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Human rhinoviruses (HRVs), the major etiologic agents of the common cold in humans, contain over a hundred distinct serotypes and belong to the picornavirus family (5). These small plus-strand RNA viruses encode a single open reading frame which is translated into a single large polyprotein with a size of 220 kDa (19, 20). Maturation cleavage of the polyprotein to generate functional viral proteins is mainly performed by two virally encoded proteases, designated 2A and 3C (19, 20). The first cleavage of the polyprotein is believed to be catalyzed by the 2A protease as a cotranslational event (19, 20). This cleavage, which takes place at the junction of capsid protein VP1 and the N terminus of the 2A protease itself, separates the viral capsid from the nonstructural proteins (19, 20). Most of the remaining cleavages are further processed by either the 3C protease or its precursor 3CD enzyme. In addition, it has been shown that these viral proteases are responsible for the cleavage of several important cellular proteins, including eukaryotic initiation factor eIF4G (p220), which may have a significant impact on host cell protein synthesis (2, 3, 7, 12, 13).

From a structural point of view, rhinovirus 2A and 3C proteins display a strong similarity to trypsin-like serine proteases, although both of them contain a cysteine as the active site nucleophile (16, 17, 21). The X-ray crystal structures of 3C proteases from both hepatitis A virus and HRV14 have been solved, revealing their structural similarity to the typical serine proteases (16, 17). However, no such study has been reported for the 2A enzymes. In spite of the similarities to the 3C enzymes, the HRV 2A protease has been proposed to be a zinc-binding protein. Sommergruber and his colleagues have demonstrated that zinc is essential for the structural integrity of the HRV2 2A protease (22, 23). Interestingly, the NS3 serine protease from hepatitis C virus (HCV) has been reported to contain a zinc-binding site comprising three cysteines and a water-histidine moiety (9, 14). Since the HRV 2A proteins contain such a conserved motif similar to the zinc-binding site of the HCV NS3 protease (6), it might be postulated that the viral 2A protease binds to the metal ion in approximately the same coordination geometry as that seen with the HCV NS3 protease (9, 14).

Great efforts have been made in the purification and characterization of the viral 2A proteases, especially the 2A enzyme from HRV2 (12, 13, 15, 18, 21-23). However, considerably less is known about the 2A proteases from other HRV strains. On the basis of sequence homology, HRV2 and HRV14 have been classified into two groups (19). Actually, HRV14 is more a polio-related enterovirus than a typical HRV strain. It is known that 2A proteases from polioviruses could not be purified with such a yield and purity as those reported for typical HRV 2A proteases (10, 13). In the present study, we describe the overexpression of the HRV14 2A protease in bacteria and the successful refolding of the enzyme in the presence of certain transition metals. The enzymatic parameters of the highly purified HRV14 2A protease were compared to those of its counterpart from HRV2. Furthermore, a simple colorimetric assay for the 2A protease was developed on the basis of its peptide substrate specificity, which should prove useful for the 2A enzyme characterization and high-throughput screening of 2A protease inhibitors.

Requirement of divalent cation for generation of active HRV14 2A protease expressed in Escherichia coli. To produce a large quantity of recombinant 2A protein in E. coli, the gene encoding the HRV14 2A protease, derived from a biologically active cDNA clone of the virus pWR40, was amplified out of the cDNA with NdeI- and BamHI-modifying primers at the 5' and 3' ends, respectively. The coding sequence for the 2A protease was then inserted into a heat-inducible expression vector, pH10, described previously for the overexpression of human immunodeficiency virus type 1 protease and HRV14 3C protein in E. coli (1, 11). Since the 2A protease gene (total of 438 bp) was ligated into the vector at the 5' NdeI and 3' BamHI site, a start codon ATG from the NdeI recognition sequence was added in front of the 2A protease cDNA sequence. As a result, a methionine was expected at the N-terminal side of the recombinant 2A protein. The resulting construct, pH10/2A, was transformed into the competent E. coli strain RV308 cells, and the transformants were selected on tryptone-yeast extract (TY) plates containing tetracycline (10 mg/ml). A positive pH10/2A clone was selected and incubated at 30°C in 2× TY broth plus tetracycline (10 mg/ml) until the optical density at 600 nm reached approximately 0.7. The cell culture was induced to produce HRV14 2A protease by shifting the temperature to 42°C for 3 h. The cells were then harvested and analyzed for the presence of 2A protease by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An overexpressed protein with an apparent molecular mass of ~16 kDa was clearly seen in the crude extracts of transformed E. coli cells (see Fig. 2B, lane 1). This protein was not present in the control bacterial cells transformed with the vector with no insert (not shown). Further analysis showed that the 2A protein was present predominantly in the inclusion bodies as expected.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Metal requirement for generation of active HRV14 2A protease. (A) Refolding HRV14 2A protein. Urea-solubilized HRV14 2A protein was refolded through dialysis against buffer B with addition of EDTA (2 mM) or the specified divalent cations at 0.1 mM. After refolding, samples were all dialyzed against fresh buffer B to remove excess metals or chelator. The 2A protease activity was then detected with the pNA peptide RKGDIKSY-pNA as a substrate as described in the text. (B) Absorbance spectra of Ni2+ substituted 2A protease. Purified urea-denatured HRV14 2A protease (10 µM) was refolded with 0.1 mM Ni2+ in buffer B. DTT-Ni2+ and EDTA-Ni2+ complex were prepared freshly before the assay by mixing 15 µM DTT or 2 mM EDTA in buffer B with 0.1 mM Ni2+. Absorbance scanning of the samples was performed against a blank containing buffer B plus 0.1 mM Ni2+.

Cleavage of chromogenic peptides by purified HRV14 2A protease. Purification of the 2A protein refolded in the presence of Zn²⁺ was achieved by chromatography on an ion-exchange column followed by size-exclusion separation. Briefly, the refolded proteins were loaded onto a Mono Q 5/5 column (Pharmacia) and then eluted with a linear gradient of 0.15 to 1 M NaCl in buffer A. Fractions containing the active 2A protease were identified by the colorimetric assay as described below, pooled, and loaded onto a Superdex-75 Hiload 26/60 column (Pharmacia). The proteins were then eluted with buffer B. The fractions containing the protease activity were identified, pooled, and used for further analyses. Most of the contaminants after the first column had sizes over 45 kDa, which could be readily separated from the 2A protease by gel filtration. Figure 2A shows the protein elution profile from the gel filtration column along with the peptide cleavage activity. The HRV14 2A protease eluted at the position corresponding to the calculated molecular mass of 16.1 kDa (Fig. 2A), which coeluted with the protease activity. Only one band was identified from the pooled fractions on a silver-stained gel (data not shown), and N-terminal amino acid analysis confirmed the presence of HRV14 2A protease in this sample (not shown). The purity of the isolated HRV14 2A was greater than 95%, as determined by high-performance liquid chromatography analysis and SDS-PAGE (Fig. 2B). The high-level expression of the HRV14 2A protease and its enrichment in inclusion bodies simplified its purification. Using the two-step purification protocol, we could obtain 5.7 mg of 2A protease per g of the transformed E. coli cells.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Purification of HRV14 2A protease. (A) Elution profile of HRV14 2A protease from Superdex-75 Hiload 16/60. Five milliliters of the 2A protein preparation or mixed gel filtration standards (1 mg/ml each) was loaded onto the column. Proteins were eluted at a flow rate of 1 ml/min with buffer B. Peaks were monitored at 280 nm (dashed line). The elution positions of the standards are labeled with OV (ovalbumin [43 kDa]) and CT (chymotrypsinogen A [19.5 kDa]). Fractions were examined for the pNA peptide cleavage activity as shown (). (B) SDS-PAGE analysis of the purified HRV14 2A protein. Protein samples (~1 µg) were separated by electrophoresis on a 16% gel and then stained with Coomassie blue. Lanes: 1, transformed bacterial cell lysate; 2, urea-solubilized inclusion bodies; 3 and 4, Mono Q and Superdex-75 column pooled fractions containing 2A protease activity, respectively.

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Comparison of HRV14 2A protease with other HRV proteases. With the assay described above, the enzymatic parameters of HRV14 2A protease were determined and compared to those of the 2A proteases from HRV2 and polioviruses. The purified HRV14 2A protease was very stable at 4°C for weeks without significant loss of activity at low concentrations. HRV14 2A protease activity was not significantly affected by high concentrations of NaCl, showing similar cleavage activity in the range of 15 mM to 1.5 M. Similar results were observed with the 2A protease from HRV2 (21). The HRV14 2A enzyme activity was slightly inhibited by organic solvents such as dimethyl sulfoxide and ethanol, even at low concentrations (5% [vol/vol]), being 27 and 25% less active than the control, respectively. HRV14 2A protease had an optimum pH range of 7 to 9, which was similar to that of the 2A enzyme from serotype 2. Inhibition studies of HRV14 2A protease with classic group-specific protease inhibitors gave results similar to those reported previously for the 3C protease from the same strain and the 2A protease from HRV2 (10, 21, 24, 25). For example, thiol alkylating reagents such as iodoacetamide and N-ethylmaleimide inactivated the enzyme, implying an important role for the cysteine residue in the 2A protease catalytic reaction. HRV14 2A protease was not sensitive to E-64, a specific inhibitor of papain-like cysteine proteases, even at a concentration of 0.1 mM (data not shown). However, the enzyme was found to be inhibited by elastatinal, a serine protease inhibitor, with a 50% inhibitory concentration of 90 µM (data not shown). Therefore, our inhibitor studies support the hypothesis that HRV 2A protease belongs to a novel class of cysteine proteases.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Effect of temperature on the protease activity of the 2A proteases from HRV2 and HRV14. The peptide substrates (250 µM) used for the HRV2 2A () and HRV14 2A () proteases are TRPIITTA-pNA and RKGDIKSY-pNA, respectively. Reactions were carried out at the indicated temperature for 30 min under the conditions described in the text, with no preincubation between the enzyme and the corresponding substrate involved.

Inhibition of refolded HRV14 2A protease activity by transition metals present in the reactions. In contrast to their positive impact on generation of active 2A protease during the refolding process, metals, including Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺, were not required for its enzymatic activity. In fact, this group of metals strongly inhibited the 2A protease cleavage activity, even at low micromolar concentrations, as illustrated in Fig. 4. Interestingly, extensive dialysis of the 2A enzyme against EDTA or addition of chelating agents directly into the cleavage reactions did not significantly affect the 2A protease activity. For example, EDTA did not inhibit 2A protease cleavage activity at concentrations of 5 mM; 85% activity remained at the highest concentration tested (25 mM). Dialysis of the active 2A protease against the buffer containing 2 mM EDTA for 48 h did not affect the enzyme activity (not shown). Additionally, no detectable 2A enzyme inhibition was found when EGTA (up to 25 mM) was present in the reaction mixtures.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Effect of divalent cations on the HRV14 2A protease activity. Peptide RKGDIKSY-pNA at 250 µM was incubated at 22°C with the purified 2A protease (0.2 µM) for 30 min under the standard assay conditions. Different cations were included in the 2A cleavage reaction at three different concentrations. The effect of the divalent cations on the HRV14 2A activity was expressed as the percentage of activity of the control sample containing no metal ions. Shown is the average of two independent measurements with variation less than 3%.