Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli

Protein Expression and PuriWcation 37 (2004) 144–153 www.elsevier.com/locate/yprep

PuriWcation and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli Luyun Huang,a Elena V. Sineva,a Michele R.S. Hargittai,a Suresh D. Sharma,a Mehul Suthar,a Kevin D. Raney,b and Craig E. Cameron¤ b

a Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

Received 3 March 2004, and in revised form 30 April 2004 Available online 25 June 2004

Abstract We have employed a pET-ubiquitin expression system to produce two his-tagged forms of hepatitis C virus (HCV) non-structural protein 5A (NS5A) in Escherichia coli. One derivative contains the full-length protein extended to include a carboxy-terminal hexahistidine tag; the other derivative contains an amino-terminal hexahistidine tag in place of the 32 amino acid amphipathic helix that mediates membrane association. At least 1 mg of each derivative at a purity of 90% could be produced from a 1-L culture. The puriWed derivatives produced high titer antibody that recognized both p56 and p58 forms of NS5A in Huh-7.5 cells expressing an HCV subgenomic replicon. The NS5A derivatives were eYciently phosphorylated by casein kinase II, leading to at least 5 mol of phosphate incorporated per mole of protein. Interestingly, this level of phosphorylation did not alter the migration of the protein in an SDS–polyacrylamide gel, suggesting that hyperphosphorylation alone is not suYcient to generate the p58 form of NS5A observed in Huh-7 cells. Neither NS5A derivative was capable of inhibiting the eIF2
-phosphorylation activity of the activated form of the double-stranded RNA-activated protein kinase, PKR, suggesting that NS5A phosphorylation may be required for this function of NS5A. However, both unphosphorylated derivatives were shown to interact with NS5B, the HCV RNA-dependent RNA polymerase, in solution by using a novel kinase-protection assay. The availability of puriWed HCV NS5A will permit rigorous biochemical and biophysical characterization of this protein, ultimately providing insight into the function of this protein during HCV genome replication.  2004 Elsevier Inc. All rights reserved. Keywords: Hepatitis c virus; ns5a

Hepatitis C virus (HCV) infection is currently a signiWcant worldwide health problem. Most infected individuals develop chronic hepatitis, with many progressing to hepatocellular carcinoma [1]. HCV non-structural protein 5A (NS5A, amino acids 1973–2419 of the polyprotein) has been implicated in a variety of interactions with viral and host proteins [2,3]. However, a clear replication function for NS5A is not known. As a serine-rich phosphoprotein, NS5A puriWed from yeast and other eukaryotic systems was phosphorylated variably, leading to substantial heterogeneity of the as-

¤

Corresponding author. Fax: 1-814-865-7927. E-mail address: [email protected] (C.E. Cameron).

1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.05.005

isolated protein [4]. In addition, it is clear that kinases co-purify with NS5A isolated from mammalian cells [5,6] or insect cells [4,7], making it diYcult to study NS5A phosphorylation in vitro. A good bacterial expression system for NS5A is desirable because this recombinant protein would be unphosphorylated, given the absence of serine/threonine protein kinases in bacteria. In this report, we describe a method for expressing and purifying his-tagged HCV NS5A protein from the soluble fraction of Escherichia coli. This method permits the isolation of 1 mg of NS5A that is 90% pure from a 1L culture. Antibodies to this protein react with a protein of the expected size in a sub-line of Huh-7 cells stably replicating and translating HCV RNA. As expected, the

L. Huang et al. / Protein Expression and PuriWcation 37 (2004) 144–153

protein is a substrate for casein kinase II (CKII) [7] and is capable of forming a complex in solution with NS5B [8]. This advance sets the stage for rigorous biochemical and biophysical studies of HCV NS5A.

Materials and methods Materials All DNA oligonucleotides for PCRs were from Integrated DNA Technologies (Coralville, IA). Sequences of the oligos used for PCR are listed in Table 1. All restriction enzymes, T4 polynucleotide kinase, and Deep Vent DNA polymerase were from New England Biolabs, T4 DNA ligase and NZCYM media were from Invitrogen. [-32P]ATP (17000 Ci/mmol) was from ICN. Ni–NTA– agarose resin and TransMessenger RNA transfection reagents were from Qiagen. DE52-cellulose resin was from Whatman. Q-Sepharose resin was from Amersham. All other reagents were of the highest grade available from Sigma, Fisher or VWR.

145

Construction of NS5A expression plasmids ModiWed NS5A-coding sequence was constructed by using PCR and the plasmid pHCVbart.rep1b/Ava-II [9] as template. Oligos 1 and 2 (Table 1) were used to construct NS5A-His. The PCR product was cloned into SacII and HindIII sites in the pET-UbCHis vector (Fig. 1B). Oligos 3 and 4 (Table 1) were used to construct His--NS5A (amino acids 2005–2419 of the polyprotein). The PCR product was cloned into NcoI and HindIII sites in the pET-UbNHis vector (Fig. 1A). Oligos 1 and 4 were used to construct NS5A. The PCR product was cloned into SacII and HindIII sites in the pETUbNHis vector. Native NS5A expressed from this construct in E. coli was used in the Western blot experiment. Expression and puriWcation of NS5A-His The two NS5A derivatives expressed from the pETUb-based plasmids are fused to yeast ubiquitin at the amino terminus. Overexpression of protein in this system is performed in the BL21(DE3)pCG1 strain of

Table 1 Oligonucleotides used in this study Oligo No.

Oligo name

Sequence

1 2

HCV-5A-SacII-for HCV-5A-6CHis-HindIII-rev

3 4 5 6

HCV-del32-5A-NcoI-NHis-for HCV-5A-HindIII-rev PKR-for-BamHI-Nhis PKR-rev-XhoI-Nhis

5⬘-GCG GGT ACC CCG CGG TGG ATC CGG CTC GTG GCT AA-3⬘ 5⬘-GCG GGT ACC AAG CTT CTA TTA ATG GTG GTG ATG GTG GTG ACC AGA GGA TCC GCA GCA GAC GAC GTC CTC ACT-3⬘ 5⬘-GCG GGT ACC CCA TGG ATC CTC TGG TGG AGT CCC CTT CTT C-3⬘ 5⬘-GCG GGT ACC AAG CTT CTA TTA GCA GCA GAC GAC GTC CTC ACT-3⬘ 5⬘-GCG CGA TCG GGA TCC TCT GGT GCT GGT GAT CTT TCA-3⬘ 5⬘-GCG CAG CTG CTC GAC TTA CTA ACA TGT GTG TCG TTC A-3⬘

Fig. 1. pET-ubiquitin expression vectors. This system was developed to permit production of viral proteins that are generated by proteolytic cleavage of a polyprotein [10]. These vectors have been modiWed to permit production of proteins in this system that contain a hexahistidine tag. (A) pETUbNHis. This vector will direct expression of a protein with an amino-terminal hexahistidine tag. The tag is separated from the protein of interest by a Gly-Ser-Ser-Gly linker. Cloning can employ either the NcoI or BamHI sites. Italicized nucleotides are not in the vector sequence and must be included in the oligonucleotide primer. (B) pET-UbCHis. This vector will direct expression of a protein with a carboxy-terminal hexahistidine tag. The tag is separated from the protein of interest by a Gly-Ser-Ser-Gly linker. Cloning can only employ either the SacII or BamHI site. Italicized nucleotides are not in the vector sequence and must be included in the oligonucleotide primer.

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E. coli; this strain is derived from BL21(DE3) cells and carries the pCG1 plasmid, which constitutively expresses a yeast ubiquitin protease that processes the ubiquitin fusion protein to produce mature protein [10]. BL21(DE3)pCG1 cells containing the NS5A-His expression plasmid were grown overnight in 25 mL NZCYM supplemented with 25 g/mL kanamycin (K25), 20 g/mL chloramphenicol (C20), and 0.1% dextrose (D) at 37 °C. The overnight culture was used to inoculate 1 L of K25, C20, D-supplemented media. The cells were grown at 37 °C to an OD600 of 0.8–1.0 before isopropyl-D-thiogalactopyranoside (IPTG) was added to a Wnal concentration of 0.5 mM. The cells were grown for an additional 4 h at 20 °C. The cells were harvested by centrifugation in a Sorvall GS-3 rotor at 7000 rpm for 10 min. The cell pellet was suspended in 25 mL lysis buVer (100 mM Tris, pH 8.0, 200 mM NaCl, and 10 mM 2mercaptoethanol (BME)), supplemented with protease inhibitors pepstatin A (7.5 g/mL) and leupeptin (7.5 g/ mL). Using protease inhibitor cocktail tablets (Roche) substantially enhanced the stability of NS5A. The cell suspension was lysed by passing through a French press (SIM-AMINCO) twice at 16,000 psi. PMSF was added to a Wnal concentration of 1 mM and NP-40 to 0.5%. The extract was clariWed by ultracentrifugation in a Beckman Ti-60 rotor for 30 min at 30,000 rpm (100,000g) at 4 °C. The supernatant was loaded onto a 40-mL DE52-cellulose column connected to a 0.75-mL Ni–NTA–agarose column at a Xow rate of 0.5 mL/min. The columns were washed with 1 column volume of lysis buVer. The Ni– NTA–agarose column was washed further with 10 mL of the lysis buVer, followed by 5 mL lysis buVer containing 5 mM imidazole. The puriWed NS5A-His was eluted by using lysis buVer containing 0.5 M imidazole. Fractions (0.75 mL) were collected and analyzed for purity by SDS–PAGE. The fractions containing the majority of the protein were pooled and dialyzed against 50 mM Hepes, pH 7.5, 1 mM DTT overnight. Glycerol was added to a Wnal concentration of 20% after dialysis. The sample was aliquoted and stored at ¡80 °C. Protein samples from each puriWcation step were monitored by both Bradford assay and SDS–PAGE analysis. PuriWcation of His--NS5A The puriWcation procedure for the full-length NS5AHis was modiWed for His--NS5A. The clariWed lysate was loaded onto a 40-mL DE52-cellulose and a 1-mL QSepharose columns connected in tandem, both pre-equilibrated with 5A lysis buVer containing 0.1% NP-40. After washing the columns with 40-mL lysis buVer, the DEAE column was removed. His--NS5A was eluted from the Q column directly onto a 0.75-mL Ni–NTA– agarose column by using 15 mL lysis buVer containing 1 M NaCl. The Ni–NTA–agarose column was then washed and eluted as described for NS5A-His.

Cell culture and transfection of HCV subgenomic replicon RNA Huh-7.5 [11] cells (1.6 £ 106) were transfected with 2 g of the in vitro transcribed replicon RNA using TransMessenger transfection system (Qiagen). Cells were harvested 12–14 h post transfection and boiled in one equivalent volume of 2£ SDS–PAGE sample buVer (225 mM Tris, pH 6.8, 5% SDS, 50% glycerol, 5% BME, and 0.05% bromophenol blue) for Western blot analysis. Western blot analysis Rabbit polyclonal antibody for NS5A was produced at Covance Research Products (Denver, PA) using the puriWed recombinant protein NS5A-His as the antigen. The antibody was aYnity puriWed by incubating antisera with NS5A blotted nitrocellulose membrane strips followed by elution with 0.2 M glycine, 1 mM EGTA buVer, pH 2.5 [12]. Mouse monoclonal antibody for NS5A was purchased from Santa Cruz Biotechnology. For the Western blot detection of the NS5A expressed in the subgenomic replicon system with our polyclonal NS5A antibody, Huh-7.5 cells expressing the wild-type HCV replicon (0.5 £ 106 cells per lane) and Huh-7.5 cells were loaded onto a SDS–polyacrylamide (8%) gel. After electrophoresis, proteins were transferred to nitrocellulose membrane with a Genie Blot apparatus (IDEA ScientiWc Company). The membrane was probed with 1:1000-dilution of the puriWed anti-NS5A polyclonal antibody, and 1:3000-dilution of goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology), or 1:1000-dilution of the mouse antiNS5A monoclonal antibody and 1:2000-dilution of the rabbit anti-mouse IgG-HRP (Santa Cruz Biotechnology). The NS5A bands were detected by ECL Western blot detection reagents (Amersham) and exposed to a BioMax MR X-ray Wlm (Kodak). Expression and puriWcation of PKR and eIF2
The human PKR gene was ampliWed by PCR from a pUC19 clone (a gift from Dr. Ron Wek, Indiana University) using oligos 5 and 6 (Table 1) and cloned into pET-UbNHis vector (Fig. 1A). The resulting plasmid, pET-UbNHis-PKR, was transformed into BL21(DE3)pCG1 cells. Cells were grown at 37 °C to an OD600 of 1.2 and induced at 25 °C for 15 h. His-PKR was puriWed as described for NS5A-His. The bacterial expression plasmid, pGST-SUI2 (a gift from Dr. Tom Denver, NIH), contains the yeast eIF2
gene [13]. This plasmid was transformed to BL21(DE3) cells. The cells were grown and induced as described above for PKR; however, the induction time was 3.5 h. The cell pellet was suspended in PBS buVer supplemented with protease inhibitors and lysed by using a French press. PMSF and NP-40 were added to Wnal concentrations of

L. Huang et al. / Protein Expression and PuriWcation 37 (2004) 144–153

2 mM and 0.5%, respectively. The cell lysate was clariWed by ultracentrifugation at 100,000g for 30 min. The clariWed lysate was loaded on a glutathione–Sepharose column pre-equilibrated with the PBS buVer. The column was washed with 20 volumes of PBS buVer, and then the protein was eluted with 100 mM Tris buVer, pH 8.0, containing 10 mM glutathione. Glycerol was added to 40% before the protein was aliquoted and stored at ¡80 °C. Casein kinase II phosphorylation assay Kinase reactions (20 L) were performed in 50 mM Hepes, pH 7.5, 100 mM KCl, 10 mM BME, and 10 mM MgCl2. NS5A protein was added to a Wnal concentration of 2 M. 100 M cold ATP was spiked with 1 M [32 P]ATP. The reaction was initiated by the addition of 500 U of CKII (New England Biolabs). After a 30-min incubation at 30 °C, reactions were quenched by addition of 2£ SDS–PAGE sample buVer. All the samples were incubated at 65 °C for 3 min prior to SDS–PAGE. The electrophoresis was stopped when the free nucleotide in the samples migrated near to the bottom of the gel. The gel was subsequently analyzed by phosphorimaging. The levels of phosphorylation were normalized to the total radioactivity loaded on each lane. Kinetics data were Wt to the equation for a single exponential. For gel mobility comparison experiment, kinase reaction was performed without the addition of [-32P]ATP. The reaction was quenched as described above prior to SDS–PAGE analysis. The unphosphorylated and phosphorylated NS5A were visualized by Coomassie blue stainning. PKR kinase-protection assay The PKR kinase-protection assay was performed in reaction buVer containing 50 mM Hepes, pH 7.5, 100 mM KCl, 10 mM BME, 2 mM MgCl2, and 2 mM MnCl2. NS5B was puriWed as previously described [14]. For assays containing both eIF2
and NS5B, the Wnal concentrations of these proteins were 0.25 and 0.75 M, respectively. For assays containing only NS5B, the Wnal concentration of NS5B was 1 M. NS5A protein was titrated into reactions from 0 up to 4 M (Wnal). The 20 L reactions also contained 100 M cold ATP spiked with [-32P]ATP. The reaction mixtures were incubated at 30 °C for 5 min allowing NS5A and kinase substrate(s) to bind, and then the reactions were initiated by the addition of 5 U of PKR. One unit is deWned as the amount of PKR required to catalyze the transfer of 1 pmol of phosphate to eIF2
(1 M), in 20 min at 30 °C in a total reaction volume of 20 L. After 20 min incubation at 30 °C, the reactions were stopped by the addition of one equivalent volume of 2£ SDS–PAGE sample buVer and incubated at 65 °C for 3 min. The samples were analyzed by SDS–PAGE followed by phosphorimaging. Kinetics data were Wt to the equation for a single exponential.

147

Results Expression and puriWcation of HCV NS5A containing a carboxy-terminal hexahistidine tag Because our laboratory has successfully expressed and puriWed several viral proteins using a pET-ubiquitin expression system [10], we employed this system to produce NS5A extended to encode a hexahistidine tag on its carboxyl terminus. Upon induction by addition of IPTG, the ubiquitin–NS5A-His fusion protein was produced and processed co- and/or post-translationally by a ubiquitin protease that was constitutively expressed in the BL21(DE3) cells from a second plasmid, thereby releasing the free his-tagged NS5A protein (NS5A-His). To obtain soluble protein, the cells were induced at 20 °C. As shown in Fig. 2A, NS5A-His was induced 2 h after the addition of IPTG at 20 °C, with maximal accumulation observed after 4 h of induction. After lysis of the induced cells, NP-40 was added to a Wnal concentration of 0.5%. Throughout the subsequent puriWcation procedure, 0.5% NP-40 was required in the puriWcation buVers to maintain solubility of the NS5A-His protein. Nucleic acids apparently can protect NS5A-His from proteolytic degradation. NS5A-His in the clariWed lysate was quite stable; however, NS5A-His in the DEAE passthrough was rapidly degraded. Therefore, it was necessary to connect a Ni–NTA column directly to the DEAE column to reduce the exposure time of NS5A-His to the proteases and thus decrease the amount of degradation. The Ni–NTA column was always washed with buVers containing 5 mM imidazole; however, impurities were never detectable when these fractions were evaluated by SDS–PAGE. After elution with the buVer containing 500 mM imidazole, NS5A-His protein was normally concentrated in the second fraction (Fig. 2B). From a 1L of cell culture, we were able to obtain approximately 1 mg of the puriWed protein (Table 2). The purity of the protein was approximately 85%. Expression and puriWcation of HCV NS5A containing an amino-terminal deletion and hexahistidine tag The full-length NS5A protein requires a high concentration of detergent for solubility. It was reported recently that the amino-terminal 30 amino acids of NS5A form an
-helical domain that anchors NS5A to the ER membrane [15]. This observation suggests that deletion of the membrane association domain may improve the solubility of the protein and the truncated protein may behave better in biochemical assays. To test this hypothesis, the amino-terminal 32 amino acids were deleted and the hexahistidine tag was moved from the carboxyl terminus to the amino terminus. This derivative will be referred to as His--NS5A. One drawback of this design, however, is that the amino-terminally

148

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Fig. 2. Expression and puriWcation of NS5A-His. (A) SDS–polyacrylamide (10%) gel of bacterial cell lysates before IPTG induction and after 2 and 4 h of induction. The induced NS5A migrates between the 45 and 66 kDa markers. (B) Coomassie-stained SDS–polyacrylamide (10%) gel of the samples from each puriWcation step. Lane 1, molecular weight markers; lane 2, clariWed lysate; lane 3, passthrough from the Ni–NTA–agarose column; lane 4, 5 mM imidazole buVer wash; lane 5, the second fraction from the Ni–NTA–agarose column after addition of 500 mM imidazole; and lane 6, the third fraction from the Ni–NTA–agarose column after addition of 500 mM imidazole. Twenty microliter of samples from each fraction was boiled with one equivalent of 2£ SDS sample buVer, and 10 L was loaded on the gel. Approximately 7.5 and 1.5 g of total protein was loaded in lanes 5 and 6, respectively. Table 2 PuriWcation of NS5A-His by DEAE–Sepharose and Ni–NTA chromatography

ClariWed lysate DEAE–Ni–NTA

Total protein (mg)

NS5A-His (mg)

Purity (%)

Yield (%)

250 1.5

6a 1.3

2 85

100 21

a NS5A-His was quantitated by densitometric analysis of a Coomassie blue-stained gel.

tagged protein permits isolation of products shorter than full-length that originate from abortive translation. To remove these NS5A fragments, the puriWcation procedure was modiWed. The pass-through fraction from the DEAE column was loaded onto a Q-Sepharose column (Fig. 3); only the full-length His--NS5A binds to this column (data not shown). Interestingly, NS5A-His did not bind to the Q column under the same condition. As expected, His--NS5A required less detergent (0.1% NP-40) for solubility. From a 1-L of cell culture, we were also able to obtain approximately 1 mg of the puriWed protein (Table 3). The purity of the protein was approximately 90%. A polyclonal antibody raised against puriWed NS5A recognizes p56 and p58 forms of NS5A in Huh-7.5 cells expressing an HCV subgenomic replicon The rabbit polyclonal antibody raised against our puriWed NS5A-His was used to probe extracts prepared from Huh-7.5 cells expressing an HCV subgenomic replicon. As a positive control, the same cell extracts were

Fig. 3. PuriWcation of His--NS5A. The samples from each puriWcation step were analyzed on a Coomassie-stained SDS–polyacrylamide (10%) gel. Lane 1, molecular weight marker; lane 2, uninduced cells; lane 3, cells after 4 h of induction; lane 4, cell lysate; lane 5, clariWed lysate; lane 6, passthrough from the Q column; lane 7, passthrough from the Ni–NTA–agarose column; lane 8, 5 mM imidazole buVer wash; lane 9, the second fraction from the Ni–NTA–agarose column after addition of 500 mM imidazole; and lane 10, the third fraction from the Ni–NTA–agarose column after addition of 500 mM imidazole. Twenty microliter of samples from each fraction was boiled with one equivalent of 2£ SDS sample buVer, and 10 L was loaded on the gel. Approximately 5 and 1 g of total protein was loaded in lanes 8 and 9, respectively.

probed with a commercially available mouse monoclonal antibody that recognizes HCV NS5A. As shown in Fig. 4A, our polyclonal antibody can recognize viral

L. Huang et al. / Protein Expression and PuriWcation 37 (2004) 144–153 Table 3 PuriWcation of His--NS5A by DEAE–Sepharose, Q-Sepharose, and Ni–NTA chromatography

ClariWed lysate DEAE-Q–Ni–NTA

Total protein (mg)

NS5A-His (mg)

Purity (%)

Yield (%)

220 1.1

6a 1.0

3 90

100 16

a

His--NS5A was quantitated by densitometric analysis of a Coomassie blue-stained gel.

NS5A with high speciWcity. Both p56 and p58 forms of the protein were detected. Because the polyclonal antibody recognizes epitopes in diVerent regions of NS5A, it has the advantage over the monoclonal antibody in terms of detecting various cleavage or phosphorylation products of NS5A. The crude polyclonal antibody can detect 50 ng of the puriWed NS5A at a 1:200,000 dilution without cross-reaction with E. coli proteins (data not shown). Therefore, when used in biochemical assays, this antibody does not require further puriWcation. The unphosphorylated NS5A-His appeared to migrate between 45 and 66 kDa on Coomassie-stained gels. To determine the gel mobility of NS5A-His and NS5A more precisely, another Western blot analysis was performed for E. coli expressed NS5A, the puriWed NS5A-His, and NS5A expressed in Huh-7.5 cells (Fig. 4B). NS5A co-migrated with the p56 band in the replicon sample, while NS5A-His migrated as p58. CKII can phosphorylate both NS5A derivatives in vitro We further conWrmed the identity of the puriWed NS5A by performing a phosphorylation assay with CKII. As reported by Choe’s group using GST-NS5A

149

[7], our puriWed NS5A-His can also be phosphorylated by CKII (Fig. 5A). When CKII was not added to the reaction, phosphorylation was not observed. This experiment demonstrated that NS5A is not capable of autophosphorylation. The in vitro CKII assays were performed under conditions that permitted the stoichiometry of phosphorylation to be determined. The kinetics of 2 M His--NS5A phosphorylation (Fig. 5B) indicated that maximum phosphorylation was achieved after a 10-min incubation. Quantitative analysis of the phosphorylation indicated that at least 5 mol of phosphate were added per mole of His--NS5A (Fig. 5C); however, this level of phosphorylation did not change the mobility of the protein in SDS–polyacrylamide gels (Fig. 5D). Addition of phosphates alone does not explain the origin of the p58 form of NS5A observed in mammalian cells. Phosphorylation of eIF2
by PKR is not inhibited by His--NS5A Because studies performed in other laboratories suggested that NS5A may be involved in the regulation of PKR activity and confer interferon resistance through its interaction with PKR [16–18], we expressed and puriWed PKR from E. coli and tested its activity with eIF2
, a natural substrate of PKR, and HCV non-structural proteins. The amino-terminal his-tagged PKR puriWed from E. coli was already activated, or at least partially activated, because autophosphorylation can be seen when incubated with [-32P]ATP in the absence of RNA (Fig. 6A). SUI2, the yeast eIF2
protein, was expressed as a GST fusion and puriWed easily by glutathione aYnity chromatography [13].

Fig. 4. (A) Antibody raised against recombinant NS5A detects both p56 and p58 forms of NS5A in replicon-expressing cells. Western blot analysis of NS5A expressed from an HCV subgenomic replicon in Huh-7.5 cells. Lysates prepared from Huh-7.5 cells containing the replicon (replicon cells) were probed by using a rabbit polyclonal antibody produced using puriWed NS5A-His as antigen (lane 2) or by using a commercially available mouse monoclonal antibody to NS5A (lane 4). The parental Huh-7.5 cells were also probed as a negative control in both experiments (lanes 1 and 3). (B) Unphosphorylated NS5A migrates as p56. Western blot analysis of recombinant NS5A-His (lane 1), bacterial extract containing unmodiWed NS5A (lane 2), forms of NS5A observed in replicon-expressing Huh-7.5 cells (lane 3), and the parental Huh-7.5 cells (lane 4).

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Fig. 5. PuriWed NS5A-His is phosphorylated by CKII. (A) NS5A-His (1 M) was incubated with [-32P]ATP in the presence and absence of CKII (100 U) for 30 min and quenched by addition of 2£ SDS–PAGE sample buVer. Phosphorylation of NS5A was determined by phosphorimaging of quenched reaction samples run on an SDS–polyacrylamide (8%) gel. (B) Kinetics of His--NS5A phosphorylation by CKII. (C) Quantitation of the data shown in (B). (D) Complete phosphorylation of His--NS5A does not alter its mobility in an SDS–polyacrylamide (10%) gel. His--NS5A (2 M) and ATP (100 M) were mixed with CKII (500 U) for 30 min. Reaction mixtures were evaluated by SDS–PAGE and visualized by Coomassie blue staining.

Our results showed that the E. coli expressed PKR does phosphorylate eIF2
, but it does not phosphorylate either derivative of NS5A (Fig. 6A and data not shown). When we titrated His--NS5A into the phosphorylation reaction, neither PKR-catalyzed autophosphorylation nor phosphorylation of eIF2
was aVected (Fig. 6). A kinase-protection interaction

assay

for

the

NS5A–NS5B

During our evaluation of non-structural proteins as substrates for PKR, we noted that NS5B was phosphor-

ylated quite eYciently by PKR (Fig. 7A). Surprisingly, PKR-catalyzed phosphorylation of NS5B was inhibited by the addition of NS5A-His (Figs. 7A and B). This experiment was performed in the presence of eIF2
, a natural substrate for PKR. Again, eIF2
phosphorylation was not inhibited by NS5A-His. Together, these data suggest that NS5A-His does not inhibit the kinase activity of PKR directly. The observed protection of NS5B from phosphorylation likely results from the interaction between NS5A-His and NS5B (Fig. 8). Consistent with this interpretation was the Wnding that, while PKR was capable of phosphorylating NS5B from the

Fig. 6. His--NS5A does not inhibit PKR activity. (A) eIF2
(1 M) was incubated with PKR (5 U) in the presence of an increasing amount of His-NS5A (0–4 M) for 20 min. Phosphorylation was determined by phosphorimaging of quenched reaction samples run on an SDS–polyacrylamide (8%) gel. (B) Quantitation of the data shown in (A). The percent of eIF2
(䊉) or PKR (䊏) phosphorylated as a function of NS5A-His concentration is plotted.

L. Huang et al. / Protein Expression and PuriWcation 37 (2004) 144–153

151

Fig. 7. NS5A inhibits phosphorylation of NS5B by PKR. (A) EVect of NS5A-His on PKR-catalyzed phosphorylation of eIF2
and NS5B. NS5AHis (0–4 M)was employed. The positions of the phosphorylated eIF2
and NS5B and the autophosphorylated PKR are indicated. (B) Quantitation of the data shown in (A). The percent phosphorylations relative to no-NS5A control of each phosphorylation event, NS5B (䊉), eIF2
(䊊), and PKR (䉱) are plotted against the concentration of NS5A-His. (C) His--NS5A also prevents NS5B phosphorylation. Symbols are as described in B. (D) Kinetics of NS5B phosphorylation by PKR. NS5B was present at a concentration of 1 M in this experiment. The amount of the phosphorylated product is plotted versus time.

as the kinetics of the phosphorylation of 1 M NS5B by 5 U of PKR (Fig. 7D) showed that the 20-min incubation time falls in the linear range.

Discussion

Fig. 8. Model for the kinase-protection assay employed to evaluate the NS5A–NS5B interaction. PKR can phosphorylate NS5B in the absence of NS5A. Formation of the NS5A–NS5B complex protects NS5B from phosphorylation either by masking the site of interaction or providing a steric block, limiting access of PKR to the susceptible site(s) on NS5B. The ability of NS5A to completely inhibit NS5B phosphorylation suggests formation of an extraordinarily stable complex.

related pestivirus, bovine viral diarrhea virus, HCV NS5A-His was incapable of inhibiting this reaction (data not shown). The same experiment was repeated with His--NS5A. A stoichiometry of 1:1 was obtained for His--NNS5A–NS5B interaction (Fig. 7C). The kinase-protection assays were performed under optimized conditions

Production of recombinant HCV NS5A has been diYcult. By using a pET-ubiquitin expression system, we were able to express a large amount of soluble NS5A protein in E. coli, permitting the development of an eYcient puriWcation procedure for this protein. In addition, this system permitted the production of a derivative with enhanced solubility relative to wild-type NS5A. Because little is known about the biochemical properties of NS5A, adding a small his-tag to NS5A to facilitate eYcient puriWcation was a reasonable Wrst strategy. However, this puriWed protein will be useful for developing a strategy to isolate the unmodiWed protein. NS5A has a calculated molecular mass of 49 kDa. Interestingly, NS5A-His migrates as a 56 kDa protein on the SDS–polyacrylamide gel. The gel mobility of this unphosphorylated protein is the same as the phosphorylated p56 form of NS5A detected in the cell culture system [19,20]. The aberrant mobility of NS5A may result from the proline-rich nature of this protein. The authenticity of the puriWed NS5A-His was supported by the Wnding that a polyclonal antibody produced by using this recombinant protein as antigen was able to detect

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NS5A in Huh-7.5 cells expressing the subgenomic replicon with high speciWcity and avidity. We also tested the capacity of this protein to function as a substrate for CKII [7]. We found that CKII eYciently phosphorylated NS5A, incorporating at least 5 mol of phosphate per molecule. Remarkably, this level of phosphorylation had no impact on the gel mobility of NS5A. This Wnding suggests that hyperphosphorylation alone is insuYcient to produce the p58 form of NS5A. It is possible that production of p58 requires phosphorylation by another kinase(s) at sites other than those used by CKII. Alternatively, p58 may be the product of some other post-translational modiWcation that requires NS5A phosphorylation. The capacity to produce unphosphorylated NS5A will permit interrogation of the mechanism of p58 formation and evaluation of the role of this form of NS5A in HCV multiplication. During our investigation of NS5A–PKR interaction, we made the observation that PKR was capable of phosphorylating HCV NS5B, the viral RNA-dependent RNA polymerase. Interestingly, the capacity of PKR to phosphorylate NS5B was diminished by NS5A. The observed eVect of NS5A was due to speciWc “protection” of NS5B rather than inhibition of PKR, as the capacity of PKR to phosphorylate eIF2
, the natural substrate for PKR, was not aVected when present in the same reaction. While it is impossible to attribute any biological signiWcance to PKR phosphorylation of NS5B at this time, the kinase-protection assay (Fig. 8) provides a convenient means to interrogate NS5A–NS5B interaction in solution. Previous functional studies of the NS5A–NS5B interaction suggested that formation of this complex inhibited polymerase activity [8]. It is possible that phosphorylation of NS5B is required to disrupt the NS5A– NS5B interaction to permit NS5B to function after replicase assembly. It is intriguing to speculate that NS5A may have nucleic-acid-binding activity. During development of the puriWcation procedure, it became clear that the presence of nucleic acid antagonized the binding of NS5A-His to Ni–NTA agarose. In addition, most attempts to remove the nucleic acid by using precipitation approaches resulted in co-precipitation of NS5A. Finally, removal of nucleic acid from extracts containing NS5A increased the susceptibility of NS5A to degradation by cellular proteases. While the argument made above rests on interpretation of biochemical observations, many biological observations also point to this possibility. For example, the amino-terminal domain of the Xavivirus NS5 protein has an RNA 2⬘-O-methyltransferase activity and is therefore an RNA-binding protein [21,22]. Deletion of the aminoterminal amphipathic helix of HCV NS5A permits the protein to enter the nucleus and activate transcription [23,24]. The NS5B polymerase lacks template speciWcity [25–27]. SpeciWc RNA binding by NS5A could confer

template speciWcity to NS5B given the existence of a very stable NS5A–NS5B interaction. The methods described herein to produce milligram quantities of high purity HCV NS5A permit all of these possibilities to be tested.

Acknowledgments This study was supported, in part, by a Grant (AI66919) from NIAID, NIH to KDR, and CEC. CEC thanks Mr. Louis A. Martarano for his generous support of research in the Eberly College of Science.

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