A Proteases Cleavage efficiency kcat/Km (M21 s21) Code
Peptide sourcea
1 2 3 4 5 6 7
HRV14 HRV14 HRV14 HRV14 HRV14 HRV2 eIF4G
Peptide sequence RKGDIKTY(NO2) -GLGPK RKGDIKTY(NO2) -GPGPK RKGDIKTY(NO2) -APGPK RKGDIKAY(NO2) -GPGPK RKGDIKSY(NO2) -GPGPK TRPIITTY(NO2) -GPSDK GRTTLSTY(NO2) -GPPRK
HRV2 2A (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y (abz) Y
NC 14.5 6 2.5 NC NC 10.0 6 3.3 1424 6 78 397 6 25
HRV14 2A ,10.0 6 9 NC NC 8.0 6 3.5 154 6 11 418 6 35 126
Note. Lysine–anthranilide and 3-nitrotyrosine within the peptides are shown as K(abz) and Y(NO2), respectively. Peptide 6 was derived from the HRV2 2A native cis-cleavage site which has an original amino acid sequence of -TRPIITTY//GPSDMYVHon the viral polyprotein. Peptide 7 was from the HRV2 2A hydrolysis site on human eIF4G p220 protein containing an original 2A recognition sequence of 478GRTTLSTR//GPPRGGPG493 (5). All other peptides were derived from the VP1/2A site (P8/P89 as shown in Table I) of the HRV14 polyprotein with the indicated substitutions (in bold). NC, no cleavage was seen. a
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
FIG. 2. Time-dependent cleavage of fluorogenic peptides by viral 2A proteases. Peptide (100 mM) hydrolysis by the purified HRV14 2A (0.5 mM, A) or HRV2 2A enzyme (0.2 mM, B) was performed at 30°C under the conditions described under Materials and Methods. Only 10% of the collected data points are shown. The peptides (with their sequences shown in the text) used in the assays are labeled as peptide 1 (D), peptide 7 (h), and peptide 6 (E). Reaction containing no HRV2 2A enzyme (F) is also shown for peptide 6.
We also examined the amino acid requirements of HRV14 2A at positions P2 and P19. Single substitution of the amino acids at these positions caused significant effects on the HRV14 2A cleavage efficiency. As summarized in Table II, peptides with alanine substitution at either P19 or P2 were no longer substrates for HRV14 2A enzyme. Interestingly, HRV14 2A protease was capable of distinguishing between serine and threonine at P2; replacement of the P2 threonine with serine (peptide 5) resulted in a more than 12-fold reduced kcat/Km value (Table II). Taken together, our data indicated that amino acids at P2, P19, and P29 were important determinants for an efficient cleavage by HRV14 2A to occur. Based on the 2A substrate specificity studies, fluorogenic peptide 7, representing the 2A cleavage site on
15
human eIF4G p220 protein, was synthesized and tested as a 2A substrate (5). This peptide was cleaved by HRV14 2A with a kcat/Km of 418 M21 s21, which manifested a roughly eight-fold fluorescence intensity increase as seen in Fig. 2A. Hydrolysis of peptide 7 by HRV14 2A was dependent on enzyme concentration (Fig. 3). Incubation of this peptide with 1 mM HRV14 2A protease for 3 h resulted in complete cleavage as determined by HPLC analysis (data not shown). Analysis of the reaction products by mass spectrometry indicated that the peptide was cleaved at the expected scissile bond (results not shown). Interestingly, HRV2 2A was also found to cleave peptide 7 with a catalytic rate similar to that for HRV14 2A enzyme (Table II). Since peptide 7 was identified as the most efficient peptide substrate for HRV14 2A, a number of groupspecific protease inhibitors were screened against HRV14 2A protease using this fluorogenic peptide as a substrate. HRV14 2A was strongly inactivated by thiol-alkylating reagents such as iodoacetamide and N-ethylmaleimide, implying an essential role for cysteine residue in the 2A catalytic reaction. A series of homophthalimide compounds, previously identified as the 2A protease inhibitors (25), inhibited the HRV14 2A at low-micromolar range when tested using the fluorogenic peptide 7 as the substrate (Table III). Overall, the HRV14 2A enzyme inhibition profile shown in Table III was very similar to that generated using the chromogenic pNA peptide as a substrate (data not shown). Therefore, these results indicated that the internally quenched small peptides could be used for 2A protease inhibitor screening and evaluation.
FIG. 3. Peptide cleavage versus HRV14 2A enzyme concentration. Peptide 7 at 100 mM was incubated with the purified HRV14 2A protease at the indicated concentration for 30 min at 30°C under the conditions described under Materials and Methods. Readings were taken against the control which contained no enzyme. Shown is an average of two measurements.
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WANG ET AL. TABLE III
Evaluation of HRV14 2A Protease Inhibitors Using Internally Quenched Peptide as Substrate Inhibitor Control EDTA E-64 Iodoacetamide N-Ethylmaleimide Pepstatin Aprotinin Elastatinal TLCK LY338387 LY343813 LY343814 LY353350
Inhibitor type No addition Metalloprotease Cys protease Cys protease Cys protease Asp protease Ser protease Ser protease Ser/Cys protease HRV14 3Ca HRV14 2A HRV14 2A HRV14 2A
Concentration
25 100 1 1 0.1 15 100 1 25 25 25 25
mM mM mM mM mM mM mM mM mM mM mM mM
2A inhibition (%) 0 0 0 100 100 0 23 6 3 68 6 6 100 0 61 6 2 59 6 5 67 6 4
a
LY338387 (N-Cbz-Phe-Met-SO2) is a peptide-based inhibitor designed specifically for HRV14 3C protease (32). LY343813, LY343814, and LY353350 are homophthalimide-type inhibitors identified for HRV14 2A (25). Protease assays were performed using 100 mM fluorogenic peptide 7 as a substrate under the conditions described under Materials and Methods. Control was run under the identical conditions except only the solvent (no inhibitor) was included in the reaction. E-64, L-trans-epoxysuccinylleucylamide-4-(guanidino)-butane; TLCK, tosyllysylchloromethyl ketone.
DISCUSSION
Both 2A and 3C proteases encoded by HRVs are important for viral polyprotein maturation and replication. In the case of HRV14, previous studies have been very much focused on the 3C protease. For example, detailed expression, purification, assay development, structural analysis, and inhibitor studies have been conducted for this enzyme (14, 16–20, 25–27). As a comparison, much less attention has been paid to the 2A protease from the same serotype. Our current understanding about the viral 2A protease arises from the extensive studies with the 2A protein from either HRV2 or poliovirus (4–6, 28–30). It is generally accepted that HRV2 and HRV14 belong to different subgroups (2, 11). Amino acid sequence comparison of the 2A proteases from HRV2 and HRV14 reveals only 40% identity and 57% similarity (data not shown). Actually, HRV14 2A shares even higher similarity to the 2A protease from coxsackievirus (data not shown). Therefore, it is interesting to investigate the 2A protease from distinct HRV serotypes, and such biochemical comparison studies would generate a better understanding of these viral enzymes. As the 2A protein crystal structure is not available, substrate-specificity studies of the 2A protease will facilitate rational design and synthesis of potent 2A inhibitors. To address these issues, we decided to purify active HRV14 2A protease. With a bacterial cell expression system similar to that used for HRV 14 3C protease (16), we have successfully expressed, refolded, and purified the active HRV14 2A protease, which was used for enzyme characterization studies. It is surprising to see that small peptides representing the native HRV14 2A cis-cleavage region could not be effectively processed in vitro by the purified enzyme. Al-
though peptide cleavage efficiency of HRV14 2A could be elevated by introducing a P29 proline in these peptides, the data raised a question about how the HRV14 2A processes the polyprotein precursor in vivo. In the case of poliovirus, substrate secondary and tertiary structures are believed to be the important determinants for the enzyme–substrate interaction in vivo (31). This conclusion may extend to HRV14 polyprotein cleavage by the 2A protease in vivo. Since the 2A maturation cleavage has been thought of as a cis or an autocatalytic event (2, 3), it is possible that protease folding and its ways to access the protein substrate might be quite different from those seen with the in vitro peptides. Another explanation is that the specific activity of the 2A protease in the HRV-infected cells could be regulated by certain cellular factors. Nevertheless, this observation marks the most significant difference between HRV14 2A and its counterpart from HRV2. Since the HPLC-based methods are usually inconvenient and time consuming, we have used fluorogenic peptides to study the 2A protease substrate specificity. Considering that the fluorophore attached to the peptides might affect the interaction between the enzyme and its substrate, we put the fluorophore and the quencher at the same positions for all the peptides in order to generate valid comparison results. Overall, our data suggest that in vitro substrate requirements of HRV14 2A are similar to those described for 2A proteases from HRV2 and coxsackievirus (21, 24, 29) in terms of their preference for a glycine at P19, a proline at P29, and a threonine at P2. The two 2As demonstrated very similar cleavage efficiency toward the peptide derived from the eIF4G p220, indicating that this
IN VITRO PEPTIDE SUBSTRATES FOR HUMAN RHINOVIRUS-14 2A PROTEASE
cellular protein might also be a potential target for HRV14 2A enzyme. Although the identity of the residues at P19 and P29 is important to the cleavage of peptides with amino acids occupying both sides of the scissile bond, the N-terminal 8 amino acids seem to be sufficient for HRV14 2A cleavage to take place (Table I). Substitution of the original prime-side amino acids with a single pNA molecule generates a new chromogenic peptide P8-pNA, which was a much better HRV14 2A substrate than the native 16-mer peptide P8/P89 (Table I). We have observed a dramatic rate difference between HRV2 2A and HRV14 2A proteases on processing the pNA peptides. HRV2 2A hydrolyzed its pNA peptide TRPIITTA-pNA with a kcat/Km of 19,500 M21 s21 (22). In comparison, HRV14 2A exhibited 65-fold less efficiency toward its own P8-pNA peptide and had a kcat/Km value of 335 M21 s21. The observed low pNA peptide cleavage rate of HRV14 2A could not be explained solely by the absence of an essential threonine at P2 in its peptide because the pNA peptide (TRPIITTApNA) designed for HRV2 2A was an even worse substrate for HRV14 2A. Apparently, the other residues upstream of the scissile bond are also important for the HRV14 2A cleavage to occur. It is possible that the substrate binding pocket at the prime side (especially S19 and S29) of HRV2 2A is more suitable for the pNA moiety than for Gly-Pro residues, because HRV2 2A prefers pNA peptides to its 16-mer P8/P89 peptide as determined in a competition cleavage assay (20). Similar observations have been made on the basis of the 2A protease inhibition profiles using homophthalimides (25). Taken together, these data suggest that HRV2 2A and HRV14 2A share a similar active-site conformation, although amino acids involved in substrate binding might be very different. The 2A protease of HRVs has been viewed as an important enzyme target for antiviral intervention, therefore, development of quantitative and convenient assays for its enzymatic activity measurement would be essential for facilitating antiviral drug discovery efforts. Parallel to the chromogenic pNA peptide specifically designed for the HRV14 2A enzyme (Table I), we have also developed several fluorogenic substrates for both HRV2 2A and HRV14 2A enzymes using small internally quenched peptides. HRV2 2A was able to cleave the fluorogenic peptide derived directly from its cis-cleavage site, while HRV14 2A preferred the peptide with the sequence derived from the human eIF4G p220 protein. Compared to the HPLC method, fluorescence assays are more intrinsically sensitive, especially for those using internally quenched peptides based on resonance energy transfer (23). For peptides with anthranilyl moiety and 3-nitrotyrosine as the fluorescence donor/quencher pair, another advantage is that these peptides can be prepared directly with the solid-phase method as described here. It is hoped that these assays developed for both HRV2 2A and HRV14 2A protease will facilitate not only the 2A
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characterization studies but also the protease inhibitor evaluation. Additionally, the peptides generated in this study may provide valuable insight into the HRV 2A protease structural determinants and help the rational design of specific 2A protease inhibitors. ACKNOWLEDGMENTS We thank John Richardson for performing mass spectrometry experiments and Drs. J. Colacino and B. A. Heinz for critically reading the manuscript.
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