The DNA fragment encoding all residues (Ala1 to Glu181) of the Chiba virus 3C-like protease was amplified by PCR, in that NdeI and Aor51HI restriction sites were introduced at the 5′ and 3′ ends, respectively. The NdeI–Aor51HI fragment was placed downstream of the T7 promoter with a linker encoding a sequence containing six consecutive His residues. The resultant plasmid, pT7ProHis, encodes a 3C-like protease with a Met residue in the N-terminal and with a SAGHHHHHHG sequence in the C-terminal. The NdeI–SphI fragment from pUCHis3CD (Someya et al., 2000) encoding the 3CD region was placed downstream of the T7 promoter to construct pT7His3CD. Next, the C139A mutation of the 3C-like protease was introduced and designated pT7His3CD-C139A. The DNA fragment encoding glutathione S-transferase (GST) was amplified by PCR, in that ApaI and SphI restriction sites were introduced at the 5′ and 3′ ends, respectively. The ApaI–SphI fragment encoding GST was ligated to the ApaI–SphI fragment of pT7His3CD-C139A. The resultant plasmid was designated pT7His3Cd-GST. This plasmid encodes the N-terminal His-tag sequence (MGGHHHHHHGASA), followed by the whole sequence of the C139A mutant 3C-like protease and Gly1 to Pro19 of 3D RNA-dependent RNA polymerase, and then followed by GST.

E. coli BL21-CodonPlus (DE3)-RIL cells (Stratagene, La Jolla, CA) were transformed with pT7ProHis or pT7His3Cd-GST. Cells were grown on medium A (McMurry et al., 1980) supplemented with 0.2% glucose and 0.1% casamino acids at 37 °C. When the absorbance at 530 nm reached 0.5, IPTG was added to a final concentration of 1 mM for induction of gene expression. After 2 h, cells were harvested, then washed and resuspended with 20 mM Tris–HCl (pH 8.0) buffer containing 0.1 M NaCl. Cells were disrupted by sonication. After the removal of cell debris, the supernatant was subjected to ultracentrifugation (150,000 ×  g, 1 h). The resultant supernatant was mixed with TALON Metal Affinity Resin (CLONTECH, Palo Alto, CA) equilibrated with 20 mM Tris–HCl (pH 8.0) buffer containing 0.1 M NaCl. The resin was washed with 20 mM Tris–HCl (pH 8.0) buffer containing 0.1 M NaCl and 0.1 M imidazole for removal of non-specifically bound proteins. The proteins of interest were eluted with 20 mM Tris–HCl (pH 8.0) buffer containing 0.1 M NaCl and 0.5 M imidazole and concentrated using Ultrafree-15 unit with Biomax-10 membrane (MILLIPORE, Bedford, MA). Proteins were determined by the method of Bradford (Bradford, 1976).

Usually, 5 μM enzyme and 5 μM substrate were reacted in 20 μl of the mixture containing 50 mM bis–tris–propane–HCl buffer (pH 8.62) for 16 h. Details of reaction mixtures were described in the respective sections. The reaction was stopped by the addition of an equal volume of 2× sample buffer, and the proteins were then analyzed by SDS-polyacrylamide (13%) gel electrophoresis. Gels were stained with Coomassie Brilliant Blue R-250. In order to estimate the efficiency of the proteolytic cleavage, density of the respective stained bands was scanned and calculated using NIH Image for Macintosh version 1.62.

The viral non-structural proteins and the proteolytic cleavage sites within the ORF1 polyprotein are depicted in Fig. 1A. In order to purify the Chiba virus 3C-like protease, we constructed pT7ProHis with the gene fragment encoding the protease with His-tag in its C-terminus (Fig. 1B). The ProHis protein (calculated molecular mass of 20.6 kDa) expressed from pT7ProHis was used as an enzyme in this study. In order to observe proteolysis at the cleavage site between the 3C and 3D, we at first used the His-3CD-C139A mutant protein expressed from pT7His3CD-C139A as a substrate, which was the N-terminal His-tagged 3CD fragment containing the Ala mutation of active-site Cys139 of the 3C-like protease (Fig. 1B). However, this protein tended to aggregate at higher pH after prolonged incubation. Other parts from the native Chiba virus ORF1 polyprotein could not be effectively produced in E. coli cells. Therefore, the His3Cd-GST protein (calculated molecular mass of 48.0 kDa) was used as a substrate, which retains the cleavage site between the 3C and 3D. The region covering the entire 3C and the N-terminal 19 amino acids of 3D was placed under the control of T7 promoter with the N-terminal His-tag and the C-terminal glutathione S-transferase (GST), the resultant plasmid being designated pT7His3Cd-GST (Fig. 1B). Although this protein itself has the 3C-like protease moiety, it never undergoes autocatalytic cleavage because the C139A mutation is introduced. If His3Cd-GST is cleaved at the 3C/3D junction (LE/GG) by the active ProHis, the His3C-C139A moiety of 20.7 kDa and the 3D′-GST moiety of 27.3 kDa are produced. Enzymatic reaction was initiated by mixing enzyme (ProHis) and substrate (His3Cd-GST). We could not obtain clear results when the reaction was performed in a short period (2–4 h) (data not shown). However, even if a substrate was mixed with an enzyme for a long period (more than 24 h), complete cleavage could not be achieved. One possibility for the incomplete cleavage is that the cleavage site of the substrate (His3Cd-GST) could not be accessible by the protease because the substrate was not properly folded. Alternatively, part of the cleavage site in the substrate might be masked by its own catalytic site or the catalytic site of other substrate molecule, so that the enzyme might have competed with the substrate for the cleavage site.Fig. 1

Construction of plasmids. (A) Map of the proteolytic cleavage sites within the ORF1 polyprotein. The numbers indicate the positions of the amino acids. Slashes indicate the cleavage sites recognized by the protease. The calculated molecular masses of the cleavage products are indicated. (B) Construction of the expression plasmids. Details are described in Section 2. Amino acids derived from the Chiba virus sequence are underlined.

Five micromolar enzyme and 5 μM substrate were mixed in 50 mM bis–tris–propane–HCl buffer of various pH containing 25 mM NaCl, and reacted at 37 °C for 16 h. Fig. 2 shows that maximum cleavage was obtained at pH 8.62, although the proteolytic cleavage occurred over a wide range of pH. Faint bands of 80 and 60 kDa were thought to be dimers of the substrate and its cleaved product (3D′-GST), respectively. When the efficiency of cleavage at pH 8.62 is 100%, those at pH 7.22, 7.66, 8.12, and 9.08 were estimated as 70, 86, 89, and 79%, respectively. A slope of relative activity was gentle at acidic pH below 8.62, while it was sharp at alkaline pH above 8.62. In the subsequent experiments, a buffer of pH 8.62 was used.Fig. 2

Effects of pH on protease activity. Five micromolar enzyme (ProHis) and 5 μM substrate (His3Cd-GST) were reacted in the 50 mM bis–tris–propane–HCl buffer of various pH (indicated at the top of the respective lanes) containing 25 mM NaCl at 37 °C for 16 h. M and C indicate molecular weight marker and reaction mixture at 0 time, respectively. His3Cd-GST was cleaved into 3D′-GST and His3C-C139A by ProHis.

Five micromolar enzyme and 5 μM substrate were mixed in 50 mM bis–tris–propane–HCl (pH 8.62) containing 25 mM NaCl, and reacted for 16 h at 4, 15, 26, 37, or 48 °C. As shown in Fig. 3 , optimal temperature for the activity was 37 °C. The activity at 4, 15, 26, and 48 °C was estimated as 23, 31, 55, and 80% of that at 37 °C, respectively.Fig. 3

Effects of temperature on protease activity. Five micromolar enzyme (ProHis) and 5 μM substrate (His3Cd-GST) were reacted in the 50 mM bis–tris–propane–HCl (pH 8.62) buffer containing 25 mM NaCl for 16 h at the indicated temperature.

We next examined the effects of monovalent and divalent cations on the proteolytic activity. The activity was compared with that under the condition where 50 mM bis–tris–propane–HCl (pH 8.62) containing 5 mM EDTA without any cations was used. The addition of 25, 50, or 100 mM NaCl decreased the proteolytic activity to 83, 52, or 36%, respectively (Fig. 4A). KCl also had an inhibitory effect on the activity (data not shown). As for the divalent cations, 5 mM MgCl₂ or 5 mM CaCl₂ did not affect the activity (108 and 105%) (Fig. 4B). In contrast, 5 mM ZnCl₂ or 5 mM HgCl₂ completely inhibited the activity (Fig. 4B). This result was consistent with Cys being a critical residue of the active site (Someya et al., 2002), and inhibition by Zn²⁺ indicated that Norovirus 3C-like protease was different from metalloprotease. When Hg²⁺ was used, two faint bands of 30 and 15 kDa slightly appeared (lane HgCl₂, Fig. 4B). It was possible that high concentrations of Hg²⁺ allowed the enzyme to alternatively cleave the substrate and/or the enzyme itself. With the addition of 5 mM MnCl₂, part of the proteins used for reaction appeared to be aggregated since Coomassie-stained materials stayed in the well (Fig. 4B). Because Mn²⁺ oxidized the proteins, the part may have remained in the well and the other part may be flown from the gel after degradation into small molecular weight peptides.Fig. 4

Effects of NaCl (A) and various divalent cations (B) on protease activity. Five micromolar enzyme (ProHis) and 5 μM substrate (His3Cd-GST) were reacted in the 50 mM bis–tris–propane–HCl (pH 8.62) buffer containing the indicated components at 37 °C for 16 h. (A) Reaction mixture contained 5 mM EDTA and the indicated concentration of NaCl. Essentially the same result was obtained when KCl was added instead of NaCl (data not shown). (B) Reaction mixture contained the indicated reagent of 5 mM.

Most of the 3C and 3C-like proteases have a cysteine residue as a nucleophile of the active site (Dougherty and Semler, 1993, Gorbalenya et al., 1989). These proteases should therefore be sensitive to sulfhydryl reagents. In fact, as shown in Fig. 4B, high concentrations (5 mM) of Hg²⁺ and Zn²⁺ blocked the activity of the 3C-like protease. In Fig. 5 , the effects of several sulfhydryl reagents were tested. Five micromolar substrate was reacted with 5 μM enzyme in the presence of 10 μM sulfhydryl reagents. Ten micromolar Hg²⁺ or Zn²⁺ almost completely inhibited the activity. p-Chloromercuribenzoate (PCMB), which includes an Hg(II) within the molecule, was also a good inhibitor of the 3C-like protease (82% inhibition). In contrast, p-chloromercuribenzenesulfonate (PCMBS) slightly inhibited the activity (27% inhibition), despite PCMBS also having an Hg(II) similarly to PCMB. Methyl methanethiosulfonate (MMTS), N-ethylmaleimide (NEM), and N-phenylmaleimide (NPM), which form a covalent bond with a sulfur atom of the cysteine residue, almost completely inhibited the activity. The reaction with NEM and NPM slightly altered the mobility of the enzyme molecules, possibly because a bulkier modified side chain of the active-site cysteine residue affected the structure of the enzyme, or because more than one molecule of the inhibitors attached to an enzyme. Neither 10 μM iodoacetate (IAA) nor iodoacetamide (IAM) inhibited the activity. These two reagents may be less sensitive to an SH group than other modifying reagents. Phenyl methylsulfonylfluoride (PMSF) is well known as an irreversible inhibitor of serine proteases. Because the 3C and 3C-like proteases have a structural architecture similar to chymotrypsin, and with essentially the same proteolysis mechanism (Dougherty and Semler, 1993, Gorbalenya et al., 1989), PMSF may inhibit the activity of the 3C and 3C-like proteases. Ten micromolar PMSF was less effective but significantly inhibited the activity of the Chiba virus 3C-like protease (20% inhibition). The effect of β-mercaptoethanol (β-ME) was also examined here. The residual activity of the 3C-like protease was approximately 80% of the control in the presence of 10 μM β-ME (Fig. 5). Even in the presence of much higher concentrations (5 mM) of β-ME, approximately 60% activity remained (data not shown). These results suggest that a disulfide bond is not essential to the activity. However, we cannot exclude the possibility that a disulfide bond is buried inside the folded protein and cannot be reduced by high concentrations of β-ME.Fig. 5

Effects of various sulfhydryl reagents on protease activity. Five micromolar enzyme (ProHis) and 5 μM substrate (His3Cd-GST) were reacted in the 50 mM bis–tris–propane–HCl (pH 8.62) buffer containing the indicated sulfhydryl reagent of 10 μM at 37 °C for 16 h. PCMB, p-chloromercuribenzoate; PCMBS, p-chloromercuribenzenesulfonate; MMTS, methyl methanethiosulfonate; NEM, N-ethylmaleimide; NPM, N-phenylmaleimide; IAA, iodoacetate; IAM, iodoacetamide; PMSF, phenylmethylsulfonylfluoride; β-ME, β-mercaptoethanol.