Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli

Virus Research 53 (1998) 141 – 149

Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli Ronald H. Al, Yiping Xie, Yuhuan Wang, Curt H. Hagedorn * Department of Medicine, Genetics Program of the Winship Cancer Center, and the Program in Biochemistry and Molecular Biology, Emory Uni6ersity School of Medicine, Atlanta, GA 30322, USA Received 3 August 1997; received in revised form 14 November 1997; accepted 14 November 1997

Abstract The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template. The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30°C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Plus-strand RNA viruses; HCV Non-structural protein 5B; RNA-dependent RNA polymerase; Chronic hepatitis C

Abbre6iations: HCV, hepatitis C virus; NS5B, HCV nonstructural protein 5B; RDRP, RNA-dependent RNA polymerase; IPTG, isopropylthiogalactoside; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction. * Corresponding author. 2101 Woodruff Mem. Res. Building, 1639 Pierce Drive, Emory University School of Medicine, Atlanta, GA 30322, USA. Tel.: + 1 404 7275638; fax: + 1 404 7275767; e-mail: [email protected]

1. Introduction Hepatitis C virus (HCV) is the major causative agent for posttransfusion and sporadic non-A, non-B hepatitis (NANBH) infections worldwide (Bradley et al., 1983; Kuo et al., 1989; Choo et al., 1989, 1991), and accounts for approximately 25% of the acute viral hepatitis (Alter, 1995). In the

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USA the prevalence of chronic hepatitis C is estimated to be 1.4% while in South America and Japan the prevalence is approximately 2 and 4%, respectively (Yano et al., 1991; Alter, 1995). In addition, HCV has a high rate of progression to chronic infection (80 – 90%), and eventually produces liver failure or hepatocellular cancer in many patients (Alter, 1995). HCV is a member of the family Fla6i6iridae, which are enveloped viruses containing a singlestranded positive-sense RNA genome (Miller and Purcell, 1990; Choo et al., 1991). Although the 9.5-kb RNA genome of HCV has been studied in detail, little is known about the HCV proteins required for viral replication. Studies using a vaccinia virus expression system have identified HCV proteins produced by the proteolytic cleavage of the HCV polyprotein (Grakoui et al., 1993, Kolykhalov et al., 1994). One of these proteins, the NS5B gene product, has a Mg + + /nucleotide binding site (Gly-Asp-Asp), that is conserved among viral RNA-dependent RNA polymerases (RDRP) (Koonin, 1991). The polymerase of the prototype-plus-stranded RNA virus, poliovirus, provides a framework to consider the mechanism of HCV genome replication. Recombinant poliovirus RDRP (3Dpol), expressed in Escherichia coli, has been useful for both enzymatic characterization and structural studies (Rothstein et al., 1988; Burns et al., 1989; Cho et al., 1993; Pata et al., 1995). Recent studies have identified additional poliovirus proteins, 3AB and 3CD, that participate in viral RNA replication and RDRP – template interactions (Andino et al., 1993; Xiang et al., 1995). In addition, a 36-kDa cellular protein that binds a 5%stem-loop structure of the poliovirus RNA genome appears to play a role in regulating RNA replication in trans (Andino et al., 1993). The poliovirus model emphasizes the role of specific protein–RNA and protein – protein interactions with a RDRP catalytic subunit in regulating viral RNA replication. Because an easily reproducible system to propagate HCV in tissue culture is not available, current efforts to understand HCV replication have focused on preparing recombinant NS5B to approach this problem. One report described the

expression of HCV NS5B in insect cells that synthesized RNA products in a template-primed reaction (Behrens et al., 1996). This study demonstrated that NS5B alone is able to catalyze RDRP activity. In addition, it provided evidence that with heteropolymeric templates NS5B uses a template-primed ‘copy back’ mechanism. The optimal incubation temperature of this enzyme was 22°C and it became relatively inactive after 20 min of incubation. Another recent report suggested that a histidine-tagged NS5B protein produced in E. coli could be denatured, purified, and refolded into an active RDRP (Yuan et al., 1997). However, the activity of this recombinant enzyme was non-linear with time, which suggests a possible problem with its stability. In this report, we describe a non-tagged recombinant HCV NS5B protein produced in E. coli that can be solubilized under non-denaturing conditions, has typical RDRP activity, and has had the aminoterminus of the recombinant protein confirmed by Edman degradation analysis.

2. Expression and preparation of HCV RDRP under non-denaturing conditions The NS5B-region of the prototype HCV (type 1a) was amplified with polymerase chain reaction (PCR) methods. HCV cDNA was obtained from Dan Bradley and Michael Beach of Centers for Disease Control and Prevention, Atlanta (Choo et al., 1991). The PCR product was digested with NheI and BamHI at sites engineered in the primers and directionally cloned into pET-11a (Novagen) to produce a recombinant protein with additional Met-Ala residues added to the NS5B amino-terminal Ser-Met-Ser-Tyr identified by polyprotein processing studies (Studier et al., 1990; Kolykhalov et al., 1994; Hagedorn et al., 1997). The naturally occurring stop codon of HCV type 1a was present at the 3%-end of this construct. The pET-11a-NS5B construct was used to transform E. coli (Oneshot™, Invitrogen). Transformants were analyzed by restriction enzyme mapping of miniprep plasmid DNA. The insert region of the plasmid was sequenced using Sequenase (US Biochemicals) and no changes

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were found that altered the predicted NS5B amino acid sequence. E. coli BL21(DE3) containing the pET-11aNS5B construct produced a protein of approximately 68 kDa under appropriate conditions (Fig. 1). Cells were grown in M9ZB media with antibiotic at 37°C. Isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM when cultures reached an OD600 of 0.6 – 0.8. Expression of the 68-kDa protein reached a maximum 2 h after the addition of IPTG (Fig. 1). Sera of some patients with chronic hepatitis C contained antibodies that reacted with the 68-kDa protein in immunoblots whereas control sera did not (a representative immunoblot is shown in Fig. 2). The samples of sera were provided by Drs Bradley and Beach of the Centers for Disease Control and Prevention (Atlanta). Control studies using parent and uninduced cell lysates indicate that the smaller proteins observed in immunoblots are most likely proteolytic fragments (not shown).

Fig. 1. Expression of HCV NS5B (RDRP) in E. coli. Cells containing pET-11a-NS5B were incubated at 37°C until an OD600 of 0.6 was reached. IPTG was added to a final concentration of 1 mM and samples were collected before and 1, 2 and 3 h after induction. Whole cells were lysed in 1X sample buffer, analyzed by 10% SDS-PAGE, and stained with Coomassie blue. Lane 1 represents molecular mass markers; lane 2, the uninduced control (0 h); lane 3, 1 h; lane 4, 2 h; and lane 5, 3 h after IPTG induction. The location of recombinant NS5B (RDRP) is indicated by an arrow.

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Fig. 2. Immunoblotting of recombinant NS5B (RDRP) with serum from patients with chronic hepatitis C. E. coli expressing NS5B were harvested and lysed by heating in SDS-PAGE sample buffer. Soluble proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with human sera using an Immunetics Miniblotter template (Cambridge, MA). Immunoblots were developed using enhanced chemiluminescent methods (ECL, Amersham). The photograph shows a representative immunoblot where lysates of cells expressing NS5B were probed with normal human serum (lane 1, 1:300 dilution) and serum from a patient with chronic hepatitis C (lanes 2, 1:500; 3, 1:300; and 4, 1:200). The location of recombinant NS5B (RDRP) is indicated by an arrow.

Current studies are determining the incidence of circulating antibodies against NS5B in patients with chronic hepatitis C. The aminoterminal sequence of the recombinant NS5B protein was determined by microsequencing methods to confirm its identity. Cells were lysed in 6 M urea, 50 mM Tris (pH 8.0), 1% NP-40, 0.5 mM EDTA, 100 mM KCl, and 1 mM DTT by sonication, and a freeze–thaw cycle. Lysates were centrifuged at 21 000×g for 30 min. Fractions enriched with the recombinant protein were separated by sodium dodecyl sulfate (SDS)PAGE, and transferred to PVDF membranes (Graves et al., 1993). The recombinant protein

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bound to PVDF membranes (ProBlott, Applied Biosystems) was subjected to ten Edman degradation cycles (Dr Jan Pohl, Microchemical Facility, Emory University). The aminoterminal sequence of the protein was identified as AlaSer-Met-Ser-Tyr-Ser-Trp-Thr-Gly, verifying that the protein induced by IPTG was indeed HCV NS5B. The absence of a terminal Met (less than 1%) in the recombinant protein is compatible with its removal by methionine aminopeptidase during the cotranslational processing of polypeptides having an Ala, Gly, Ser, Cys, Thr, Pro, or Val as the second residue (Ben-Bassat et al., 1987; Arfin et al., 1995). To solubilize NS5B (RDRP) under non-denaturing conditions cells were harvested 2 h after IPTG-induction and lysed on ice for 20 min in 50 mM Tris pH 7.5, 100 mM KCl, 0.5 mM EDTA, 1 mM DTT, 0.1% NP-40, 15% glycerol with 30 mg/ml lysozyme. Samples were sonicated in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml each of aprotinin, leupeptin and pepstatin A, and centrifuged (21 000×g at 4°C for 30 min). Preparations of NS5B were obtained from the supernatant fraction (referred to as lysate) and ion exchange chromatography (see below).

3. Enzymatic activity of recombinant NS5B (RDRP)

3.1. Poly(U) polymerase assay The ability of recombinant NS5B, expressed in E. coli, to catalyze a RDRP reaction was determined with a poly(U) polymerase assay that has been used in studying the poliovirus RDRP (Hey et al., 1986). Samples of lysate were assayed in 50 ml incubations containing 50 mM HEPES pH 8.0; 500 mM each of ATP, CTP, and GTP; [3H]UTP at 11 mM (specific activity: 37 Ci/mol, DuPont NEN); 4 mM DTT, 5 – 30 mM Mg(C2H3O2)2; 60 mM ZnCl2; and 4 mg/ml of actinomycin D. In addition, each incubation contained 1 mg of poly(A)460 – 600 (Pharmacia) as template and 0.5 mg oligo(U)5 – 25 as primer (Hey et al., 1986). The same salt concen-

trations were present in assays comparing different samples or controls. Incubations were done at 30°C for 30–60 min and [3H]poly(U) was quantitated as previously described (Hey et al., 1986). Assays were done under conditions where [3H]poly(U) formation was linear with time for at least 60 min and when the quantity of protein was varied between 2 and 35 mg. The temperature, pH and Mg + + optima were 30°C, 8.0 and 20 mM, respectively. Unfractionated lysates of E. coli that expressed recombinant HCV NS5B were active up to 60 min of incubation at 30oC, whereas control lysates from cells not expressing NS5B produced only background activity in poly(U) polymerase assays. After 30 min of incubation NS5B lysates exhibited 11669 16 compared with 226946 fmol UMP incorporation per mg protein for parent cell lysates (mean9S.E.M.). Following 60 min of incubation NS5B lysates exhibited 30409 84 compared with 416997 fmol UMP incorporation per mg protein for control lysates. Adsorption of NS5B to DEAE Sepharose and stepwise elution with increasing concentrations of salt yielded a preparation with a 4-fold increase in a poly(U) polymerase specific activity. In brief, lysates (500 mg of protein) were diluted to a final concentration of 20 mM Tris pH 8.0, 20 mM KCl, 1 mM DTT, 0.5 mM EDTA, 0.01% Triton X-100 or NP-40, 10% glycerol, plus protease inhibitors (binding buffer) and mixed with 60 ml of DEAE Sepharose (Pharmacia). The resin was poured into a column and washed with five column volumes of binding buffer. Proteins were eluted stepwise with 100, 300, 500 mM, and 1 M NaCl. Proteins eluted from the column were concentrated using Centricon concentrators, diluted with binding buffer containing no NaCl and reconcentrated to achieve a final concentration of 100 mM NaCl and a protein concentration of at least 1 mg/ml. The fraction eluted with 300 mM NaCl exhibited a 4-fold increase in poly(U) polymerase specific activity as compared with starting material. This preparation was used in subsequent studies that examined recombinant NS5B activity.

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3.2. Polymerase assay using globin mRNA as template

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Gly-Asp-Asp RDRP motif, however the carboxyl terminal 144 amino acids were replaced by 20 amino acids encoded in the expression vector.

In order to characterize the products of the HCV RDRP reaction, we utilized a model heteropolymeric RNA (globin mRNA) as a template in incubations containing oligo(U) primers and 10 mCi of [32P]UTP (18 Ci/mmol). The radiolabeled RNA produced in such incubations was approximately the size of the globin mRNA template (Fig. 3, lanes 2 and 3). The partially purified enzyme preparation yielded, as expected, larger quantities of radiolabeled RNA in comparison with the RDRP lysate (Fig. 3, lane 3 versus 2). The reaction was highly primer-dependent under these incubation conditions (Fig. 3, lane 5) and oligo(dT)12 could substitute for oligo(U) but was less efficient (not shown). When either template or enzyme was omitted from incubations, as expected no radiolabeled RNA products were observed. In addition, similar results were observed when [a-32P]UTP was replaced with [a-32P]CTP or [a-32P]ATP (data not shown). The products were destroyed by RNase but not DNase (not shown). Enzyme preparations had essentially no activity (B2% of controls) in the absence of magnesium. In addition, they showed maximal activity at 20 mM Mg + + and the RNA products were approximately the size of the globin mRNA template at all magnesium concentrations tested (data not shown).

4. Expression and activity of a carboxyl terminal deletion mutant of HCV NS5B (RDRP) As a control a carboxyl terminal deletion mutant of NS5B was constructed, expressed and assayed. This construct (NS5B-DC) was prepared by limited digestion of the NS5B expression vector with BamHI, and BglII. After digestion a 7-kb DNA product was isolated by agarose gel electrophoresis and ligated. This resulted in the predicted mutant NS5B gene product that was truncated after Ile-447 of the full-length 591amino-acid-long recombinant protein. The truncated gene still contained the conserved

Fig. 3. Characterization of RNA products synthesized by recombinant HCV NS5B when globin mRNA was used as template. Incubations were as described for the poly(U) polymerase assay except globin mRNA (0.25 mg/incubation) was used as a template, [a-32P]UTP was used instead of [3H]UTP, and each incubation contained 25 mg of protein. After 60 min at 30°C samples were phenol/chloroform extracted and RNA was precipitated with ethanol in the presence of carrier tRNA. RNA products were analyzed by 1.5% agarose/formaldehyde gel electrophoresis (Sambrook et al., 1989). An autoradiogram of a dried gel is shown. The first two lanes represent incubations containing control E. coli lysate (cells not expressing NS5B) (lane 1) and NS5B E. coli lysate (cells expressing NS5B) (lane 2). Lanes 3 – 6 represent RNA products from incubations containing partially purified recombinant NS5B; complete incubation with enzyme, template and primers (lane 3); minus template (lane 4); minus primers (lane 5); and minus enzyme (lane 6). The migration of 1.5 mg of globin mRNA and an RNA ladder, both visualized by ethidium bromide staining, are indicated.

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Fig. 4. Expression of NS5B-DC, a carboxyl terminal deletion mutant of NS5B. Panel a, Coomassie blue stained SDS-PAGE analysis of whole E. coli lysates prepared from IPTG-induced control cells containing the expression vector without an insert (lane 1); cells containing the full-length NS5B construct (lane 2); and cells containing the carboxyl terminal deletion mutant (DC) construct (lane 3). The location of full-length NS5B is labeled A (lane 2) and NS5B-DC is labeled B (lane 3). Panel b, immunoblotting of full-length and NS5B-DC. An immunoblot, using rabbit anti-HCV NS5B, of E. coli expressing full-length (uninduced, lane 1; IPTG-induced, lane 2) and the COOH deletion mutant (uninduced, lane 3; IPTG-induced, lane 4) is shown. The full-length NS5B is labeled A and the deletion mutant is labeled B. Rabbit anti-NS5B was raised as described in detail previously (Hagedorn et al., 1997) and immunoblots were developed following the methods used in Fig. 2. Panel c, enzyme assays of NS5B-DC (COOH deletion mutant) and controls. Lysates of E. coli (7.9 mg each) expressing full-length NS5B (lane 1), NS5B-DC (lane 2) and controls expressing no RDRP (lane 3) were assayed using globin mRNA as a template as described in Fig. 3. An autoradiogram of radiolabeled RNA products separated by agarose/formaldehyde gels is shown.

Expression of NS5B-DC was verified by SDSPAGE and immunoblotting analysis (Fig. 4, panels a and b). Cells that expressed NS5B-DC had no poly(U) polymerase activity above paired controls (cells containing vector without an insert) at different protein concentrations (for example, 5, 9 and 17 mg, data not shown). In addition, in assays using globin mRNA as a template no activity was observed with NS5B-DC (Fig. 4, panel c). The lack of RDRP activity of NS5B-DC was not due

to differences in its ability to be solubilized as compared with full-length NS5B as verified by Western blotting studies (not shown). This provided additional evidence that full-length NS5B was indeed responsible for the activity observed in the polymerase assays. Moreover, the shift in the primary immunoreactive protein to the lower molecular mass form in cells expressing NS5B-DC provided additional evidence that the antiserum was specific.

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Fig. 4. (Continued)

The high degree of dependence on primers that recombinant NS5B (RDRP) showed under different conditions is similar to that observed for poliovirus RDRP (3Dpol) (Rothstein et al., 1988). This is also in agreement with the properties of HCV NS5B when expressed in insect cells based on poly(U) polymerase assays that showed less than 5% of control activity in the absence of primers (Behrens et al., 1996). However, when more structured heteropolymeric RNA templates were presented to recombinant NS5B isolated from insect cells it showed a high degree of template-primed (‘copy back’) activity. Another difference between NS5B expressed in insect cells and E. coli was that their temperature optima were 22 and 30°C, respectively. A consequence of these temperature differences may be the observed relative amounts of primer-dependent RNA polymerase activity due to different conformations of the RNA templates. Differences in the molar ratio of primers and templates may also be a factor in the observation of predominantly template-

primed or primer-dependent RNA synthesis. Since neither NS5B expressed in E. coli nor insect cells has been purified to homogeneity the possibility of other factors affecting enzyme activity cannot be excluded. Further studies of both enzymes, including the use of full-length HCV templates containing the recently identified 3% structural motif, may provide insight into these apparent differences (Tanaka et al., 1995; Kolykhalov et al., 1996). The observation that HCV NS5B (RDRP) expressed in E. coli will replicate globin mRNA in a primer-dependent reaction suggests that additional HCV proteins or host-cell factors are required for the specific interaction between NS5B and HCV RNA templates in vivo. In addition to regulating template specificity, such subunits might regulate other aspects of enzyme activity such as the rate of product formation. Nevertheless, our results indicate that such regulatory subunits are not essential for the catalytic subunit of the HCV RDRP, NS5B, to copy a primed RNA

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template. Several possible subunits are the HCV RNA helicase encoded within the NS3 gene and/ or the approximately 58-kDa NS5A protein (Suzich et al., 1993). In addition to interacting with NS5B, such proteins may interact directly with a structural motif within the 3% region of a negative strand template RNA or act in trans with a 5% structural motif in a resident positive strand. Indeed, 3AB and 3CD represent two such proteins in the poliovirus system (Andino et al., 1993; Xiang et al., 1995). Alternatively, host-cell proteins that bind structural motifs within viral RNA may provide template specificity or regulate polymerase activity. One example is the ribosomeassociated 36-kDa cellular protein that binds a stem-loop structure in the 5% untranslated region of poliovirus RNA, and regulates the synthesis of positive (+ ) strands in trans (Andino et al., 1993). A different example is the cis-acting cellular protein that regulates the polymerase activity of rubella virus. In this case phosphorylation of the host protein (calreticulin) appears essential for protein binding to the viral RNA 3% stem-loop structure (Singh et al., 1994). The well described 5% untranslated region and the recently identified 3% structural motif within the HCV genome are likely targets for such protein-RNA and subsequent protein–protein interactions (Tanaka et al., 1995; Honda et al., 1996; Kolykhalov et al., 1996; Reynolds et al., 1996). The ability to produce enzymatically active HCV NS5B (RDRP) should allow us to experimentally approach the question of what viral proteins or host-cell factors regulate template specificity or relative synthetic rates of plus and minus strands of HCV RNA. The identification of regulatory subunits may also be critical to developing molecular-based therapeutics that selectively inhibit HCV replication in vivo.

Acknowledgements This work was supported by an Elsevier Research Initiative Award from the American Gastroenterology Association Foundation, the Glaxo Institutes of Digestive Health and NIH grant AI41424 (CHH). RHA was the recipient of an

American Liver Foundation Student Research Fellowship and scholarships from the ‘Stichting de Drie Lichten’ and ‘Bekker-La-Bastide Fonds’ of the Netherlands. We thank E. Ehrenfeld and O.C. Richards for their helpful discussions and their gifts of purified poliovirus RDRP and oligo(U). We also thank J.M. Taylor and E.H. Van Beers for carefully reading the manuscript.

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