Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase

Protein Expression and Purification 71 (2010) 147–152

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Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase Kisun Ryu a, Kyun-Hwan Kim b,c, Seong-Yeon Yoo a, Eun-Young Lee a, Keo-Heun Lim a, Mi-Kyung Min d, Hajeong Kim d, Seong Il Choi e, Baik L. Seong a,e,* a

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, Republic of Korea Department of Pharmacology, School of Medicine, and Center for Cancer Research and Diagnostic Medicine, IBST, Konkuk University, Seoul 143-701, Republic of Korea Institute of Functional Genomics, Konkuk University, Republic of Korea d Mogam Biotechnology Research Institute, 341 Pojung-Dong, Yongin-City, Gyeonggi-Do 446-799, Republic of Korea e Translational Research Center for Protein Function Control, Yonsei University, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 19 October 2009 and in revised form 28 December 2009 Available online 8 January 2010 Keywords: NS5B HCV High-throughput screening RNA-dependent RNA polymerase LysN fusion

a b s t r a c t The non-structural protein 5B (NS5B) is an essential component for the genome replication of hepatitis C virus (HCV). Thus, its activity holds the potential of being a target for therapeutic actions against HCV. The availability of large amount of functionally active NS5B enzyme may facilitate the identification of NS5B inhibitors via high-throughput screening (HTS). Here, we expressed the C-terminal 20-amino acids truncated NS5B in a bacterial system using the N-terminal domain of Escherichia coli lysyl-tRNA synthetase (LysN) as a solubility enhancer. The fusion protein (LysN-NS5B) was purified in a yield of 6.2 mg/L. The activity of LysN-NS5B was confirmed by in vitro RNA-dependent RNA polymerase (RdRp) activity assay, and the biochemical properties of LysN-NS5B were further characterized by kinetic analysis. The optimal RdRp activity was shown at 30 °C with 5 mM of Mg2+ or 10 mM of Mn2+, while the Km value for UTP was determined as 5 lM. The RdRp activity of LysN-NS5B was strongly inhibited by phenyldiketoacid, a specific inhibitor of HCV NS5B activity. Our results suggest that the LysN fusion system is a suitable approach for producing an active form of NS5B that can be used for HTS of NS5B inhibitors. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Hepatitis C virus (HCV),1 a member of the Flaviviridae, is a positive-stranded RNA virus affecting over 170 million people worldwide [1–3]. HCV infection can cause liver cirrhosis, which may eventually lead to hepatocellular carcinoma [4]. Currently, the availability of prophylactic measures, e.g., vaccine, against HCV infection is quite limited. The most general therapeutic action against HCV relies on the treatment with interferon-a, ribavirin or the combination of both. However, the inefficiency and accompanying adverse effects of these approaches have been noted [5,6]. Accordingly, the discovery of an effective and safe anti-HCV agent is necessary. The HCV genomic RNA is 9.6 kb in length, encoding a single open reading frame (ORF) flanked by untranslated regions (UTRs). The ORF is translated to a polyprotein of 3011 amino acids. This * Corresponding author. Address: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, Republic of Korea. Fax: +82 2 362 7265. E-mail address: [email protected] (B.L. Seong). 1 Abbreviations used: HCV, hepatitis C virus; NS5B, non-structural protein 5B; HTS, high-throughput screening; LysN, N-terminal domain of Escherichia coli lysyl-tRNA synthetase; RdRp, RNA-dependent RNA polymerase; ORF, open reading frame; UTR, untranslated region. 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.01.004

polyprotein is processed into structural proteins C, E1, and E2 and non-structural (NS) proteins p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The NS proteins have an essential role in the viral life cycle [7–9]. In particular, the NS5B functions as a RNA-dependent RNA polymerase involved in the genome replication process. Structurally distinct from human DNA and RNA polymerases, the NS5B polymerase has been considered as a target for antiviral drug development [10,11]. This enzyme is composed of finger, palm, thumb domains and a GDD motif as of other RNA-dependent RNA polymerases (RdRps). Additionally, the HCV polymerase contains C-terminal hydrophobic amino acid residues, known as the membrane anchoring domain [12]. The three dimensional crystal structure of the NS5B polymerase [13–15] provides a structural basis for designing of novel inhibitors for this enzyme. Because of the limitations in the utilization of in vivo HCV models, the significance of developing a reliable in vitro assay system, especially for high-throughput screening (HTS) of NS5B inhibitors, has been emphasized [16,17]. For in vitro HTS, a prerequisite is to obtain a substantial amount of the active form of NS5B. Several NS5B expression systems producing either full-length or C-terminus truncated NS5B forms in insect cells or E. coli are available from previous studies [18–28]. However, most of these systems hold limitations related to their productivity.

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Recently, we have developed a novel solubility enhancing vector system to produce aggregation-prone heterologous proteins in soluble and active forms by using E. coli [29,30]. This expression vector utilizes the N-terminal domain (1–154 aa) of E. coli lysyltRNA synthetase (named LysN) as a fusion partner. Here, HCV NS5B was fused to LysN in order to increase the expression level and solubility. The LysN-NS5B fusion protein was expressed predominantly as a soluble form and was purified in a yield higher or comparable to those of other systems previously reported [20,26,27,31]. Based on the results of an in vitro RdRp assay, the fusion system and its product, LysN-NS5B, presented here may be useful for the functional assessment of candidate compounds holding antiviral entities against HCV. Materials and methods Materials The Ex Taq polymerase, dNTP mix, and T4 DNA ligase were purchased from Takara (Shiga, Japan). The E. coli HMS174 (DE3) pLysE was obtained from Invitrogen (Carlsbad, CA). The restriction endonucleases were purchased from New England Biolabs (Beverly, MA). The HiTrap Chelating HP column, Poly(A), Oligo(dT)12–18, and UTP were from Amersham-Pharmacia Biotech (Piscataway, NJ). The [a-32P]UTP was purchased from PerkinElmer (Wellesley, MA). Amicon Centriprep YM-10 was obtained from Millipore (Bedford, MA). The other commonly used chemical reagents including IPTG, DTT, imidazole, PMSF, DMSO, and glycerol were obtained from Sigma (St. Louis, MO). Construction of plasmids The pLysN-NS5B expression plasmid was constructed from pLysN vector, which is composed of T7 promoter-NdeI-LysN-H6ENLYFQ and a multiple cloning site (MCS) containing sites for KpnI, BamHI, EcoRV, SalI, and HindIII [29]. The DNA sequence of genotype 3a HCV NS5B was amplified via PCR using oligonucleotide primers of 50 -TCAAGGTACCTCGATGTCCTACACATGG-30 (sense) and 50 -TTAAGTCGACTTATTAGCGGGGTCGGGCACG-30 (antisense). The amplified gene was cloned into the pLysN vector at restriction sites of KpnI and SalI, and the identity of the clone was confirmed by sequencing. Expression and purification of LysN-NS5B The E. coli HMS174 (DE3) pLysE harboring pLysN-NS5B was culture at 37 °C. At the optical density of 0.6 at 600 nm, expression of LysN-NS5B was induced by IPTG at the final concentration of 1 mM. After incubated for 8 h at 30 °C, the cells in 500 mL of culture medium were pelleted and dissolved into 10 mL of lysis buffer (20 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM b-mercaptoethanol, 1% NP-40, 10% glycerol, and 10 mM imidazole) supplemented with 1 mM PMSF, 1 mM reduced glutathione and 0.2 mM oxidized glutathione. After sonication, the insoluble portion was removed from the cell suspension by centrifugation at 20,000g for 12 min. The supernatant was filtered (0.2 lm) and loaded onto a Ni2+ ion charged HiTrap Chelating HP column (column volume of 5 mL) at a flow-rate of 0.5 mL/min. The LysN-NS5B protein was eluted with imidazole gradient of 10–500 mM at the flow-rate of 2 mL/min. Fractions containing LysN-NS5B were identified by Western blot analysis using anti-His antibody [32]. After concentrated by Amicon Centriprep YM-10, the purified protein was dialyzed against storage buffer (50 mM NaPO4, pH 6.8, 300 mM NaCl, 10 mM DTT, 1 mM EDTA, and 1% Tween 20). One volume of glycerol was added for storage at 80 °C.

In vitro RNA-dependent RNA polymerase activity assay and kinetic analysis The activity of purified LysN-NS5B was measured by RdRp assays. Reaction mixtures were prepared in a total volume of 20 lL with 50 mM HEPES, pH 8.0, 25 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 10 lM UTP, 2 lCi [a-32P]UTP (800 Ci/mmol), 50 ng/lL actinomycin D, 20 lg/mL rifamycin, 20 U RNasin, 100 lg/mL Poly(A), 25 lg/mL Oligo(dT)12–18, and 300 nM of purified LysN-NS5B. After 60 min of incubation at 30 °C, RdRp activities were terminated by adding EDTA at a final concentration of 10 mM. Thereafter, each reaction mixture was loaded on a DE81 filter disc (Whatman). Air-dried filter discs were washed three times with a buffer containing 500 mM of Na2HPO4 and once with 100% ethanol. Incorporation of radiolabeled UTP by LysN-NS5B was evaluated by using a liquid scintillation counter (Packard). For the determination of the Km value of LysN-NS5B, 20-lL reaction mixtures were prepared with 2 lg of Poly(A) and 0.5 lg of Oligo(dT)12–18 plus 300 nM of the fusion protein. After adding various concentrations of UTP (0.8–30 lM), samples were incubated for 20 min at 30 °C. Km values were determined from the Michaelis–Menten steady-state kinetics. Inhibition of LysN-NS5B by phenyldiketoacid Phenyldiketoacid is reported as an active site-directed inhibitor of HCV NS5B polymerase with the IC50 of 5.7 lM [33]. Polymerase reaction mixtures containing 50 nM of purified LysN-NS5B were treated with either 0, 1, 10 or 100 lM of phenyldiketoacid, and were incubated for 2 h at 30 °C. Subsequently, each reaction was terminated by adding 20 lL of RNA gel loading buffer (80% formamide, 100 mM EDTA, 0.05% SDS, 0.025% bromophenol blue, and 0.025% xylene cyanol FF). Reaction products were resolved by a 7 M urea–PAGE (5% acrylamide), and visualized by Phosphor Imager BAS2500 (Fuji Photo Film, Japan). Quantification was conducted with densitometric values obtained from Image Gauge v3.46 software (Fuji Photo Film, Japan). Results and discussion Expression and purification of LysN-NS5B Previous reports have shown that the direct expression of the full-length NS5B gives relatively low yield which may be caused by its insoluble domain [23,28]. The truncation of the C-terminal hydrophobic amino acids of NS5B promoted the solubility to some extent [20,26,27]. Based on these findings, we initially attempted to express the truncated form of HCV NS5B in E. coli. However, the truncated NS5B still remained mostly insoluble under the expression condition of 30 °C (data not shown). To increase the expression level and solubility at the equivalent condition, we fused NS5B to the C-terminus of LysN, an efficient solubility enhancer [30]. The enhancement in the solubility of the fusion protein, LysN-NS5B, expressed at 30 °C, was confirmed by SDS–PAGE (Fig. 1A) and Western blot analysis (Fig. 1B) with the molecular weight of 82 kDa. The protein was fractionated by nickel-affinity chromatography with a linear gradient of imidazole ranging from 10 to 500 mM (Fig. 1C). Collectively, we obtained approximately 6.2 mg of purified LysN-NS5B from 1 L of E. coli culture broth (from about 2.8 g of wet cell weight). This is higher or at least comparable to the yield of other systems developed for NS5B production by others [20,26,27,31]. Previously, the C-terminal 21 amino acids truncated NS5B was produced as the glutathione S-transferase (GST)-fused form by two independent groups. The productivity of GST-NS5B was less than 1 mg from 1 L of E. coli culture [20,27]. Tomei et al. reported that the NS5B-DC21 of the HCV-BK was obtained with the yield of 6 mg/L [26]. Recently, Ivanov et al.

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Fraction number

Sup

Ppt

Tot

IPTG

3 19 25 27 31 33 35 37 49

205 116 97

LysN-NS5B (82 kDa)

66

45

29

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Marker

B

The optimal RdRp conditions of LysN-NS5B

3 19 25 27 31 33 35 37 49

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75

50

35

500

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750

375

500

250

250

125

Imidazole (mM)

A280 (mAU)

C

firms that the RdRp activity of purified protein is ascribed to the NS5B, but not to the modulated activity of E. coli RNA polymerase. The primer dependence of HCV NS5B in vitro has been documented previously [20,23,28]. Under the condition of Poly(A) used as a template, the RdRp activity of NS5B was dramatically reduced by 50-fold in the absence of Oligo(U) primer [23]. In another reports, the RdRp activities of the recombinant NS5B were almost negligible in the absence of Oligo(U) primer [20,28]. To confirm that the observed RdRp activity of LysN-NS5B is strictly dependent on the primer, Oligo(dA) was used a control primer, which is not complementary to the Poly(A) RNA template. Consistent with the previous studies, RdRp activity was not detected under this condition (Fig. 2). All these data show that the LysN-NS5B exhibit is an active RdRp activity with strict primer dependence, suggesting the suitability of this fusion protein for the functional studies or inhibitor screening of HCV NS5B.

Previous reports showed that divalent ions such as Mg2+ and Mn2+ are required for the RdRp activity of HCV NS5B [18,19]. However, the effects of the divalent ions on the RdRp activity are somewhat controversial among various reports [21,22,24,25,27,34]. We investigated the effect of divalent ions such as Mg2+ and Mn2+ (0–20 mM) on the RdRp activity of LysN-NS5B (Fig. 3A). The results showed that effectiveness of Mg2+ ions were approximately twofold of that of Mn2+ on coordinating the RdRp activity of LysN-NS5B. The maximal induction of activity by Mg2+ and Mn2+ were observed at 5 and 10 mM, respectively. In previous studies with poliovirus 3D polymerase and HCV RNA-dependent RNA polymerase, Mn2+ has been shown to be more effective than Mg2+ on the RdRp activity [21,24,27,34,35]. On the other hand, higher activity has also been documented for Mg2+ than Mn2+ as well [22,25]. This discrepancy might be due to the difference in genotypes, purification steps involved and the conditions for the activity assays. The optimal reaction temperature of the recombinant NS5B polymerase is shown to be 30 °C (Fig. 3B), which is consistent with the findings from previous studies [20,22]. Thus, subsequent experiments for further characterization of LysNNS5B were carried out at 30 °C in the presence of 5 mM Mg2+.

0

0 3

10

19 25 31 37

49

Fraction number Fig. 1. Expression and purification of recombinant LysN-NS5B from E. coli. (A) Analysis of recombinant LysN-NS5B protein by SDS–PAGE. NI, total cell extract of non-induced cells; T, total extract; P, cell pellet after centrifugation; S, supernatant of cell extract after centrifugation. Elution fractions from nickel affinity column are indicated. (B) Western blot analysis of protein samples. Samples used in (A) were subjected to Western blot analysis. (C) Chromatographic profile of LysN-NS5B purification. Protein purification by Ni-column using imidazole gradient was monitored at a wavelength of 280 nm.

reported that HCV NS5BD55 could be obtained at the yield of 3.5 mg/ L by utilizing rare codon tRNA supplement and two cistrons system [31]. Thus, we suggest the present expression system as an alternative system that can be used for yielding a substantial amount of NS5B. In vitro RdRp activity of purified LysN-NS5B We next verified the RdRp activity of LysN-NS5B using Poly(A) as an RNA template and Oligo(dT)12–18 as the primer, as described previously. The treatment of rifamycin, a specific inhibitor of E. coli RNA polymerase, showed no influence on the RdRp activity of LysN-NS5B; whereas, the activity of E. coli RNA polymerase, measured as a control, was strongly inhibited (Fig. 2). The data con-

Kinetic analysis of LysN-NS5B The LysN-NS5B from our system was further characterized via an experiment using three different concentrations of the product,

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Marker

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60.000 40.000 20.000 0

LysN-NS5B

E. coli RNA polymerase

Fig. 2. RNA-dependent RNA polymerase (RdRp) activity of purified LysN-NS5B. The RdRp activity was measured by [a-32P]UMP incorporation using an artificial Poly(A) template with primers; Oligo(dA) or Oligo(dT)12–18. The activity assay was performed at 30 °C for 60 min under the conditions described in Materials and methods. E. coli RNA polymerase (0.1 U) was added to the control reactions.

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A

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Activity (cpm)

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0 30

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Fig. 3. The optimal RdRp reaction conditions of LysN-NS5B. (A) Requirement of divalent metal ions for the RdRp activity of LysN-NS5B. Induced RdRp activity by Mg2+ and Mn2+ are shown in diamonds and squares, respectively. (B) Effects of reaction temperature on the RdRp activity of LysN-NS5B. All reactions were performed in presence of 5 mM of Mg2+.

i.e., 60, 300, and 600 nM (Fig. 4A). The 60 min time-course revealed the dose-dependence of the incorporation rate of UTP on the amount of enzyme added, particularly 40 and 60 min after the reaction was initiated (Fig. 4B). The Km value of the fusion protein, LysN-NS5B, for UTP was determined via an RdRp reaction. The plot of initial velocity versus substrate concentration showed a typical rectangular hyperbola relationship (Fig. 5A). When transformed into a Lineweaver–Burk plot of double-reciprocal presentation, we were able to estimate the Km to be about 5 lM (Fig. 5B). In previous reports, the Km values of non-tagged NS5B and histidine-tagged NS5B, expressed in insect cell, were 5.4 and 22 lM, respectively [26,36]. Our results indicate that the LysN-NS5B shows a similar biochemical property to those of the recombinant enzymes from these reports.

Inhibition of LysN-NS5B by known inhibitor To test the relevance of LysN-NS5B for HTS of inhibitors, the effects of phenyldiketoacid on the activity of our recombinant LysN-NS5B was further investigated. This molecule has been known to inhibit the HCV NS5B polymerase (IC50 = 5.7 lM) and the reversetranscriptase of human immunodeficiency virus (IC50 = 54 lM) [33]. A dose-dependent pattern of the inhibition of RdRp activity by phenyldiketoacid against LysN-NS5B is comparable to the NS5B (Fig. 6). The basal activity of N-terminally fused NS5B exhibited 86% of that of the intact form (compare lanes 1 and 5 in Fig. 6A). The slight

Fig. 4. Effects of LysN-NS5B concentrations on UTP polymerization. (A) Timecourse of polymerase activity of purified LysN-NS5B. The polymerization reaction was carried out at 30 °C using 60 (diamond), 300 (square), and 600 nM (triangle) of LysN-NS5B. Aliquots were collected at each time-point and analyzed for polymerase activity. (B) Dose-dependent polymerase activity of LysN-NS5B. The polymerization reaction was performed at 30 °C for 40 min (dashed line) and 60 min (solid line).

A

3.500 3.000

V (cpm)

23

Reaction temperature (°C)

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[UTP] (µM)

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16

0.0012 0.0009 0.0006 0.0003 0

-0.2

0

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0.4

0.6

0.8

1

1.2

1.4

1/[UTP] (1/µM) Fig. 5. Kinetic analysis of LysN-NS5B. (A) Effect of substrate (UTP) concentration on the velocity of polymerization reaction. The reaction was performed at 30 °C with various concentrations of UTP, ranging from 0.8 to 30 lM. (B) Lineweaver–Burk (LB) plot of polymerization reaction. The data obtained from (A) were transformed into an LB plot and the Km value was estimated to be 5 lM.

K. Ryu et al. / Protein Expression and Purification 71 (2010) 147–152

reduction of activity of LysN-NS5B might be due to steric constraints by its N-terminal fusion partner. The IC50 value was approximately 4 lM for LysN-NS5B (Fig. 6B). The enzyme activity were determined by using an artificial Poly(A) template, which has been widely used for high-throughput screening of HCV NS5B inhibitors [37,38]. Overall, the recombinant LysN-NS5B from our system exhibited comparable enzymatic behaviors to NS5B, demonstrating its suitability for the screening of antiviral compounds against HCV. In conclusion, here we introduce a novel and efficient method for the production of an active form of HCV NS5B using LysN as a solubility enhancer. The functional properties of the product LysNNS5B was characterized by in vitro RdRp activity assay. The results imply that LysN-NS5B can function as the intact NS5B, indicating that the fusion did not significantly alter the known properties of the enzyme. The yield of final purified LysN-NS5B, approximately 6.2 mg/L of flask culture, was higher or at least equivalent to the yields obtained from other approaches [20,26,27,31]. Considering its yield and functionality, the recombinant LysN-NS5B in this report

1

10

100

0

NS5B 1

10

100

LysN-NS5B 0

A

Phenyldiketo acid (µM)

B 100

Relative activity (%)

80

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40

20

0

0

10

20

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Inhibitor concentration (µM) Fig. 6. Inhibition of the polymerase activity of LysN-NS5B by phenyldiketoacid. (A) Effects of phenyldiketoacid on the RdRp activity of LysN-NS5B and NS5B. Reaction mixtures were prepared with various concentrations of phenyldiketoacid. After 2 h, reaction mixtures were resolved by a 7 M urea–PAGE (5% acrylamide). Polymerization products were visualized by phosphor imager BAS2500. (B) Semi-quantification of RdRp products by densitometric analysis. Each value for LysN-NS5B (diamond) and NS5B (square) represent the data obtained from (A). The estimated IC50 value of phenyldiketoacid against LysN-NS5B is 4 lM.

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could be a useful means for the identification of anti-HCV drugs via HTS.

Acknowledgments We thank Moon-Suhn Ryu, Food Science and Human Nutrition Department, University of Florida, for helpful discussion and preparation of the manuscript. The work has been supported in part by the National Strategic Research Grant from the Ministry of Knowledge Economy of the Korean Government (Grant No. 10031969) and the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (2009-0092970). This work was also supported in part by the Yonsei University Research Fund of 2009 (to K. Ryu and S.I. Choi).

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