Oligomeric Interaction of Hepatitis C Virus NS5B Is Critical for Catalytic Activity of RNA-dependent RNA Polymerase*

HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the “palm and fingers” substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001)Hepatology 33, 728–737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutionsin vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivoand in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.

Hepatitis C Virus (HCV) 1 is the major causative agent of parenterally transmitted hepatitis (1)(2)(3). HCV infection frequently leads to chronic hepatitis, liver cirrhosis, and eventually hepatocellular carcinoma (4,5). In the case of HCV-associated hepatocellular carcinoma, there is often prolonged active inflammation manifested by high alanine aminotransferase level generally associated with high virus load. Therefore, eradication of replication of HCV would be expected to reduce or even prevent incidence of hepatocellular carcinoma. HCV has a positive-sense single-stranded RNA genome of approximately 9.6 kb, which contains a large open reading frame encoding a polyprotein of ϳ3,000 amino acid residues and two highly conserved untranslated regions flanking the 5Ј and 3Ј ends of the genome. The viral encoded polypeptide precursor is cotranslationally or posttranslationally processed by host and viral proteases into at least 10 distinct products: NH 2 -C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (6 and the references therein). The non-structural proteins NS2-5B are thought to be required for viral genome replication.
The exact mechanism of replication of the HCV genome remains to be elucidated (7,8), but NS5B is an RNA-dependent RNA polymerase (RdRP), a core enzyme for HCV replication. NS5B belongs to a large family of nucleic acid-dependent nucleic acid polymerases having the palm and fingers substructure and harboring the motifs (ABCDEF) conserved among RdRPs. Several residues of NS5B within the conserved motifs have been identified as critical for RdRP activity by introducing mutations within the motifs (7)(8)(9)(10)(11)(12)(13)(14). In addition, NS5B has several unique structural characteristics, including two loops from the fingers and a thick thumb ( Fig. 1) (12), which contribute to a flattened spherical shape as revealed by the crystal models (15)(16)(17). Recently we identified five novel residues located outside of the conserved motifs as critical for RdRP activity by two-step scanning for alanine, clustering, and then point mutation of alanine. These include Tyr-191 near the catalytic center, Cys-274 and Tyr-276 at the fingertips, Glu-18 in the long loop, and His-502 on the outer surface of the thick thumb. The latter two seem to be far from the pocket for catalytic activity (12). We suspected that such a defect in RdRP activity suggests some conformational changes in NS5B. The oligomerization of several enzymes has been reported among the nucleic acid polymerase family with the palm and fingers substructure. Oligomerized forms of poliovirus 3D (18 -20) and the heterodimer of HIV reverse transcriptase (RT) (21,22) have been studied extensively and demonstrated to be critical for catalytic activity. A certain conformation seems to be prerequisite to the catalytic activity of these enzymes described as closed or open and inactive or active forms.
Here we report that HCV NS5B has an intrinsic ability to oligomerize or dimerize, and that two residues, Glu-18 and His-502, are critical for the oligomerization, which is prerequisite for RdRP activity. The two exposed residues might be directly involved in the interaction, and the enzyme activity since these residues are exchangeable in a strict sense.

EXPERIMENTAL PROCEDURES
Plasmids and Mutagenesis-The bacterial expression vectors pGENKS, pYFLAG, and pLHis and the mammalian expression vectors pNKFLAG and pNKGST were reported previously (23)(24)(25). An alaninescanning method was applied to construct NS5B mutations to minimize the effects of substituted amino acid residues by site-directed mutagen-* This work was supported in part by a grant-in-aid for scientific research (Category B) and development, a grant-in-aid for scientific research on priority areas (Category C) in oncogenesis of Ministry of Education, Sports, Culture, and Technology, and a grant-in-aid for cancer research of Ministry of Health, Labor, and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Expression and Purification of Bacterially Recombinant NS5B Proteins-GST-fused NS5B mutant proteins and FLAG-tagged NS5B protein were expressed and purified as reported previously (11,12). His-5Bt protein was expressed and purified by the method of Oh et al. (7) with some modification. Escherichia coli strain BL21 pLysS(DE3) transformed by pLHis-NS5Bt was grown to an A 600 of 0.5 at 37°C, and protein expression was induced by 1 mM isopropyl-␤-D-thiogalactopyranoside at 25°C for 6 h. The cells from 250 ml of suspension were harvested by centrifugation and resuspended in binding buffer (50 mM sodium phosphate, pH 8.0, 500 mM NaCl, 10% glycerol, 0.1% Triton X-100, 10 mM ␤-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (Sigma), and 10 mM leupeptin (Roche Molecular Biochemicals). Histidine-tagged NS5Bt was purified by incubating the sonication supernatant with nickel resin (Invitrogen) followed by extensive washing with binding buffer containing 0 -20 mM imidazole and then eluted with binding buffer containing imidazole in a step-gradient manner (ϳ40 -300 mM). The NS5Bt peaks were combined and dialyzed against buffer A (50 mM Tri-HCl, pH 8.0, 1 mM DTT, 150 mM NaCl, and 10% glycerol) before being frozen at Ϫ80°C in small aliquots.
Poly(A)-dependent UMP Incorporation Assay-RdRP activities of wild type GST-NS5Bt and mutants and the combined GST-NS5Bt mutants were examined by UMP incorporation assay as reported previously (9,11,12,27). The incorporation of [␣-32 P]UMP was measured using poly(A) and oligo(U) 14 as template and primer, respectively.
Preparation of Cell Extracts, Coprecipitation with GST Resin, and Western Blot Analysis-Transient transfection of COS-1 cells was carried out as described previously (24). COS-1 cells were cotransfected with pNKGST-NS5B (with or without mutations in NS5B) together with pNKFLAG-NS5B. The cells were harvested and washed with PBS and sonicated in PBS lysis buffer (PBS containing 240 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1 mM DTT) with 10 g each of aprotinin and leupeptin/ml. Total cell lysates were diluted 5-fold with PBS lysis buffer and mixed with 10 l of glutathione resin and then incubated for 3 h on a rotator at room temperature. After a wash with PBS containing 0.5% Triton X-100, the bound proteins were eluted, fractionated by SDS-10% PAGE, transferred onto nitrocellulose membranes, and subjected to Western blot analysis with anti-FLAG monoclonal antibody. The proteins were visualized by enhanced chemiluminescence according to instructions by the manufacturer (Amersham Biosciences, Inc.). The nitrocellulose membranes used for Western blot analysis with anti-FLAG monoclonal antibody were reprobed with anti-GST mono-clonal antibody (Zymed Laboratories Inc.) as recommended by the manufacturer (Amersham Biosciences, Inc.).
GST Pull-down Assay in Vitro-Approximately 1 g of GST-NS5Bt or mutated bacterial recombinant protein immobilized on 1 l of glutathione-Sepharose 4B resin was preblocked with 1% bovine serum albumin and then incubated with 0.2 g of FLAG-tagged NS5B in 0.5 ml of phosphate-buffered saline containing 1% Triton X-100 for 4 h on a rotator at room temperature. Alternatively, the GST-NS5B and FLAG-NS5B lysates were combined and incubated for 16 h at 4°C and then applied to glutathione-Sepharose 4B resin. After a wash with phosphate-buffered saline containing 1% Triton X-100, the bound proteins were solubilized with SDS loading buffer, fractionated by SDS-10% PAGE, and subjected to Western blot analysis with anti-NS5B IgG antibody .
Glycerol Gradient and Molecular Sieving Analyses of HCV NS5B-Two micrograms of purified His-NS5Bt protein in gradient buffer (50 mM Tri-HCl, pH 8.0, 50 mM NaCl, 0.1% Triton X-100, and 1 mM DTT) containing 5% glycerol was loaded on 5 ml of 10 -35% glycerol gradient. The molecular marker proteins (each 200 g) in gradient buffer with 5% glycerol were loaded separately onto the identical gradient. The molecular marker proteins used were albumin, aldolase, and catalase from Amersham Biosciences. After centrifugation at 10,800 ϫ g for 20 h at 20°C, fractions were collected. Each pooled sample was precipitated with 10% trichloroacetic acid, washed with cold acetone, and separated by 10% SDS-PAGE. The His-NS5Bt protein was concentrated and dialyzed against buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100, and 1 mM DTT) and loaded onto Superose 6 column (PC 3.2/30). Fractionation was carried out using SMART system (Amersham Biosciences, Inc.) at a flow rate 30 l/min at 4°C, and the peaks were detected at 300 nm. Eluted sample was collected, and each fractionated sample (20 of 50 l) was subjected to 10% SDS-PAGE analysis. The standard calibration curve was prepared according to the manufacturer manual by the two elution profiles using the gel filtration calibration kits (Amersham Biosciences, Inc.) in the same running buffer.

RESULTS
Homomeric Interaction of HCV NS5B-Oligomerization of NS5B protein was investigated by pull-down assay in vitro and in mammalian cells. Bacterial lysates containing GST-NS5Bt and FLAG-NS5Bt were incubated together and were captured on glutathione beads. After extensive washing, the bound proteins were fractionated and then analyzed by Western blotting with anti-NS5B IgG antibody. As shown in Fig. 2a, FLAG-NS5Bt protein was efficiently recovered by GST-NS5Bt, indicating a specific homomeric interaction.
Next we confirmed the homomeric interaction of HCV NS5B in mammalian cells. The mammalian expression vectors pNKGST-5Bt and pNKFLAG-5B were transiently transfected into COS-1 cells, and the proteins were overexpressed. The amounts of protein were adjusted to similar levels as detected by anti-FLAG-M2 and anti-GST antibody (Fig. 2b, upper and  bottom). The supernatants of lysates were incubated with GST resin and subjected to pull-down assay and Western blotting using anti-FLAG M2 antibody (Fig. 2b, middle). The homomeric interaction of the NS5B proteins was observed in COS-1 cells since FLAG-NS5B protein was efficiently recovered by GST-NS5Bt-bound resin. These results in vitro and in vivo indicate that HCV NS5B is capable of oligomerization or homomeric interaction.
Delineation of the Homomeric Interaction Residues in HCV NS5B-A library of the clustered and point substitution mutants of HCV NS5B was used to determine the residues of HCV NS5B important for interaction with itself. The library covers one-third of all residues and two-thirds of aromatic and charged amino acid residues, which are outside of the conserved motif but conserved among HCV-isolated clones (6). All but cm20 covering amino acids 17-23 and cm2 covering amino acids 500 -506 of GSt-NS5Bt proteins exhibited the ability to recover FLAG-NS5Bt in vitro (Fig. 3a). The defect of cm20 and cm2 was confirmed with transiently coexpressed wild type FLAG-NS5B and mutant versions of GST-NS5B in mammalian cells. Point alanine substitutions covering the sequences of  (17). Glu-18 and His-502 are located at the long loop and helix T of NS5B (15)(16)(17), respectively, which are exposed toward the back side of the right-hand model. Graphics were processed by Cinema 3D version 3.0. cm20 and cm2 were examined for oligomerization in vitro and in vivo. All mutations with the exception of E18A and H502A exhibited the ability to bind wild type NS5Bt in vitro and wild type NS5B in vivo (Fig. 3, a and b, data not shown). These results definitely demonstrate that Glu-18 and His-502, indispensable for the RdRP activity as reported previously (6), are the only residues critical for the oligomerization.
Amino Acids Essential for the Homomeric Interaction of HCV NS5B Are Residual-specific and Exchangeable-To further evaluate the close relationship between the oligomerization and RdRP activity of NS5B, we introduced several substitutions at residues Glu-18 and His-502 and examined their effect on oligomerization in vivo and in vitro and RdRP activity in vitro. All the substitution mutants of NS5B including those in which the residue was replaced with a similarly charged group (E18D, H502R, and H502K) failed to exhibit RdRP activity (Table I) and bind the wild type FLAG-NS5Bt in vitro and FLAG-NS5B in vivo (Fig. 4, a and b). Therefore, the oligomerization of NS5B requires glutamic acid at amino acid 18 and histidine at amino acid 502. The electrostatic force alone can not explain such a residue-specific result.
Whether or not the oligomerization stringently requires the interaction of two residues, it may be possible to replace these residues without affecting the substructures. FLAG-NS5Bt proteins with H502E were actually pulled down by GSt-NS5Bt with E18H in vitro (Fig. 4a), and the result was confirmed in vivo (Fig. 4b). In contrast, no interaction was observed between wild type NS5Bt and that with that E18H or with H502E. The effect on RdRP activity of changing the two residues at amino acid 18 and amino acid 502 was examined in vitro. The activity   FIG. 2. Homomeric interaction of HCV NS5B in vitro and in mammalian cells. a, bacterial lysates containing GST-NS5Bt and FLAG-NS5Bt as indicated were incubated overnight and then captured on glutathione beads. The complexes were eluted and fractionated by 10% SDS-PAGE and subjected to Western blot analysis with anti-NS5B IgG antibody. Mock GST alone is shown (upper). An aliquot of each incubate mix indicating input was directly analyzed by SDS-PAGE and Western blotting analysis with anti-NS5B IgG antibody (lower). b, COS-1 cells were transiently transfected with mammalian expression plasmids pNKGST-NS5B and pNKFLAG-NS5B. Total lysates were separated by 10% SDS-PAGE and subjected to Western blotting with anti-FLAG monoclonal antibody (upper). Proteins bound to glutathione-Sepharose 4B resin were washed with PBS containing 0.5% Triton X-100, fractionated by 10% SDS-PAGE, and detected by Western blotting with anti-FLAG antibody (middle). The nitrocellulose membranes used for Western blotting with M2 antibody were reprobed with anti-GST antibody (bottom).

FIG. 3. Delineation of the residues needed for homomeric interaction in HCV NS5B in vitro and in mammalian cells. a, bacterial
lysates containing GST-NS5Bt (wild type and mutants) and FLAG-NS5Bt as indicated were incubated overnight and then captured on glutathione beads (lower). Mock, FLAG-NS5Bt alone. Pull-down assay and Western blot analysis were carried out with anti-NS5B IgG antibody as described in Fig. 2a. An aliquot of each incubated sample was directly analyzed by SDS-PAGE and Western blotting with anti-NS5B IgG antibody (upper). b, COS-1 cells were transiently transfected with mammalian expression plasmids pNKGST-NS5B (wild type and mutants) and pNKFLAG-NS5B. Mock, pNKGST-NS5B alone. Pull-down assay and Western blot analysis were carried out with anti-FLAG antibody (middle) and anti-GST antibody (upper) as described in Fig. 2b. Expressed FLAG-NS5B in the COS-1 cells is shown (upper). was recovered by 70% only when the two mutated proteins were mixed (Table I). These results clearly indicate that oligomerization of NS5B is prerequisite for RdRP activity and that Glu-18 and His-502 are critical for the homomeric interaction, which probably occurs via direct interaction of the residues, although there is no direct proof at present. HCV NS5B Is Monomeric or Oligomeric in Solution-To further characterize the homomeric interaction of NS5B, purified (His) 6 -NS5Bt at a concentration similar to that used for RdRP assay was loaded on a 10 -35% glycerol gradient. In parallel, the protein molecular markers were applied to the gradient. The samples were fractionated by centrifugation, and the fractionated samples were subjected to SDS-PAGE. The NS5Bt protein was mainly distributed in fractions 9 -17 consisting of two components, which were faster than 4.4 S (albumin) and 7.5 S (aldolase), respectively (Fig. 5). A minor portion of NS5Bt was present in the fast sedimenting fractions 22 and 23, which were faster than 11.3 S (catalase). The result suggests that the recombinant NS5Bt protein is mainly a monomer or dimer with a minor fraction in oligomer. To confirm the oligomeric formation of NS5Bt, the His-NS5Bt was loaded on Superose 6 column using SMART system, and fractionated samples were subjected to the SDS-PAGE analysis (see "Experimental Procedures"). A main peak at 70 kDa corresponds to monomeric His-NS5Bt, and a minor peak at around 140 kDa corresponds to dimeric one judging by the profiles of molecular sieving (Fig. 6, upper panel) and SDS-PAGE analysis (Fig. 6, lower panel). The former peak includes an additional protein of the bacterial chaperon copurified with recombinant NS5B (33). The S values and the molecular masses of the monomeric and dimeric forms of NS5Bt are rather proportionally plotted on the standard curves with the globular molecular markers. Therefore, the axial ratio of NS5Bt in solution is close to those of the molecular markers (the f/f 0 values range 1.25-1.34) and resembles a flattened sphere similar to the crystal models of NS5B (18,20). The dimeric His-NS5Bt is a much smaller portion (ϳ3-5% total His-NS5Bt) as compared with that detected in the glycerol gradient (Fig. 5). This result may be attributed to the considerable loss of His-NS5Bt by a brief centrifugation of the dialyzed sample before application onto the column (data not shown). DISCUSSION We report here the oligomerization of HCV NS5B in vivo and in vitro. By scanning clustered and point alanine substitution mutants, Glu-18 and His-502 were identified as critical for the homomeric interaction. Because these two residues were also found to be critical for RdRP activity in vitro, the homomeric interaction is indispensable for RdRP activity.
According to crystal models of NS5B, Glu-18 is located in the middle of a long loop connecting the fingers and thumb, which is a unique feature among RdRPs (Fig. 1). Template RNA seems to be in a position close to the connecting part of this loop. His-502 is in helix T that pairs with helix U in the thumb subdomain. The paired helices are one component of the armadillo repeats that are unique among RdRPs and contribute to the thick thumb of HCV NS5B (12, 15-17). The relative position of helix T seems to be the part of the thumb most distal to  4. Amino acids essential for homomeric interaction via Glu-18 and His-502 are residual-specific and exchangeable. a, approximately 0.2 g of the purified FLAG-NS5Bt protein was incubated with 1 g of each GST-NS5Bt mutant protein prebound to glutathione-Sepharose 4B resin (see "Experimental Procedures"). The resin-bound proteins were washed with phosphate-buffered saline containing 1% Triton X-100, fractionated by 10% SDS-PAGE, and subjected to Western blot analysis with anti-FLAG antibody. b, COS-1 cells were transfected with mammalian expression plasmids pNKFLAG-NS5B and pNKGST-NS5B wild type or mutants with a single amino acid substitution as indicated on the top. Pull-down assay and Western blot analysis were carried out with anti-FLAG antibody and anti-GST antibody as described in Fig. 1b. the catalytic center. The two residues are both exposed outside (Fig. 1) and are on the dorsal side of the "right-hand" model of NS5B. Importantly, the residues are located in the HCV-specific substructures of NS5B, a long loop and thick thumb strongly suggesting the interaction surface of NS5B to be unique, although common structural features have been predicted among six viral families of RdRPs (27).
Substitutions of the two residues of NS5B, even with residues having same kind of the charge, disrupted the RdRP activity and binding to wild type NS5B. Furthermore, only the combination of E18H and H502E resulted in RdRP activity when two mutant NS5B proteins were mixed. These results strongly suggest that the oligomerization occurs through the direct interaction of Glu-18 and His-502, although no direct evidence of this is available at present. The possibility still remains that another residue(s) may also be critical for the oligomerization, because our clustered alanine substitutions covered only around one-third of all residues and two-thirds of the aromatic and charged residues of NS5B. However, the oligomerization of NS5B occurs through a long loop and thick thumb, not between homologous substructures. In that sense, both the oligomerization of NS5B and polio 3D require regions located outside of the conserved motifs (18 -20). Because a single mutation of the two residues completely eliminated the oligomerization, the dimerized protein seems to be the catalytically active form of NS5B. This notion is supported by the finding that dimerized NS5Bt was predominant among oligomerized forms in the fractionation profile of NS5B obtained by glycerol density gradient centrifugation. It is possible that the dimerization through Glu-18 and His-502 facilitates or affords higher ordered interactions of NS5B through interaction(s) between undefined residues, which might not be enough for NS5B to dimerize.
The two residues of NS5B critical for the oligomeric interaction are also necessary for the RdRP activity. However, two active sites seem not to be prerequisite to RdRP activity, because the VDD mutant, catalytically defective but competent to oligomerize with wild type NS5Bt, did not exhibit any inhibitory effect on the RdRP activity of NS5B (9,11,14). Further study is necessary to understand the functional and biological relevance of the intrinsic ability of NS5B to oligomerize. Recently, we reported that NS5B interacts with NS5A in vitro and in vivo (6). The functional interaction of NS5B and NS5A was strongly suggested by the experiments on HCV replication using a HCV replicon in mammalian cells (28 -30). These results suggest the possible involvement of NS5A, which may modulate the oligomerization needed for RdRP activity of NS5B, although a rather weak stimulation of NS5A on RdRP activity was detected in vitro. The essential role of the oligomerization for RdRP activity of NS5B may be another target to design specific inhibitors for HCV replication as explored for HIV reverse transcriptase (31,32). FIG. 6. A fractionation profile of HCV NS5B with a molecular-sieving column. The His-tagged HCV NS5Bt was concentrated and dialyzed, and the sample was applied to Superose 6 PC 3.2/30 column and fractionated (see "Experimental Procedures"). Upper panel shows a fractionation profile of NS5Bt detected at 300 nm. The fractionated samples (fractions 16 -26) were analyzed by 10% SDS-PAGE together with the molecular markers and 10% of the sample applied to the column (Input) and visualized by Coomassie Brilliant Blue staining as shown in lower panel. During pretreatment of the sample for application, a preferential loss of His-NS5Bt was observed as compared with the yield of a bacterial chaperon (33). The calibration curve was made by two elution profiles of the molecular markers (see "Experimental Procedures").