RNA-dependent RNA Polymerase Activity of the Soluble Recombinant Hepatitis C Virus NS5B Protein Truncated at the C-terminal Region*

Tatsuya YamashitaDagger §, Shuichi Kaneko§, Yukihiro ShirotaDagger §, Weiping QinDagger , Takahiro NomuraDagger , Kenichi Kobayashi§, and Seishi MurakamiDagger

From the Dagger  Department of Molecular Oncology, Cancer Research Institute and the § 1st Department of Internal Medicine, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Hepatitis C virus (HCV)1 is a positive single-strand RNA virus that has been shown to be the major causative agent of transfusion-associated hepatitis (1, 2). From the structural similarities, it had been proposed that HCV was related to the flaviviruses and pestiviruses, but later it was classified as a separate genus in the Flaviviridae family because of its distinctive characteristics (3-5). The viral genome encodes a polyprotein precursor of about 3000 amino acids (aa), which is temporarily processed by a combination of host and viral proteases, resulting in at least 10 distinct products as follows: NH3-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (6-8). The HCV polyprotein is first cleaved by a host signal peptidase generating the structural proteins, C/E1, E1/E2, E2/p7, and p7/NS2 (9-11). The NS2/NS3 site is then cut by an HCV-encoded metalloprotease, NS2, and the remaining sites are processed by a virus-encoded serine protease, NS3, with or without NS4A as a cofactor (12-19). NS3 has protease, NTPase, and RNA helicase activities (20-22), whereas NS5A may be phosphorylated and act as a putative cofactor of NS5B (23, 24). Since HCV NS5B contains the "GDD" sequence motif, which is highly conserved among all RdRPs characterized to date (25), it has been predicted that the NS5B protein encodes an RdRP, which is the central catalytic enzyme of the HCV replicase. RdRP activity of the recombinant NS5B purified from transfected insect cells was recently reported (26, 27).

Since persistent infection of HCV is related to chronic hepatitis and eventually to hepatocarcinogenesis (28), HCV replication is one of the targets to eradicate the HCV reproduction and to prevent hepatocellular carcinoma. HCV is believed to follow a similar replication strategy to other positive-stranded RNA viruses (29-33). However, the molecular mechanism of HCV replication remains elusive mainly because the cell culture systems for HCV reproduction are far too insensitive to dissect the molecular events of viral replication (34-36). Under these limitations, we undertook an alternative approach to characterize the properties of NS5B, an RdRP which is the putative central catalytic enzyme of HCV RNA synthesis (1).

Here we present the purification method and properties of a soluble bacterial recombinant HCV NS5B protein which retains activity for RNA-dependent RNA synthesis in vitro. The purified bacterial NS5B shares several properties with the protein expressed in insect cells, but it has no terminal transferase activity. In addition, we found that the C-terminal 21 amino acid residues of NS5B harbors a putative anchoring domain that affects its subcellular localization in mammalian cells, although the region is dispensable for the RdRP activity.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Plasmids-- A plasmid-derived from pGENK1 (37, 38), pGENKS, was used to express the recombinant HCV NS5B in Escherichia coli. It encodes the consensus kination site for protein kinase A, the cleavage site for thrombin, and additional multiple cloning sites (EcoRI, SacI, KpnI, XmaI, SalI, and BamHI) (Fig. 1A). HCV JK1 cDNA (39) harboring NS5B was subcloned by PCR using the following the set of primers, NS5BFor and NS5BRev, which have an artificial initiation codon, and SacI and SalI sites (Table I, all primer sequences hereafter are shown in the table). The truncated mutant of NS5B, NS5Bt, which lacks the 21 amino acid residues at the C-terminal (aa 2990-3010), was subcloned by PCR with NS5BFor and NS5BtRev. Substitution mutants of NS5Bt-m1 to -m3 were constructed by PCR mutagenesis with overlap extension using the designed mutagenizing primers, NS5BFor and NS5BtRev (38). The resultant NS5Bt-m1 to -m3 proteins have the following substitution mutations, GDD to VDD (aa 2736-2738), YRHRAR to AAAAAA (aa 2919-2924), and CGYRRCR to AAAAAAA (aa 2693-2699), respectively. The substituted mutant at the C-terminal of NS5B, NS5B-m4, was constructed by PCR with NS5BFor and NS5Bm4Rev, which have an artificial BamHI site. NS5B-m4 has two amino acid substitutions, Leu and Val, both to Pro at residues 2998 and 3001, respectively (Fig. 1B).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Sequence of the primers used in the present study

All the mammalian expression vectors were derived from pSG5UTPL (37, 40). The green fluorescent protein (GFP) cDNA was prepared by PCR using phGFP-S65T (CLONTECH) as a template with the primer set, GFPNotFor which has the artificial EcoRV and NotI sites, and GFPEcoRev which has an artificial EcoRI site. The DNA fragment with an artificial EcoRV site and BamHI sites was inserted into the pSG5UTPL blunt and BamHI vector, whose EcoRI site was blunted using Klenow fragment before BamHI digestion (pGFP). By using this pGFP vector, another mammalian expression vector, pNKFLAG, was constructed. The sequence encoding the FLAG-tag epitope sequence in the preferable context for translation initiation was generated from pFLAGHis/p53 (gift from R. Roeder). The fragment generated by PCR with the primer set, NKFLAGFor which has the artificial NotI site, the consensus translation initiation site, and p53Rev which has an artificial BamHI site, was inserted into the pGFP NotI and BamHI vector, generating the pNKFLAG/p53. Plasmid pNKFLAG was constructed to replace the p53 insert with the multicloning sites as described above using EcoRI and BamHI sites. The various kinds of HCV NS5B cDNA were inserted into the EcoRI and BamHI sites of pSG5UTPL, pNKFLAG, and pGFP.

Another plasmid series, pGEM3zf(+)/NS5BBg, pGEM3zf(+)/poly(U), and pGEM3zf(+)/3'X were used for in vitro transcription to prepare the RNA templates. These were constructed by means of PCR with synthesized oligonucleotide primers as described below. For pGEM3zf(+)/NS5BBg, a set of primers, 5BBgFor and 5BBgRev, which have an artificial BamHI and EcoRI site, respectively, was used in the PCR with pGEM3zf(+)/HCV JK-1 as a template, which contains HCV JK-1 cDNA between the EcoRI and BamHI sites. For pGEM3zf(+)/poly(U), a set of oligonucleotides, poly(U)For which has an artificial EcoRI site and poly(U)Rev which has the artificialBamHI and BbsI sites, was annealed and subjected to PCR cloning, generating a fragment containing the poly(U) stretch of HCV. For pGEM3zf(+)/3'X, a set of oligonucleotides, 3'XFor which has the artificial EcoRI and BbsI sites and 3'XRev which has the artificial AflII and BamHI sites, was annealed and subjected to PCR cloning, generating a fragment containing 3'X (41, 42). The DNA fragments with the artificial EcoRI and BamHI sites were inserted into the pGEM3zf(+) (Promega) EcoRI and BamHI vector. All the constructs were sequenced with Taq sequencing kits and a DNA sequencer (374A, Applied Biosystems).

Expression and Purification of Bacterial Recombinant NS5B Protein-- GST-fused HCV NS5Bt was expressed as described previously (43-45). The plasmid pGENKS/NS5Bt was transformed into the E. coli strain BL21 pLysS(DE3), and the transformants were then cultured in 10 ml of LB medium with 100 µg/ml ampicillin at 30 °C overnight. Cultures were diluted into 1 liter of LB medium with 100 µg/ml ampicillin and incubated at 30 °C until the A600 reached 0.6-0.7. These cultures were then induced overnight with 0.4 mM isopropyl-beta -D-thiogalactopyranoside. The cells (from 1 liter) were harvested by centrifugation and washed once with phosphate-buffered saline (PBS). The pellet was suspended in 32 ml of PBS containing 1 mM dithiothreitol (DTT) and 1% Triton X-100 (buffer A). The suspension was sonicated on ice until it was no longer viscous and then centrifuged at 15,000 × g for 20 min. The supernatant was placed on ice (supernatant 1, S1), while the pellet was suspended in 32 ml buffer A containing 1.0 M NaCl, sonicated again, and the suspension was centrifuged. After the supernatant was collected (supernatant 2, S2) and mixed with the S1 fraction, the NaCl concentration was adjusted to 0.33 M using buffer A. This combined supernatant (supernatant 3, S3) was passed through a DEAE-Sephacel column equilibrated with buffer A. The flow-through fraction was mixed with 1 ml of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) equilibrated with buffer A, and the protein was allowed to absorb to the beads for 1 h at 4 °C. The beads were then washed thoroughly with buffer A and then with 50 mM Tris-HCl, pH 8.0, and 1 mM DTT. The GST-NS5B was eluted with 4 ml of buffer B (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM glutathione, 10 mM DTT, and 0.1% Triton X-100) and then eluted with 4 ml of buffer B containing 500 mM NaCl before being subjected to column purification (27). After the NaCl concentration was adjusted to 150 mM, the eluted solution was applied to a heparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with buffer B. The column was then washed with LG buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 20% glycerol, 0.5% Triton X-100), and the column was eluted with LG buffer containing from 100 mM to M NaCl. The elution profile was analyzed by 10% SDS-PAGE, and the GST-NS5Bt was found to elute broadly from 500 to 900 mM NaCl. These fractions were collected and diluted with LG buffer to adjust the NaCl concentration to 150 mM. The solution was then applied to a poly(U)-Sepharose column (Amersham Pharmacia Biotech) equilibrated with LG buffer containing 150 mM NaCl. After washing, the column was eluted with LG buffer containing from 200 mM to 1 M NaCl. GST-NS5Bt eluted between 500 and 700 mM NaCl. The fractions were collected and dialyzed against LG buffer containing 150 mM NaCl. For the non-fusion NS5Bt, the protein-bound glutathione resin was washed extensively with thrombin cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM CaCl2, and 1% Triton X-100). The GST-fused NS5Bt was cleaved overnight in thrombin cleavage buffer containing 50 units of thrombin (Amersham Pharmacia Biotech) to release the NS5Bt protein from the GST-bound glutathione resin. The supernatant containing the NS5Bt protein was collected and dialyzed against LG buffer containing 150 mM NaCl (45). Samples were collected at various stages in the purification, analyzed by SDS-PAGE, and assayed for polymerase activity. The protein concentration was measured by the Bradford method or Coomassie staining with bovine serum albumin as the standard.

Poly(A)-dependent UMP Incorporation Assay and the Substrate Specificity of the Activity-- The incorporation of [alpha -32P]UMP or [alpha -32P]dTTP was measured as described previously (26, 27, 46). The standard reaction (10 µl) was carried out in buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 20 units of RNase inhibitor (Wako Chemicals Co. Ltd.), 2 µCi of [alpha -32P]UTP (800 Ci/mmol, Amersham Pharmacia Biotech), 10 µM UTP, 10 µg/ml poly(A), and oligo(U)14 (1 µg/ml). After a 2 h incubation at 25 °C, the reaction solution was transferred to DE81 filters (Whatman) to stop the reaction. The filters were washed extensively with 0.5 M Na2HPO4, pH 7.0, rinsed with 70% ethanol, and air-dried before the filter-bound radioactivity was measured by a scintillation counter. For RdRP activity, both oligo(U)14 and oligo(dT) were used as primers. For reverse transcriptase activity, poly(A), oligo(dT), and [alpha -32P]dTTP were used as the template, primer, and substrate, respectively. For RNA polymerase activity, poly(dA), oligo(U), and [alpha -32P]UTP were used, whereas for DNA polymerase activity, poly(dA), oligo(dT), and [alpha -32P]dTTP were used. Rifampicin and actinomycin D (Sigma) were dissolved in ethanol and added to the reactions at the indicated concentrations. In these experiments the reactions that contained the same volume of ethanol were used as a standard.

Preparation of RNA Templates for Polymerase Assay-- The plasmids, pGEM3zf(+)/5BBg, pGEM3zf(+)/poly(U), and pGEM3zf(+)/3'X, were linearized by digestion with BamHI or EcoRI, whereas pNKFLAG was linearized by digestion with BglII to prepare the control RNA. In vitro transcription using these templates was carried out with T7 RNA polymerase or SP6 RNA polymerase according to the manufacturer's instructions (Promega). After incubation, the DNA templates were digested with RNase-free DNase. The RNA products were extracted with phenol/chloroform (1:1) and passed through a Sephadex G-50 column to remove free nucleotides before ethanol precipitation. The RNA concentrations were measured spectrophotometrically, adjusted to 1 µg/µl with water, and then stored at -20 °C. The quality of the RNA samples was confirmed by electrophoresis in a MOPS-denaturing gel or urea-denaturing polyacrylamide gel.

RNA-dependent RNA Polymerase Assay-- RNA-dependent RNA polymerase assay was performed in a total volume of 40 µl containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 20 units of RNase inhibitor, 50 µg/ml actinomycin D, 5 µCi of [alpha -32P]UTP, and 0.5 mM each of the remaining NTPs (i.e. ATP, CTP, and GTP) with 10 µg/ml RNA template (26). The concentration of the limiting nucleotide was adjusted to 10 µM. The reaction mixtures were incubated at 30 °C for 2 h. After incubation, the reaction was stopped by digestion with 50 µg of proteinase K (Boehringer Mannheim) in proteinase K buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 0.5% SDS) for 30 min. The RNA products were extracted with phenol/chloroform (1:1) before ethanol precipitation. After heat denaturation (94 °C, 2 min) these products were analyzed by 8 M urea, 8% PAGE. The gels were dried and analyzed using a BAS 1000 BioImage analyzer system (Fuji).

Subcellular Localization of Transiently Expressed NS5B-- Subcellular localization of NS5B in mammalian cells was examined by a transient expression system using HepG2, HLE, and COS-1 cells. About 1 × 105 cells were plated on a slide glass in a Quadriperm microscope slide culture well (Heraeus) 1 day before transfection with the GFP-NS5B expression plasmid, pGFP/NS5B, or the FLAG-tagged NS5B expression plasmid, pNKFLAG/NS5B. The cells were rinsed and fixed with 1.5% paraformaldehyde in PBS for 30 min before being post-fixed for 5 min in 100% cold methanol. These slides were then air-dried at -25 °C and stored at -80 °C. GFP-fused proteins were detected after counterstaining with 0.0005% Evans Blue in PBS. Samples expressing FLAG-tagged proteins were blocked with 0.5% bovine serum albumin in PBS and stained overnight with anti-FLAG M2 antibody (Scientific Imaging Systems Co. Ltd.) diluted in PBS containing 0.5% bovine serum albumin (1:330). Immunostaining was carried out according to the standard procedure, using absorbed rabbit anti-mouse IgG, biotinylated goat anti-rabbit IgG, and streptavidin-fluorescein isothiocyanate (Amersham Pharmacia Biotech) with counter staining by Evans Blue.

The processed slides were examined using a BX-50 fluorescence microscope (Olympus) with NIBA and WIB filters, and the images were visualized by digital printing (Pictrography 3000, Fuji). Similar expression levels of the GFP-fused proteins in HLE cells were immunologically detected by Western blotting using anti-NS5B IgG and anti-GFP IgG (CLONTECH) (data not shown).

Preparation of Rabbit Antisera, Human HCV Patient Sera, and Immunological Detection-- Antisera against NS5B were raised in rabbits by subcutaneous inoculation with around 200 µg of purified bacterial hexahistidine-tagged NS5B in complete Freund's adjuvant (Sigma). The IgG fraction of the antisera was purified using a protein A-Sepharose column according to the manufacturer's instruction (Amersham Pharmacia Biotech). Both the antisera and purified IgG fraction were used at a 1:3000 dilution for Western blotting as described previously (38, 40).

The human serum used in this study was obtained from a Japanese chronic HCV (genotype Ib) patient. The IgG fraction was purified as described above. The antibody was used in Western blotting at a 1:200 dilution.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Expression and Purification of the Bacterial Recombinant NS5B Protein-- Several different kinds of vectors including His-tagged and GST fusion expression plasmids were examined to obtain the soluble recombinant full-size NS5B protein from E. coli transformants (Fig. 1A). All the NS5B proteins tested were expressed at a low level, and most were recovered in the insoluble fractions as described previously (47). With the prediction programs in Genome net servers2 PSORT and SOSUI, we found that the C-terminal part of NS5B contains a highly hydrophobic region (aa 2989-3005) which is predicted to be an anchoring domain (Fig. 1B, shaded region). Therefore, the effect of the C-terminal region was examined by constructing a GST fusion expression plasmid, GST-NS5Bt, which lacked the C-terminal 21 amino acid residues (aa 2989-3010, see "Experimental Procedures"). A 95-kDa protein corresponding to the GST-fused NS5Bt was overexpressed in the E. coli transformants by isopropyl-beta -D-thiogalactopyranoside induction and then recovered in the soluble fractions (Fig. 2A). The soluble protein was bound to glutathione resin; however, most of the 95-kDa protein remained on the resin after standard elution procedure (38, 40, 44). Therefore, we modified the elution procedure to improve the yield of the soluble GST-NS5Bt and finally succeeded in recovering about 1 mg of the soluble recombinant protein from a 2-liter cell culture. Protein was first eluted with glutathione in buffer B containing 150 mM NaCl and next with glutathione in buffer B containing 500 mM NaCl (see "Experimental Procedures"). Several proteins of around 30 kDa in molecular mass, which were degradation products containing the GST portion, were eluted with buffer B containing 150 mM NaCl (data not shown), but most of the 95-kDa protein was eluted with buffer B containing 500 mM NaCl. The sample recovered in this buffer contained trace amounts of other proteins including multiple degradation products of GST-NS5Bt at 70-80 kDa and a faint band of 60 kDa in mass (Fig. 2A, lane 4). Therefore two additional affinity chromatography steps, heparin- and poly(U)-Sepharoses, were introduced to purify further the GST-NS5Bt (see "Experimental Procedures"). The 60-kDa band was removed by the poly(U) column chromatography. The final preparation contained a single 95-kDa band with no other bands detected by Coomassie Brilliant Blue staining (Fig. 2A, lane 5). The 95-kDa band was identified as the recombinant NS5Bt protein since it was specifically recognized with both rabbit anti-NS5Bt IgG and anti-GST IgG on Western blotting (Fig. 2, B and C). Also the band was immunologically detected by serum from a human HCV patient but not by that from a normal blood donor (data not shown). The GST-NS5Bt protein, which bound to the glutathione resin, was treated with thrombin through the artificial consensus sequence located at the junction of the GST portion to obtain the non-fusion NS5Bt (Fig. 1A). New bands of a doublet with a molecular mass of approximately 63 kDa were recovered in the unbound fraction of thrombin cleavage (Fig. 2A, lane 6), and these were specifically recognized by anti-NS5Bt IgG but not by anti-GST IgG (Fig. 2, B and C, lane 6). The purified GST-fused and non-fused NS5Bt proteins were dialyzed for 24 h against LG buffer containing 150 mM NaCl and stored at -80 °C for further examination.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Construction of HCV NS5B expression plasmids. A, the GST expression vector pGENKS. All the HCV subgenomic cDNA fragments were inserted into the SacI and the SalI sites of pGENKS and expressed as GST-fused proteins in E. coli. Underlined are the thrombin cleavage and kination sites for the in vitro labeling with [gamma -32P]ATP. B, upper part shows the polyprotein structure of HCV, and the hydrophobic profile (Kyte and Doolittle) of the NS5B protein is shown below. The shaded region shows a putative anchoring domain predicted by the SOSUI program. Expression constructs for the NS5B protein are shown in the lower part of the figure. Numbers indicate amino acid positions within the HCV polyprotein precursor. NS5B contains the full-size protein. NS5Bt has a deletion from aa 2989 to 3010. NS5Bt-m1 has a GDD motif substituted to VDD. NS5Bt-m2 and -m3 have an alanine substitution in the clustered regions of basic residues as described under "Experimental Procedures." NS5B-m4 has mutations in the putative anchoring region as described under "Experimental Procedures."


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2.   Purification of the bacterial expressed GST-NS5Bt protein. A, the GST-NS5Bt protein was expressed in E. coli and purified as described under "Experimental Procedures." Samples from the purification steps were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). Lanes 1-6 are total cell lysate, sonication supernatant, the solution after DEAE-Sephacel chromatography, the eluate from the glutathione-Sepharose 4B column, the eluate from poly(U)-Sepharose column, and non-fusion NS5Bt after thrombin cleavage respectively. B, Western blot analysis using anti-NS5B antibody. C, Western blot analysis using anti-GST antibody. One-tenth of the protein amounts was transferred to the nitrocellulose membrane and subjected to Western blotting.

RNA Synthesis Activity of the Bacterial Recombinant NS5B Proteins-- RNA synthesis activity of the samples from various purification steps of the bacterial recombinant NS5Bt protein was examined by the UMP incorporation assay, namely incorporation of [alpha -32P]UMP using poly(A) and oligo(U)14 as template and primer, respectively (see "Experimental Procedures"). UMP incorporation of purified GST-NS5Bt was detected, and the relative activity exceeded over 10,000 times that of the starting material (Table II). The incorporation was detected in a dose-dependent manner, but no incorporation was observed in the presence of GST alone (Fig. 3C and Table III). The incorporation was continued for at least 4 h at 25 °C; however, at 37 °C the rate was low and it continued for only 2 h when a plateau was reached (Fig. 3B, 25 and 37 °C). The RNA synthesis activity of both cleaved and uncleaved forms of NS5Bt was not inhibited by the presence of 20 µg/ml rifampicin or 50 µg/ml actinomycin D, and it remained unaffected even at concentrations of up to 200 and 500 µg/ml, respectively (Table III and data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Purification of the HCV RNA polymerase


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   RdRP activity of the purified GST-NS5Bt and thrombin cleaved NS5Bt in the UMP incorporation assay. A, substrate specificity of the protein was measured by the assays using poly(A) or poly(dA) as template and oligo(U)14 or oligo(dT) primer. Incorporation of [alpha -32P]UMP or [alpha -32P]dTTP was measured as described under "Experimental Procedures." B, time course of the reaction. The reactions were carried out at two temperatures (25 and 37 °C) using 90 ng of GST-NS5Bt and at 30 °C using 0.5 µg of thrombin-cleaved NS5Bt. C, dose effect of the GST-NS5Bt. Different amounts of GST-NS5Bt were added to the reaction.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Summary of RdRP activity in GST-NS5Bt and thrombin-cleaved NS5Bt

No incorporation was observed without either primer or template, indicating that there was no terminal transferase activity in the NS5Bt protein fractions (Table III). Optimal incorporation was observed over a broad range of pH values from pH 7.0 to 8.5; however, the incorporation was inhibited at low or high pH values such as pH 6.0 or 9.0 (Fig. 4A). The incorporation was most efficient at 30 °C and was reduced at temperatures lower or higher than 30 °C (Fig. 4B). It was inhibited when the KCl concentration was more than 100 mM (Fig. 4C) and was strictly dependent on the Mg2+ ion. The optimal concentration was between 2.5 and 5 mM (Fig. 4D). The divalent Zn2+ ion could not replace this requirement (data not shown). Ionic detergents such as 0.01% Sarkosyl or SDS completely inhibited the activity, whereas 0.1% of non-ionic detergents such as Triton X-100, Nonidet P-40, Tween 20, and CHAPS had little effect (Table IV).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Properties of the RdRP activity of the purified GST-NS5Bt and thrombin-cleaved NS5Bt. The RdRP activities of the purified GST-NS5B and thrombin-cleaved NS5Bt were measured by the UMP incorporation assay under different reaction conditions. The reactions were carried out using 90 ng of GST-NS5Bt or 0.5 µg of thrombin-cleaved NS5Bt. The incubation was basically carried out for 2 h at 25 °C. A, the reaction mixtures at different pH values were prepared by sodium phosphate buffer. Percentage incorporations of the standard reaction (Tris-HCl buffer, under "Experimental Procedures") are shown. One hundred percent of GST-NS5Bt and thrombin-cleaved NS5Bt was 47,284 and 20,841 cpm, respectively. B, the reaction was carried out at the indicated temperatures. C, KCl was added to the reaction mixture at the indicated concentrations. D, the UMP incorporation assay was carried out in the presence of the different concentrations of magnesium ion. The reactions were performed with 90 ng of GST-NS5Bt or 0.5 µg of thrombin-cleaved NS5Bt.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Effect of detergent on the activity
100% activity was 25,755 cpm in the standard reaction (no detergent).

The properties of the GST-NS5Bt polymerase activity were characterized using different templates and primers. Poly(A)-dependent oligo(U)-primed UMP incorporation in the assay was much higher than that primed by oligo(dT) (about 2 times), and the purified GST-NS5Bt retained no reverse transcriptase, RNA polymerase, or DNA polymerase activities in these reaction conditions (Fig. 3A). Since the UMP incorporation assay utilizes an artificial template and primer, we next examined whether GST-NS5Bt could utilize the HCV RNA as template and primer as reported previously (26, 27) (Fig. 5). For this purpose, the HCV 3'-UTR, which should serve as replication initiation site, was divided into three regions as follows: the 5BBg contained the region from the BglII site in NS5B to 3'-UTR before the poly(U) stretch, the poly(U) contained the poly(U) stretch, and the 3'X which was recently discovered and may have an important role in the viral replication (41, 42). These regions of the HCV subgenome RNA were synthesized by in vitro transcription and examined in the RNA synthesis assay (Fig. 5A and see "Experimental Procedures"). 32P-Labeled RNA bands of the same or a smaller size than the input RNAs were detected (Fig. 5B). The RNA synthesis required all four ribonucleotides as substrate, and no incorporation was detected when UTP or CTP alone was used as substrate (data not shown). Both HCV RNAs and control RNA could serve as template and primer (Fig. 5, lane 4). The in vitro RNA synthesis with 3'X of the HCV 3'-UTR was rather low compared with those with the other RNA regions (Fig. 5, compare lanes 1 and 4 to 3). The poly(U) stretch of HCV 3'-UTR could not serve as template and primer by itself (Fig. 5, lane 2). These results suggest that RNA was utilized as template and primer and that GST-NS5Bt had no terminal transferase activity as demonstrated with the UMP incorporation assay.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   RNA synthesis activity using in vitro transcribed RNA. A, construction of RNA templates. 5BBg contained the region from the BglII site in NS5B to 3'-UTR, poly(U) contained the poly(U) stretch of HCV, and 3'X contained 3'X of HCV. Control RNA were prepared by BglII-digested pNKFLAG plasmid. The RNA sizes are described in the figure. B, RNAs prepared by in vitro transcription were used as template and primer (see "Experimental Procedures"), and the reactions were carried out for 2 h at 30 °C. The RNA products were purified by organic extraction followed by ethanol precipitation and finally separated on 8 M urea, 8% PAGE.

The GST-NS5Bt proteins purified from transformants of E. coli JM109 and BL21 pLysS had similar properties in the UMP incorporation assay (data not shown). The glutathione-eluted fraction purified from BL21 pLysS contained no T7 RNA polymerase since it could not be detected immunologically with an anti-T7 RNA polymerase antibody.3

The possibility that the GST moiety may affect the properties of the RdRP activity of NS5Bt was addressed by the purified thrombin-cleaved non-fusion NS5Bt (see "Experimental Procedures"). The activity in the standard UMP incorporation assay was lower than that of GST-NS5Bt which was purified using heparin and poly(U) columns (about 25% of GST-NS5Bt, Table II). No incorporation was observed without either primer or template, indicating the absence of terminal transferase activity in the NS5Bt protein fractions (Table III). It was resistant to rifampicin and actinomycin D which is the same as GST-NS5Bt (data not shown). The properties of the cleaved protein were almost the same as those of GST-NS5Bt with regard to optimal temperature, KCl sensitivity, and optimal magnesium concentration (Fig. 4) except the RdRP activity of the non-fused NS5Bt was higher in the sodium phosphate buffer (Fig. 4A compared with Fig. 3B or Fig. 4, B-D), and its optimal pH was higher that of the GST-fused form (Fig. 4A). Thrombin-cleaved non-fusion NS5Bt could also utilize RNA as template and primer (data not shown).

These results show that HCV NS5Bt in the GST-fused and non-fused forms from E. coli retain their RdRP activity, which requires a template and primer in the UMP incorporation assay, but they can utilize the HCV RNAs and control RNA as template and primer and have neither terminal transferase activity nor any other kind of polymerizing activity.

RdRP Activity of Mutant NS5Bt-- The results of the RNA synthesis activity detected in the two assays indicate that the NS5Bt protein retains RdRP activity. To prove this, we examined the importance of the GDD motif of NS5B, which is highly conserved among viral RdRPs (25). The mutated GST-NS5Bt protein, NS5Bt-m1, in which the GDD sequence was substituted to VDD, was purified from the E. coli transformed cells (48). The GST-NS5B-m1 protein did not exhibit any RdRP activity in the UMP incorporation assay (Table V). This result shows that the RNA synthesis activity of the GST-NS5B protein absolutely requires the presence of the GDD motif, indicating that the purified bacterial recombinant NS5B protein exhibits the RdRP activity of HCV NS5B.

                              
View this table:
[in this window]
[in a new window]
 
Table V
The RdRP activity of the GST-NS5B mutants

We also evaluated the other mutant proteins, NS5Bt-m2 and -m3, with substitutions in clustered basic residues. The GST-NS5Bt-m2 and -m3, which were purified by the same method as for GST-NS5Bt, did not exhibit any RNA synthesis activity or any inhibitory effect on the RNA synthesis activity of the wild-type GST-NS5Bt protein in the same reaction mixture (Table V and data not shown).

The C-terminal Part of NS5B Encodes a Putative Anchoring Domain That Affects the Subcellular Localization of NS5B-- Since the C-terminal 21 amino acid residues is dispensable for the RdRP activity (Fig. 2 and Table I), we addressed the role of the C-terminal region on the subcellular localization of NS5B in mammalian cells (Fig. 6). Mammalian expression plasmids of NS5B in the green fluorescent protein (GFP)-fused forms were introduced by transient expression to a hepatoma cell line, HLE, and the subcellular localization of the GFP was examined by fluorescence microscopy (see "Experimental Procedures"). The GFP-NS5B protein was predominantly distributed in perinuclear regions and diffusely in the cytoplasm, although the GFP alone was localized diffusely in both the cytoplasmic and nuclear regions (Fig. 6, A and D). Localization of the GFP signal was dramatically affected by truncation of the C-terminal region, and the GFP-NS5Bt protein was predominantly concentrated in the nuclei (Fig. 6B). The fluorescent nuclear signal of the GFP-NS5Bt was detected as several clusters that were huge spherical and irregular forms. The GFP-NS5B-m4, harboring two amino acid substitutions in the anchoring domain, which was constructed to cancel the likelihood of an anchoring domain in the prediction programs, was localized in the nuclei as multiple clusters and diffusely in cytoplasm (Fig. 6C). A similar role for the anchoring domain on the subcellular localization was observed with COS-1- and HepG2-transfected cells which expressed the GFP-fused or FLAG-tagged NS5B proteins (data not shown). These results strongly suggest that the C-terminal region of NS5B, especially the putative anchoring domain, has a role in retaining the protein in the cytoplasm.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of the putative anchoring domain on the subcellular localization of NS5B. The GFP expression plasmids of the full-size and mutated NS5B were introduced into a hepatoma cell line, HLE, by transient transfection. The cells were processed and examined with a fluorescent microscope as described under "Experimental Procedures." Expressed proteins were GFP-NS5B (A), GFP-NS5Bt (B), GFP-NS5B-m4 (C), and GFP alone (D), respectively.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The persistent property of the HCV infection has been explained by its ability to escape from the host immune surveillance through hypermutability of the exposed regions in the envelope protein E2 (49, 50). Persistent infection of HCV causes chronic inflammation in liver, liver cirrhosis, and eventually increases the risk of hepatocellular carcinoma (28). HCV replication or the RNA-dependent RNA polymerase, a central catalytic enzyme of replication, is one of the possible targets to prevent hepatocarcinogenesis by blocking HCV infection.

We demonstrated here the expression and purification of soluble bacterial recombinant HCV NS5B in a GST-fused form. The GST-NS5Bt protein exhibited RNA synthesis activity in the UMP incorporation assay, which is completely dependent upon the template and primer and absolutely requires the GDD motif, a conserved sequence among viral RdRPs (25). The thrombin-digested non-fused form of the wild NS5Bt exhibited the similar properties of RdRP activity except for its behavior in sodium phosphate buffer. The purified mutated GST-NS5Bt proteins (m1 to m3) had no RdRP activity. These results indicate that the bacterial recombinant NS5B protein retains RdRP activity. The bacterial NS5B reported here and the baculovirus-expressed NS5B seem to share similar properties in their RdRP activity, including the requirements for template and primer, Mg2+ dependence, and optimal conditions for their reactions (26, 27). However, the bacterial recombinant NS5B did not exhibit terminal transferase activity which has been demonstrated in the insect-expressed NS5B. The GST moiety located at the N terminus is not the reason for the absence of terminal transferase activity because the thrombin-cleavaged NS5B also had no terminal transferase activity. At present, the reason for this discrepancy remains unclear. However, recently, another group has suggested that terminal transferase activity detected may be due to contamination of host protein (51).

The truncation of the C-terminal part of NS5B was first designed to prepare the soluble GST-fused form of NS5B in E. coli. However, later we found that this region also affected the subcellular localization of NS5B in mammalian cells. The C-terminal part includes two regions, an anchoring domain spanning aa 2989-3003 and a C-terminal tail (aa 3004-3010), the former is highly conserved among the HCV isolates in the data bases. This study is the first to propose the presence of an anchoring domain at the C-terminal region of NS5B. NS5Bt in the GFP-fused form was exclusively distributed in the nuclei which is in contrast to the perinuclear localization of the full-length NS5B protein (52, 53). NS5B-m4, which contained mutations in the anchoring domain, was localized in the nuclei and cytoplasm. Thus it is plausible that the anchoring domain at the C-terminal has a role in retaining the protein in the cytoplasm. RNA replication of many plus strand RNA viruses requires viral membrane proteins which may facilitate assembly of the replication complex anchored to cell membranes (5, 29, 30), and the C-terminal region of NS5B may have a similar role in HCV replication.

NS5B has no typical signal for nuclear targeting, thus the nuclear localization of NS5Bt may be through its ability to interact with nuclear components. As GST-NS5Bt exhibits RNA-binding ability as detected by UV cross-linking or electrophoretic mobility shift assays,4 the RNA binding may be contributing to the nuclear localization of the NS5Bt protein. Interestingly, the several huge clustered forms of the GFP-NS5Bt seem to be unique and distributed in nucleolar regions (54, 55). The role of the C-terminal tail flanking the anchoring domain still remains to be addressed. It should be stressed that the C-terminal region is dispensable for the RdRP activity which was detected by two assays and also for the interaction of NS5A and NS5B both in vitro and in vivo.4

The method applied here is sensitive enough to detect the RdRP catalytic activity of HCV NS5B, but it could not detect specific initiation of HCV replication since the RdRP activities detected in these systems neither require the 3'X sequence located at the end of the genome nor a specific primer as reported previously (26, 27, 51). Plus strand RNA virus replication requires a precise initiation site and a specific primer, either protein or pre-existing RNA (32, 56-58). Therefore, the RdRP activity associated with the NS5B proteins simply reflects the catalytic activity of the central protein of replication. Specific and efficient viral replication could be achieved by the help of the other non-structural proteins such as NS3, NS4A, NS4B, and NS5A which may form a dynamic and higher ordered protein complex(es), together with viral RNA and probably host factors or subcellular compartments (33, 59-62). In this context, it is noteworthy that we could observe only a weak stimulating effect of the bacterial recombinant HCV NS5A on the RdRP activity of NS5B in the in vitro assay, although modulation of HCV replication through NS5A has been suggested and the physical interaction of NS5A and NS5B is detectable in vitro and in vivo.4

The bacterial expression and purification methods we described here are simple and can efficiently purify recombinant NS5B. They will not only provide a useful tool to evaluate the effects of putative metabolic competitors on HCV RdRP activity, but will also facilitate mutation analysis of NS5B in future.

    ACKNOWLEDGEMENTS

We are grateful to M. K. Yi, M. Honda, and Y. Nakamoto for providing HCV JK1 plasmid cDNA; and to Y. Lin, D. Dorjsuren, and N. Hayashi for encouraging discussion. We are grateful to B. A. Heinz for critical comments and communicating unpublished results. We thank M. Takamatsu, C. Matsushima, F. Momoshima, M. Yasukawa, and K. Kuwabara for their technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-76-265-2731; Fax: 81-76-234-4501; E-mail: semuraka{at}kenroku.ipc.kanazawa-u.ac.jp.

1 The abbreviations used are: HCV, hepatitis C virus; RdRP, RNA-dependent RNA polymerase; DTT, dithiothreitol; PBS, phosphate-buffered saline; aa, amino acid(s); GFP, green fluorescent protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; UTR, untranslated region; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

2 The on-line address for PSORT and SOSUI is as follows: http://www.genome.ad.jp.

3 B. A. Heinz, personal communication.

4 Y. Shirota, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 359-362[Medline] [Order article via Infotrieve]
  2. Kuo, G., Choo, Q. L., Alter, H. J., Gitnick, G. L., Redeker, A. G., Purcell, R. H., Miyamura, T., Dienstag, J. L., Alter, M. J., Stevens, C. E., Bonino, F., Colombo, M., Lee, W.-S., Kuo, C., Berger, K., Shuster, J. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 362-364[Medline] [Order article via Infotrieve]
  3. Francki, R., Fauquet, C., Knudson, D., and Brown, F. (1991) Arch. Virol. 2, 223
  4. Miller, R. H., and Purcell, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2057-2061[Abstract]
  5. Simmonds, P., Alberti, A., Alter, H., Bonino, F., Bradley, D., Brechot, C., Brouwer, J., et al.. (1994) Hepatology 19, 1321-1324[Medline] [Order article via Infotrieve]
  6. Choo, Q. L., Richman, K. H., Han, J. H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina Selby, R., Barr, P. J., Weiner, A. J., Bradley, D. W., Kuo, G., and Houghton, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2451-2455[Abstract]
  7. Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Sugimura, T., and Shimotohno, K. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9524-9528[Abstract]
  8. Takamizawa, A., Mori, C., Fuke, I., Manabe, S., Murakami, S., Fujita, J., Onishi, E., Andoh, T., Yoshida, I., and Okayama, H. (1991) J. Virol. 65, 1105-1113[Medline] [Order article via Infotrieve]
  9. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., and Shimotohno, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5547-5551[Abstract]
  10. Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M., and Rice, C. M. (1993) J. Virol. 67, 1385-1395[Abstract]
  11. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W., and Rice, C. M. (1994) J. Virol. 68, 5063-5073[Abstract]
  12. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10583-10587[Abstract]
  13. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993) J. Virol. 67, 4665-4675[Abstract]
  14. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993) J. Virol. 67, 2832-2843[Abstract]
  15. Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K., and Shimotohno, K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10773-10777[Abstract]
  16. Eckart, M. R., Selby, M., Masiarz, F., Lee, C., Berger, K., Crawford, K., Kuo, C., Kuo, G., Houghton, M., and Choo, Q. L. (1993) Biochem. Biophys. Res. Commun. 192, 399-406[CrossRef][Medline] [Order article via Infotrieve]
  17. Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobsen, H. (1993) J. Virol. 67, 3835-3844[Abstract]
  18. Manabe, S., Fuke, I., Tanishita, O., Kaji, C., Gomi, Y., Yoshida, S., Mori, C., Takamizawa, A., Yosida, I., and Okayama, H. (1994) Virology 198, 636-644[CrossRef][Medline] [Order article via Infotrieve]
  19. Tomei, L., Failla, C., Santolini, E., De Francesco, R., and La Monica, N. (1993) J. Virol. 67, 4017-4026[Abstract]
  20. Suzich, J. A., Tamura, J. K., Palmer Hill, F., Warrener, P., Grakoui, A., Rice, C. M., Feinstone, S. M., and Collett, M. S. (1993) J. Virol. 67, 6152-6158[Abstract]
  21. Kim, D. W., Gwack, Y., Han, J. H., and Choe, J. (1995) Biochem. Biophys. Res. Commun. 215, 160-166[CrossRef][Medline] [Order article via Infotrieve]
  22. Tai, C. L., Chi, W. K., Chen, D. S., and Hwang, L. H. (1996) J. Virol. 70, 8477-8484[Abstract]
  23. Tanji, Y., Kaneko, T., Satoh, S., and Shimotohno, K. (1995) J. Virol. 69, 3980-3986[Abstract]
  24. Asabe, S. I., Tanji, Y., Satoh, S., Kaneko, T., Kimura, K., and Shimotohno, K. (1997) J. Virol. 71, 790-796[Abstract]
  25. Poch, O., Sauvaget, I., Delarue, M., and Tordo, N. (1989) EMBO J. 8, 3867-3874[Abstract]
  26. Behrens, S. E., Tomei, L., and De Francesco, R. (1996) EMBO J. 15, 12-22[Abstract]
  27. De Francesco, R., Behrens, S. E., Tomei, L., Altamura, S., and Jiricny, J. (1996) Methods Enzymol. 275, 58-67[Medline] [Order article via Infotrieve]
  28. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q.-L., Houghton, M., and Kuo, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6547-6549[Abstract]
  29. Neufeld, K. L., Richards, O. C., and Ehrenfeld, E. (1991) J. Biol. Chem. 266, 24212-24219[Abstract/Free Full Text]
  30. Osman, T. A. M., and Buck, K. W. (1996) J. Virol. 70, 6227-6234[Abstract]
  31. Song, C., and Simon, A. E. (1995) J. Virol. 69, 4020-4028[Abstract]
  32. Sun, J.-H., Adkins, S., Faurote, G., and Kao, C. C. (1996) Virology 226, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  33. Barton, D., Black, E. P., and Flanegan, J. B. (1995) J. Virol. 69, 5516-5527[Abstract]
  34. Shimizu, Y. K., Iwamoto, A., Hijikata, M., Purcell, R. H., and Yoshikura, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5477-5481[Abstract]
  35. Shimizu, Y. K., and Yoshikura, H. (1994) J. Virol. 68, 8406-8418[Abstract]
  36. Nakajima, N., Hijikata, M., Yoshikura, H., and Shimizu, Y. K. (1996) J. Virol. 70, 3325-3339[Abstract]
  37. Murakami, S., Cheong, J., and Kaneko, S. (1994) J. Biol. Chem. 269, 15118-15123[Abstract/Free Full Text]
  38. Yi, M.-K., Nakamoto, Y., Kaneko, S., Yamashita, T., and Murakami, S. (1997) Virology 231, 119-129[CrossRef][Medline] [Order article via Infotrieve]
  39. Honda, M., Kaneko, S., Unoura, M., Kobayashi, K., and Murakami, S. (1993) Arch. Virol. 128, 163-169[Medline] [Order article via Infotrieve]
  40. Lin, Y., Nomura, T., Cheong, J. H., Dorjsuren, D., Iida, K., and Murakami, S. (1997) J. Biol. Chem. 272, 7132-7139[Abstract/Free Full Text]
  41. Tanaka, T., Kato, N., Cho, M. J., Sugiyama, K., and Shimotohno, K. (1996) J. Virol. 70, 3307-3312[Abstract]
  42. Kolykhalov, A. A., Feinstone, S. M., and Rice, C. M. (1996) J. Virol. 70, 3363-3371[Abstract]
  43. Morrow, C. D., Warren, B., and Lentz, M. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6050-6054[Abstract]
  44. Yi, M. K., Kaneko, S., Yu, D. Y., and Murakami, S. (1997) J. Virol. 71, 5997-6002[Abstract]
  45. Tan, B.-H., Fu, J., Sugrue, R. J., Yap, E.-H., Chan, Y.-C., and Tan, Y. H. (1996) Virology 216, 317-325[CrossRef][Medline] [Order article via Infotrieve]
  46. Lama, J., Sanz, M. A., and Rodriguez, P. L. (1995) J. Biol. Chem. 270, 14430-14438[Abstract/Free Full Text]
  47. Yuan, Z.-H., Kumar, U., Thomas, H. C., Wen, Y.-M., and Monjardino, J. (1997) Biochem. Biophys. Res. Commun. 232, 231-235[CrossRef][Medline] [Order article via Infotrieve]
  48. Jablonski, S. A., Luo, M., and Morrow, C. D. (1991) J. Virol. 65, 4565-4572[Medline] [Order article via Infotrieve]
  49. Weiner, A. J., Brauer, M. J., Rosenblatt, J., Richman, K. H., Tung, J., Crawford, K., Bonino, F., Saracco, G., Choo, Q. L., Houghton, M., and Han, J. H. (1991) Virology 180, 842-848[Medline] [Order article via Infotrieve]
  50. Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., Bonino, F., Crawford, K., Marion, C. D., Crawford, K. A., Brunetto, M., Barr, P. J., Miyamura, T., McHutchinson, J., and Houghton, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3468-3472[Abstract]
  51. Lohmann, V., Korner, F., Herian, U., and Bartenschlager, R. (1997) J. Virol. 71, 8416-8428[Abstract]
  52. Hwang, S. B., Park, K. J., Kim, Y. S., Sung, Y. C., and Lai, M. M. (1997) Virology 227, 439-446[CrossRef][Medline] [Order article via Infotrieve]
  53. Lutz, P., Purion-Dutilleul, F. Y. L., and Kedinger, C. (1996) J. Virol. 70, 3449-3460[Abstract]
  54. Kennedy, B. K., Gotta, M., Sinclair, D. A., Mills, K., McNabb, D. S., Murthy, M., Pak, S. M., Laroche, T., Gasser, S. M., and Guarente, L. (1997) Cell 89, 381-391[Medline] [Order article via Infotrieve]
  55. Scinclair, D. A., Mills, K., and Guarente, L. (1997) Science 277, 1313-1316[Abstract/Free Full Text]
  56. Andino, R., Rieckhof, G. E., Achacoso, P. L., and Baltimore, D. (1993) EMBO J. 12, 3587-3598[Abstract]
  57. Pilipenko, E. V., Poperechny, K. V., Maslova, S. V., Melchers, W. J. G., Slot, H. J. S., and Agol, V. I. (1996) EMBO J. 15, 5428-5436[Abstract]
  58. Owen, K. E., and Kuhn, R. J. (1996) J. Virol. 70, 2757-2763[Abstract]
  59. Lemm, J. A., and Rice, C. M. (1993) J. Virol. 67, 1905-1915[Abstract]
  60. Lemm, J. A., Rumenapf, T., Strauss, E. G., Strauss, J. H., and Rice, C. M. (1994) EMBO J. 13, 2925-2934[Abstract]
  61. Quadt, R., Ishikawa, M., Janda, M., and Ahlquist, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4892-4896[Abstract]
  62. McBridge, A. E., Schlegel, A., and Kirkegaard, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2296-2301[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.