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

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.

Hepatitis C virus (HCV), 1 a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepa-titis (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 5Ј-untranslated region, an open reading frame, and a 3Ј-untranslated region (1,4,5). The order of the gene products of the single open reading frame is NH 2 -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)(18)(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 5Ј-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 3Ј-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
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, 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(IϩIIϩIII), and the correct direction was confirmed by PCR using the corresponding primers. The fragment from pGL3/JK(IϩIIϩIII) digested by MluI and BglII was reintroduced into the corresponding MA/JKIV site.
To construct MA/JKI, we digested pGL3/JK(IϩIIϩIII) 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 (IIϩIII). For this purpose, we digested pGL3-MluI-BglII-S232I with AatII then inserted this fragment into the corresponding part of pGL3/JK(IϩIIϩIII) to produce pGL3/JK (IIϩIII). 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 (IIϩIII) to yield pGL3/JKII. We produce MA/JKIII by moving the fragment pGL3/JK (IIϩIII) 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.
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- , 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.

Critical Regions of NS5B in HCV Replication
Huh-7-KV-C cells were prepared and grown as previously reported (39).
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).
RdRP Activity Assay in LE19 Template-RNA LE19 (38) (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 Na 2 HPO 4 (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).

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 S232Iadapted 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.
The Critical Role of the Five Residues in M1LE in RdRP Activity in Vitro -Although the five residues are well conserved among the HCV isolates, it remains uncertain that the replication incompetence of the mutants in the M1LE isolate is not due to a defect in RdRP activity. Therefore, we examined whether the five residues in M1LE are also critical for RdRP activity in vitro. The five mutant forms of NS5Bt in the M1LE isolate were expressed, purified, and subjected to three different RdRP assays (see "Materials and Methods"). A synthetic template and primer poly(A) and oligo(U) and LE19 template system with or without primer was used. LE19 is a 19-nucleotide RNA whose sequence is derived from the 3Ј-end of the minus strand of the bovine viral diarrhea virus genome. LE19 allowed us to use GTP only for initiation and not for elongation (38). The five mutant proteins exhibited very weak RdRP activities (2-7% of that of wild type M1LE) in the primer-dependent and de novo initiation assays (Table II). This result demonstrates that the five residues are critical for the RdRP activity of NS5B in the M1LE isolate as they are for those of the JK-1 isolate.
Residues Responsible for the Incompetence of JK-1 NS5B during HCV Replication-We first constructed a chimeric HCV replicon, MA/JK5B, in which M1LE NS5B in MA was replaced by JK-1 NS5B to address the role of the five residues (Fig. 2), but the chimeric replicon was defective during HCV replication (Fig. 3B). To gain an insight into this phenomenon, NS5B was divided into four parts, I, II, III, and IV, and several replicons harboring the chimeric NS5B of M1LE and JK-1 were constructed. Parts I-IV covered aa 1-108, 108 -183, 183-448, and 448 -590, respectively (Fig. 2). After transfection of these replicons and selection by G418, we found that MA/JKIII was responsible for the defectiveness of the NS5B JK-1 isolate during HCV replication, because the other three chimeric replicons, MA/JKI, MA/JKII, and MA/JKIV emerged as often in colonies as those with MA (Fig. 3B). To specify the responsible residue(s) in part III of JK-1, we compared the amino acid sequence of part III with those of JK-1, M1LE, and Con1 and found that residues Cys-213, Asp-220, Asn-231, Ser-288, and Asp-444 were conserved between M1LE and Con1 but not for JK-1 (Fig. 3C). Ala-252 was also conserved in M1LE and Con1, but previously we identified this amino acid did not affect the RdRP activity (34), and amino acids Ala and Val belong to the same group among the 20 kinds of amino acids, then we first check these five residues. The mutant MA replicons MA/ C213N, MA/D220C, MA/N231S, MA/S288N, and MA/D444G were found to harbor one of the nonconserved residues present in part III of JK-1 NS5B. After transfection and selection with G418, the replicon MA/D444G were present as often in the colonies as MA, and MA/C213N and MA/N231S were present in the colonies but at one-fourth to one-fifth that of MA. MA/ D220C and MA/S288N were defective in HCV replication similar to M1LE/VDD (Fig. 3D). This indicates that the two residues Ser-288 and Asp-220 are indispensable for HCV replication in vivo and that the two residues Cys-213 and Asn-231 are required for efficient HCV replication. Either residue Asp or Gly at aa 444 allows efficient HCV replication. To confirm this, we constructed the mutant replicon MA/JKIII4M or MA/JK5B4M by substituting the four residues of JK-1 at aa 213, 220, 231, and 288 with the corresponding M1LE NS5B amino acids (Fig. 2). After transfection and G418 selection, these replicons emerged as G418-resistant colonies as was the case for MA, indicating that the 4 JK-1 residues in part III are responsible for the defective property of JK-1 NS5B during HCV replication (Fig. 4A). To further confirm these results, we mutated one of the four M1LE residues of MA/JK5B4M back to the corresponding JK-1 residue. As expected, MA/JK5B4M D220C and MA/JK5B4M S288N could not form G418-resistant colonies, but MA/JK5B4M C213N and MA/JK5B4M N231S could but at a lower efficiency than MA/JK5B4M (Fig. 4B).
Correlation between HCV Replication in Vivo and RdRP Activity in Vitro -The incompetent property of JK-1 NS5B during HCV replication in vivo is inconsistent with the RdRP activity of JK-1 NS5B in vitro. Two possibilities may explain this phenomenon. It may be due to putative interactions between NS5B and other NS proteins through the four residues in part III of NS5B. Alternatively, the RdRP activity of JK-1 NS5B is much weaker than that of M1LE, and strong RdRP activities are required for HCV replication. We first examined the RdRP activities of the mutant proteins using GST-NS5Bt in three different assays. Mutant GST/NS5Bt proteins were expressed in E. coli and subsequently purified (see "Materials and Methods") (Fig. 5A). The mutant protein JK-GST5Bt4M with four amino acid substitutions at aa 213, 220, 231, and 288 complementary to those of M1LE exhibited similar RdRP activity to that of M1LE-GST5Bt/WT. In contrast, JK-GST5Bt/WT exhibited weak activity compared with M1LE-GST5Bt/WT under assay conditions. Similar differences in RdRP activities among these three proteins were observed using three different assay systems (Fig. 5B). We further constructed four different types of back mutation of JK-GST5Bt4M at one of the four M1LE residues analogous to the JK-1 residue. The mutant proteins JK-GST5Bt4M/D220C and JK-GST5Bt4M/S288N ex-hibited weak RdRP activities as did JK-GST5Bt/WT, whereas JK-GST5Bt4M/C213N and JK-GST5Bt4M/N231S exhibited activities greater than 25% of that for M1LE-GST5Bt/WT (Fig.  5C). Taken together, the HCV replication correlated well with RdRP activity. However, NS5Bs exhibited weaker RdRP activity in vitro and were incompetent during replication. This suggests that HCV replication requires a higher RdRP activity above a certain threshold (see "Discussion").
Heat-labile Property of JK-1 NS5B-The weak activity of JK-1 NS5Bt may be due to low concentrations in the assay. To examine this possibility, RNA incorporation was measured at different protein concentrations. The RdRP activity with JK-GST5Bt/WT or M1LE-GST5Bt4M enzyme was 11-15% of that of M1LE-GST5Bt/WT at higher protein concentrations (data not shown and Fig. 6A), but was relatively much weaker at lower concentrations (Fig. 6B). This finding may demonstrate the labile nature of JK-1 enzyme. Therefore, we next examined RdRP activities of these enzymes at different temperatures (Fig. 6). JK-GST5Bt/WT and M1LE-GST5Bt4M enzymes were found to be heat-labile at higher temperatures. The heat-labile property of the enzymes was observed evidently at the low concentration (no activity) and moderately at the high concen-tration (30 -33% of M1LE-GST5Bt/WT) at 37°C (Fig. 6). Such labile nature at lower concentrations and higher temperatures of the enzymes was similarly observed with the three RdRP assay methods (data not shown). The labile property may be a result of degradation of the labile enzyme during incubation. To test this possibility, we examined whether degradation of the proteins occurs in the high temperature. The labile protein (Fig. 6C) as well as the M1LE-GST5Bt/WT protein (data not shown) were not degraded much after incubation at 37°C in the RdRP reaction mixture, suggesting that the catalytic activity of the enzymes is labile. DISCUSSION HCV replication, RNA-dependent RNA synthesis, is distinct from the host macromolecular processes and thus has been regarded as a target for intervention to block chronic infection and eventual hepatocarcinogenesis (50,51). NS5B is a core replicating enzyme with RdRP activity (9, 13, 16), and it has  primer-dependent and primer-independent (de novo) properties (9, 10, 13-15, 21, 22, 41-45). We previously scanned about one-third of all NS5B or most of the conserved sequences among HCV isolates using two-step alanine scanning libraries and identified the five novel residues, Glu-18, Tyr-191, Cys-274, Tyr-276, and His-502, that are critical for RdRP activity using UMP incorporation assay (34). Here, we report that the five residues are critical for HCV replication in vivo in the improved M1LE replicon system (39,40).
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 GTPbinding 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 resi- dues 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)(53)(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  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. 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.