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J. Biol. Chem., Vol. 279, Issue 24, 25474-25482, June 11, 2004

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Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture^*

Yuanyuan Ma, Tetsuro Shimakami, Hong Luo, Naoyuki Hayashi, and Seishi Murakami¶

From the Department of Molecular Oncology, Cancer Research Institute and the Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa 920-0934, Japan

Received for publication, January 30, 2004 , and in revised form, March 2, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728–737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993–999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hepatitis C virus (HCV),¹ a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepatitis (1). Persistent infection with HCV is an important cause of chronic liver disease around the world. Infections with this hepatotropic flavivirus are associated with inflammatory liver injury, progressive fibrosis, and, in the most severely affected patients, cirrhosis and hepatocellular carcinoma (2, 3). The HCV genome is a single-stranded RNA molecule 9.6 kb in length with positive-sense polarity (1, 5). The genome consists of a 5'-untranslated region, an open reading frame, and a 3'-untranslated region (1, 4, 5). The order of the gene products of the single open reading frame is NH₂-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein precursor can be processed by the host and virally encoded proteases to generate mature structural and nonstructural proteins required for virus replication and assembly (6–8).

Studies on HCV replication focusing on the inhibition of HCV replication may have therapeutic significance for chronic hepatitis and also could reduce the incidence of or even prevent hepatocellular carcinoma. NS5B is an RNA-dependent RNA polymerase (RdRP), a core enzyme required for HCV replication. Recombinant forms of NS5B have been expressed and purified from bacterial and insect cells (9–16). It belongs to a large family of nucleic acid-dependent nucleic acid polymerases (NdNp) sharing finger and thumb subdomain structures with conserved motifs (17–19). NS5B also has several unique properties, including two loops connecting fingers and a thick thumb that may be the major elements responsible for the closed conformation of HCV NS5B, and may also play a role in the "clamping" motion of this enzyme (42). Similar to other viral RdRPs, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (9–16), and two RNA synthesis reaction modes have been described: RNA elongation using a pre-annealed primer, and RNA initiation through a de novo mechanism (14, 15, 20–22).

Despite the progress made in understanding the genomic organization of the virus and the functions of the viral proteins, fundamental aspects of HCV replication, pathogenesis, and persistence remain unknown. Studies on the replication of HCV have been hampered by the lack of efficient cell culture systems. Many attempts have been made to identify cell lines that allow efficient infection with HCV and virus production, but these systems suffer from low virus yield and poor reproducibility (23). Recently, a cell culture system was developed based on subgenomic selectable replicons consisting of the HCV 5'-nontranslated region directing translation segment of the neomycin phosphotransferase gene, internal ribosomal entry site of the encephalomyocarditis virus, HCV NS proteins NS3 to NS5B, and HCV 3'-nontranslated region. After transfection these RNAs into Huh-7 cells and selection with G418, cell colonies can be generated that carry persistently replicating HCV replicons (24, 25). Several replicon systems derived from a genotype 1b HCV-N isolate and a genotype 1a HCV-H77 isolate were reported to replicate in Huh-7 cells (24, 26–29). Recently, Kishine et al. (30–33) also established a replicon system using the HCV sequence derived from a cultured human T cell line MT-2C infected with HCV (genotype 1b, M1LE isolate) in vitro and isolated #50-1 cells that replicate subgenomic RNA with some amino acid mutations.

We previously searched for critical residues of RdRP activity in NS5B sequences rather than conserved sequences among HCV isolates by a two-step scanning process with clustered and point alanine-substitution libraries and identified five residues in a JK-1 isolate (34). Among them, Glu-18 and His-502 are critical for homomeric oligomerization, Tyr-276 is crucial for template/primer binding, and the role of Tyr-191 and Cys-274 remains to be elucidated (47). In this study, we evaluated the role of these residues in HCV replication in vivo using a HCV replicon system derived from an M1LE isolate (31). Here, we report that the five residues critical for RdRP activity are indispensable for HCV replication. Also, we discovered the incompetence of JK-1 NS5B in HCV replication and specified four residues within or in vicinity of the conserved motifs responsible for the incompetence that may reflect the labile property of JK-1 NS5B in diluted concentrations or at high temperatures.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of Plasmids—pNNRZ2RU (31) harbors a subgenomic replicon derived from the cell line MT-2C infected with HCV (genotype 1b, GenBank^TM accession number AB080299 [GenBank] , M1LE isolate) and contains cDNA of the wild type (WT) replicon, M1LE/wild. For convenience, pNNRZ2RU was digested with MluI and BglII, and the MluI and BglII fragments were inserted into pGL3 Basic vector (Promega) to create pGL3-MluI-BglII. An adaptive mutation S232I within NS5A, which significantly increases RNA replication, was introduced into this plasmid to create pGL3-MluI-BglII-S232I. It was then used as an intermediate vector. All mutations were introduced once into pGL3-MluI-BglII-S232I, and the MluI and BglII fragments of pGL3-MluI-BglII-S232I containing each mutation were ligated back to the MluI and BglII sites of pNNRZ2RU to create each mutant (39). MA is the replicon carrying the point mutation S232I in NS5A and was used as a wild type in this study. To generate M1LE/VDD as a negative control, a point mutation that changed the GDD motif of NS5B to VDD was introduced into M1LE/wild as previously reported (39).

We followed two steps to construct MA/JK5B. First, HCV JK-1 cDNA (36) harboring NS5A and NS5B was subcloned by PCR using the primers JK5BBglII For and 5BAgeI Rev, and then this fragment was inserted into BglII and AgeI sites of MA to generate MA/JKIV. Next, HCV JK-1 cDNA was subcloned by PCR using the primers 5AAatII For and JK5BBglII Rev. We digested this fragment using AatII and BglII to generate two fragments, AatII-AatII and AatII-BglII, because there are two AatII sites and one BglII site within the fragment. These two fragments were inserted into the AatII and BglII sites of pGL3-MluI-BglII-S232I to create pGL3/JK(I+II+III), and the correct direction was confirmed by PCR using the corresponding primers. The fragment from pGL3/JK(I+II+III) digested by MluI and BglII was reintroduced into the corresponding MA/JKIV site.

To construct MA/JKI, we digested pGL3/JK(I+II+III) using AatII, inserted it into pGL3-MluI-BglII-S232I to yield pGL3/JKI, and confirmed the correct direction by PCR. To generate MA/JKII and MA/JKIII, we first constructed pGL3/JK (II+III). For this purpose, we digested pGL3-MluI-BglII-S232I with AatII then inserted this fragment into the corresponding part of pGL3/JK(I+II+III) to produce pGL3/JK (II+III). We confirmed the correct direction by PCR. To generate MA/JKII, we moved the fragment pGL3-MluI-BglII-S232I digested by Eco81I and BglII into the corresponding sites of pGL3/JK (II+III) to yield pGL3/JKII. We produce MA/JKIII by moving the fragment pGL3/JK (II+III) digested by Eco81I and BglII into pGL3-MluI-BglII-S232I to create pGL3/JKIII.

M1LE/E18A from NS5B was generated by PCR using primers carrying nucleotide alternations, and 5AAatII For or NR5Bt Rev. The PCR fragment containing this mutation was digested by AatII then inserted into the AatII sites of pGL3-MluI-BglII-S232I, and the direction was confirmed by PCR. MA/Y191A, C274A, Y276A, C213N, D220C, N231S, S288N, D444G, and MA/4M(C213N/D220C/N231S/S288N) of NS5B were individually generated by PCR using primers carrying the necessary nucleotide alternations along with 5BSEF5 or NR5Bt Rev. PCR fragments containing each mutation were digested with Eco81I and BglII and inserted into the Eco81I and BglII sites of pGL3-MluI-BglII-S232I. MA/H502A from NS5B was also generated by PCR using primers carrying the corresponding mutation and 5BSEF5 or 5BAgeI Rev. The PCR fragment containing this mutation was digested using BglII and AgeI and then inserted into the corresponding site of MA.

To create MA/JKIII4M(N213C/C220D/S231N/N288S of NS5B), MA/JKIII was subcloned by PCR using primers with the necessary mutation and Eco81I For or NR5Bt Rev. This PCR fragment was digested by Eco81I and BglII and inserted into pGL3-MluI-BglII-S232I. To produce MA/JK5B4M(N213C/C220D/S231N/N288S), MA/JK5B4MC213N, MA/JK5B4MN231S, MA/JK5B4MD220C, and MA/JK5B4MS288N, mutations were introduced by PCR using primers with the necessary mutations and 5BSEF5 or NR5Bt Rev, and PCR fragments containing each mutation were inserted into the Eco81I and BglII sites of pGL3/JK(I+II+III).

A plasmid derived from pGENK1, pGENKS (16, 35), was used to express recombinant glutathione S-transferase (GST)-fused protein in Escherichia coli, which contains multiple cloning sites (EcoRI, SacI, KpnI, XmaI, SalI, and BamHI) downstream of the sequences encoding the GST protein. HCV JK-1 and M1LE cDNA harboring NS5B were subcloned by PCR with JK5B For/JK5Bt Rev and NS5B For/NS5Bt Rev, respectively, which have artificial SacI and BamHI sites. The PCR fragments were inserted into the SacI and BamHI sites of pGENKS to yield pGENKS-JK-GST5Bt/WT and pGENKS-M1LE-GST5Bt/WT, which encode wild type GST-fused NS5B proteins. pGENKS-M1LE-G-ST5Bt/Y191A, pGENKS-M1LE-GST5Bt/C274A, pGENKS-M1LE-GST-5Bt/Y276A, pGENKS-M1LE-GST5Bt4M(C213N/D220C/N231S/S-288N), pGENKS-JK-GST5Bt4M(N213C/S231N/C220D/N288S), pGEN-KS-JK-GST5Bt4M/C213N, pGENKS-JK-GST5Bt4M/N231S, pGENKS-JK-GST5Bt4M/D220C, and pGENKS-JK-GST5Bt4M/S288N, each encoding mutant GST-fused NS5B protein, were constructed by digestion and transfer of the corresponding pGL3-MluI-BglII-S232I DNA containing each mutation. First, pGL3-MluI-BglII-S232I containing each mutation was digested with MunI and BglII, and these fragments were introduced into pGENKS-M1LE-GST5Bt/WT or pGENKS-JK-GST5Bt/WT, respectively. To generate pGENKS-M1LE-GST5Bt/E18A and pGENKS-M1LE-GST5Bt/H502A, mutations were introduced by PCR using the primers with the necessary mutation and NR5B For or NR5Bt Rev. The PCR fragments were individually inserted into the SacI/MunI and MunI/BamHI sites of pGENKS-M1LE-GST5Bt/WT. The sequences of all the constructs were confirmed using the dideoxy sequence method. The main primers used for plasmid construction are shown in Table 1.

In Vitro Transcription and Purification of RNA—The replicon RNAs were synthesized in vitro and purified as previously reported (39).

RNA Transfection and Selection of G418-resistant Cells—Transfection into Huh-7 cells was carried out by electroporation as previously reported (39). For the selection of G418-resistant cells, the medium was replaced with fresh medium containing 1 mg/ml G418 (Geneticin, Invitrogen) 24–48 h after transfection, and the medium was changed twice a week thereafter. Three weeks after transfection, G418-resistant cell colonies were stained with Coomassie Brilliant Blue (CBB) (0.6 g/liter in 50% methanol-10% acetic acid).

Expression and Purification of Bacterial Recombinant NS5Bt Protein—GST-fused HCV NS5Bt protein was expressed and purified as previously described (16).

Poly(A)-dependent UMP Incorporation Assay—The RdRP activity of GST-NS5Bt was examined by a UMP incorporation assay as previously reported (16). 20 ng of GST-NS5Bt protein was incubated at 25 °C for 60 min in a reaction solution (20 µl) containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 1 mM dithiothreitol, 1 mM EDTA, 1% (v/v) Triton X-100, 40 units of RNase inhibitor, 4 µCi of [-³²P]UTP (800 Ci/mmol), 10 µM UTP, 10 µg/ml poly(A), and 1 µg/ml oligo(U)14. The reaction was stopped by transferring the reaction solution to a DE81 filter (Whatman), and the filter was then extensively washed with 0.5 M Na₂HPO₄ (pH 7.0) and briefly rinsed with 70% ethanol. The filter-bound radioactivity was measured using a scintillation counter.

RdRP Activity Assay in LE19 Template—RNA LE19 (38) was chemically synthesized. The standard RdRP assay consisted of 0.125 µM concentration of template in a 20-µl reaction mixture containing 20 mM sodium glutamate (pH 8.2), 12.5 mM dithiothreitol, 1% (v/v) Triton X-100, 200 µM ATP and UTP, 200 µM GTP, 250 nM [-³²P]CTP (Amersham Biosciences), and 20 ng of protein. MgCl₂ or MnCl₂ was added to a final concentration of 2 mM, and the RNA synthesis reaction mixtures were incubated at 25 °C for 60 min (38). The reaction was stopped by transferring the reaction solution to a DE81 filter (Whatman), and the filter was then extensively washed with 0.5 M Na₂HPO₄ (pH 7.0) and briefly rinsed with 70% ethanol. The filter-bound radioactivity was measured using a scintillation counter.

Western Blotting Assay—20 or 160 ng of JK-GST5Bt/WT, M1LE-GST5Bt4M, and M1LE-GST5Bt/WT was analyzed using Western blotting assay prior to or after incubation at 37 °C in the presence of RdRP reagent (as shown above) for 1 h. We used the anti-NS5B (14–5) monoclonal antibody, which was kindly provided by Dr. M. Kohara (Tokyo Metropolitan Institute of Medical Science, Rinshoken, Japan).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Five NS5B Residues Are Critical for HCV Replication in Vivo —Previously, we identified five amino acid residues in NS5B of a JK-1 isolate that are critical for RdRP (Figs. 1 and 2). Alanine substitution at these residues abolished RdRP activity of purified bacterial recombinant NS5B protein in a glutathione S-transferase (GST)-fused form, GST-NS5Bt, that was missing the C-terminal membrane recruitment domain (34). The one or more roles of these residues remain to be evaluated in vivo, because these properties of the residues were only studied in vitro. We then applied the M1LE HCV replicon to Huh-7 cells (40). The components of the replicon are similar to a construct previously reported (25), but NS3–NS5B were derived from an M1LE isolate (Fig. 2). For enhanced HCV replication efficiency, we utilized an improved HCV RNA replicon system using an M1LE HCV replicon harboring the S232I-adapted mutation (conventionally designated MA in this report), and Huh-7-KV-C cells, a Huh-7 cell line cured from replicating HCV replicon by interferon treatment (39). In vitro transcribed replicon RNA with one of the five mutations of NS5B (E18A, Y191A, C274A, Y276A, or H502A) was transfected by electroporation into the Huh-7-KV-C subline, and cells were cultured in the presence of G418. As a negative control, a replicon M1LE/VDD in which the GDD motif of NS5B was mutated to VDD was used. The GDD motif is highly conserved among viral RdRPs, and even NdNPs and is critical for RdRP activity of NS5B, whereas NS5B/VDD has no RdRP activity (16). After G418 selection for 3 weeks, more than 900 colonies with the MA replicon were visible, and no colonies were observed with these five mutant replicons or the replicon M1LE/VDD (Fig. 3A), indicating that all five residues are critical for HCV RNA replication in vivo.

View larger version (49K): [in this window] [in a new window]   FIG. 1. Residues in HCV NS5B critical for RdRP activity in vivo and in vitro. Five previously reported critical residues, aa 18, 191, 274, 276, and 502 (yellow), two critical residues, aa 220 and 288 (green), and two important residues, aa 213 and 231 (blue) that were identified in this study are shown in secondary structures according to a crystal model of HCV NS5B (Protein Data Bank accession number 1QUV [PDB] ) as reported by Ago et al. (18). Graphics were processed using Cinima 3D version 4.0.

View larger version (56K): [in this window] [in a new window]   FIG. 2. Schematic presentation of HCV mutant replicons. NS5B was separated into four regions by convenient restriction sites: I (aa 1–108), II (aa 108–183), III (aa 183–448), and IV (aa 448–590). The amino acids analyzed are shown as follows: critical residues (dashes), important residues (arrow), and replaceable residue (double dashes). MA is the M1LE wild type with the adaptive mutation S232I in NS5A (39). We prepared several kinds of chimeric replicons as shown here. MA/JKI-MAJKIV are chimeric replicons harboring region I, II, III, or IV of JK-1 NS5B, respectively. MA/JKIII4M is a replicon that harbors the region III of JK-1 NS5B with four mutations, N213C/C220D/S231N/N288S, and is derived from M1LE. MA/JK5B4M is a replicon that harbors JK-1 NS5B with four mutations, N213C/C220D/S231N/N288S, and is derived from M1LE. The open boxes show the sequence derived for M1LE NS5B. The hatched boxes show the sequence derived for JK-1 NS5B.

View larger version (36K): [in this window] [in a new window]   FIG. 3. Effect of a point mutation in NS5B and JK-1 5B on HCV RNA replication. Huh-7-KV-C cells were transfected with 1 µg of in vitro transcribed replicon RNAs. G418-resistant cells were selected for at a G418 concentration of 1 mg/ml. G418-resistant cell colonies were stained by CBB 3 weeks after transfection. This figure shows the mean number of G418-resistant cell colonies isolated per 10-cm-diameter cell culture dish, per 1 µg of RNA. Error bars indicate the standard deviations of at least three independent experiments. A, RNA replicons with five critical residues as previously reported. B, RNA replicons are MA, MA/JK5B, MA/JKI, MAJKII, MAJKIII, MAJKIV, and M1LE/VDD. C, comparison of the sequence of NS5B part III among JK1, M1LE, and Con1. The residues with a black background are those conserved between M1LE and Con1 but not in JK1. D, RNA replicons with four newly identified amino acid residues. MA, positive control; MILE/VDD, negative control.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Effect of the four residues in region III of NS5B on HCV replication. Huh-7-KV-C cells were transfected with 1 µg of in vitro transcribed replicon RNAs. The G418-resistant cells were selected for at a G418 concentration of 1 mg/ml. G418-resistant cell colonies were stained by CBB 3 weeks after transfection. This figure shows the mean number of G418-resistant cell colonies isolated per 10-cm-diameter cell culture dish, per 1 µg of RNA. Error bars indicate the standard deviations of at least three independent experiments. A, transfected replicons are MA, MA/JKIII4M, MA/JK5B4M, and M1LE/VDD. B, transfected replicons are MA/JK5B4M and MA/JK5B4Ms with one JK-1 residue as indicated.

View larger version (40K): [in this window] [in a new window]   FIG. 5. Purification of NS5Bt proteins and RdRP activities of mutant NS5Bts. A, the GST-5Bt proteins used in this study were expressed in E. coli and purified as described under "Materials and Methods." Each purified protein was analyzed by 10% SDS-PAGE and stained with CBB. The expected molecular mass of the GST-5Bt proteins was about 95 kDa. Lanes: 1, JK-GST5Bt/WT (NS5B was derived from a JK-1 isolate); 2, M1LE-GST5Bt/WT (NS5B was derived from an M1LE isolate); 3, M1LE GST5Bt/E18A; 4, M1LE-GST5Bt/Y191A; 5, M1LE-GST5Bt/C274A; 6, M1LE-GST5Bt/Y276A; 7, M1LE-GST5Bt/H502A; 8, JK-GST5Bt4M (JK-GST5Bt/WT with four combined mutations N213C, C220D, S231N, and N288S); 9, JK-GST5Bt4M/C213N; 10, JK-GST5Bt4M/D220C; 11, JK-GST5Bt4M/N231S; 12, JK-GST5Bt4M/S288N; and 13, M1LE-GST5Bt4M. B, the RdRP activity of JK-GST5Bt/WT, M1LE-GST5Bt/WT, and JK-GST5Bt4M were measured using three different assay systems as described under "Materials and Methods." Error bars indicate the standard deviations of at least three independent experiments. 1) [-32P]UMP incorporation assay with poly(A) and oligo(U) as a template and primer; 2) LE19 de novo initiation; and 3) LE19 primer extension. C, RdRP activities of the purified GST-5Bt proteins JK-GST5Bt4M, JK-GST5Bt4M/C213N (JK-GST5Bt4M with aa 213 changed back to the JK-1 isolate), JK-GST5Bt4M/D220C (JK-GST5Bt4M with aa 220 changed back to the JK-1 isolate), JK-GST5Bt4M/N231S (JK-GST5Bt4M with aa 231 changed back to the JK-1 isolate), and JK-GST5Bt4M/S288N (JK-GST5Bt4M with aa 288 changed back to the JK-1 isolate), were measured using three different systems as shown in Fig. 5B (see "Materials and Methods"). Error bars indicate the standard deviations of at least three independent experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 6. Properties of the RdRP activity of the purified JK-GST5Bt/WT, M1LE-GST5Bt4M, and M1LE-GST5Bt/WT. The RdRP activities of the purified proteins were measured by an LE19 de novo synthesis assay under different reaction conditions (see "Materials and Methods"). A, the reactions were carried out for 60 min at different temperatures using 1% Triton X-100 and 160 ng of protein. B, the reactions were carried out for 60 min at different temperatures using 1% Triton X-100 and 20 ng of protein. C, the different amounts of three kinds of proteins JK-GST5Bt/WT (lanes 1, 2, 7, and 8), M1LE-GST5Bt4M (lanes 3, 4, 9, and 10), and M1LE-GST5Bt/WT (lanes 5, 6, 11, and 12) were checked by Western blotting using anti-NS5B prior to (lanes 1, 3, 5, 7, 9, and 11) or post (lanes 2, 4, 6, 8, 10, and 12) incubation in 37 °C for 1 h in the presence of RdRP reagents.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

According to the NS5B crystal models (Fig. 1), Glu-18 is located in the middle of a unique A1-loop that provides many contact points during the proposed "closed" conformation of the enzyme (42). His-502 is in helix T, the part of the thumb most distal to the catalytic center, and close to the second GTP-binding pocket, which may be critical for a conformation change in NS5B (37). We previously identified these two residues in the HCV-specific substructures, which are critical for homomeric oligomerization, a prerequisite of RdRP activity (47). Tyr-191 is located between helix H and I in the palm, and this bulky residue may contribute to the proper confirmation of template and primer as suggested by Bressanelli et al. (19) and Ago et al. (18), or to NTP substrate binding. The critical residues, Cys-274 and Tyr-276, are proximal to motif B and on the very edge of the fingertips (Fig. 1). Tyr-276 might play a critical role in template or template/primer binding, because NS5Bt/Y276A is defective in partial double-strand RNA-binding (34). The exact role of Cys-274 remains uncertain. The incompetence of alanine substitution of the five residues in NS5B in the M1LE replicon might reflect the weak RdRP activity of NS5B. The RdRP activities of the mutant proteins are around 5% of the M1LE-GST5Bt/WT. If HCV replication ability reflects RdRP activity, then detectable colonies should appear due to M1LE replicon with a substitution at one of the five residues, because the wild M1LE replicon emerged in more than 900 colonies. However, no colony was reproducibly observed bearing MA/JK5B.

We found that NS5B of the JK-1 isolate can not replicate in the chimeric HCV replicon in Huh-7 cells. Mapping the residues responsible for this incompetence identified two critical residues, aa 220 and 288, and two important ones, aa 213 and 231 (Fig. 1). We confirmed this using several chimeric and mutant replicons, which showed that these 4 JK-1 residues are sufficient in explaining the incompetence of JK-1 NS5B during HCV replication. Asp-220 is located in the catalytic pocket and is important for substrate binding (13). It is also close to Arg-222, which is involved in NTP trafficking (49). aa 288 is located in motif B, near the catalytic center of NS5B, and is also near aa 286, 287, and 291, of which mutations were reported to result in defective RdRP activity (11, 13, 46). Mutations at aa 287 and 291 might also be involved in RNA binding (11). aa 213 is located in motif A in the palm, and aa 231 is located at the edge of motif A. Interestingly, all four residues are in the catalytic pocket and within or in the vicinity of the motifs conserved among NdNp. Furthermore, the abilities of HCV replication correlate well with the RdRP activities of mutant NS5Bt proteins. These results support the hypothesis that the critical or important properties of the four residues during HCV replication in vivo reflect the RdRP activities of NS5B proteins.

The results showing the RdRP activities of GST-NS5Bt protein of JK-1, M1LE, and their mutant forms indicate that HCV replication in vivo correlates well with RdRP activity in vitro. M1LE NS5B proteins harboring one substitution mutation among the five residues are defective during HCV replication but exhibit weak RdRP activity, suggesting that HCV replication requires RdRP activity above a certain threshold. The correlation between the abilities in vivo and in vitro of mutated M1LE NS5B at the four newly identified residues suggests that these residues are responsible for the catalytic activity and not the interactions between NS5B and other NS proteins in vivo.

When RdRP assays were carried out at higher enzyme concentrations, JK-1 NS5B protein exhibited low RdRP activity (Fig. 6A). Interestingly, JK-1 NS5B and M1LE NS5B, which harbor the four JK-1 residues share several common features, are defective during HCV replication in vivo (Fig. 3B and data not shown), have weak RdRP activity, and are labile at low concentrations and high temperatures in vitro (Fig. 6). It is not certain whether the labile property of these NS5B proteins is the cause of the weak RdRP activity. However, the labile property of these NS5B proteins seems to well explain their incompetence during HCV replication, because HCV replication may occur at lower enzyme concentrations and at a higher than optimal temperature than that used for RdRP assays. HCV RNA replication takes place in a distinctly altered membrane structure of the endoplasmic reticulum, a membranous web or membrane raft, where NS proteins are recruited through their own membrane association domains and protein-protein interactions (48, 52–54). Therefore, further comparative studies on HCV replication in vivo and in vitro are required.

In summary, we identified several residues that are important or critical for HCV replication in vivo and for RdRP activity in vitro. We found that HCV replication using the HCV replicon correlates well with the RdRP activity, although HCV replication seems to require RdRP activity above a certain threshold. Because HCV RdRP activity of NS5B is a potential target for antiviral drugs, the unique structural and functional properties of HCV NS5B described here may shed light on the mechanism of HCV replication and could also be useful in the design of specific inhibitors.

	FOOTNOTES

 
^* This work was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety, by research grants-in-aid for scientific research (B) and development, and by a grant-in-aid for scientific research on priority areas (C) in oncogenesis from the Ministry of Education, Sports, Culture, and Technology. 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.

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

¹ The abbreviations used are: HCV, hepatitis C virus; RdRP, RNA-dependent RNA polymerase; aa, amino acid(s); NS, nonstructural; WT, wild type; GST, glutathione S-transferase; CBB, Coomassie Brilliant Blue; NdNp, nucleic acid-dependent nucleic acid polymerases.

	ACKNOWLEDGMENTS

 
We are grateful to K. Shimotohno for providing the M1LE HCV replicons and to the members of our division for critical discussion. We thank M. Yasukawa and K. Kuwabara for their technical assistance.

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