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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Chapter 10Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase

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Abstract

Structural and functional studies of the hepatitis C virus (HCV) RNA-dependent RNA polymerase have contributed to our understanding of polymerase mechanism, viral RNA replication, and have generated targets for antiviral development. This review summarizes recent studies on the properties of the HCV polymerase.

Introduction

HCV, like other (+)-strand RNA viruses, uses its viral genomic RNA as a template for both translation and generation of a complementary (−)-stranded RNA intermediate. The (−)-stranded RNA is then used as the template for the synthesis of molar excess of (+)-stranded progeny RNA molecules. (For a good general review on RNA virus replication, see Buck, 1996.) A membrane-associated replicase enzyme complex consisting of virally encoded and host proteins is responsible for the replication of viral RNA. The catalytic subunit of the replicase complex is the HCV encoded nonstructural 5B protein (NS5B), which contains all the sequence motifs highly conserved among all the known RNA-dependent RNA polymerases (RdRps) (Poch et al., 1989). By extension of studies from the human immunodeficiency virus (HIV), where the reverse transcriptase is a primary target for effective antivirals, the HCV RdRp is considered an important target for drug development (Beaulieu and Tsantrizoa, 2004; Wu and Hong, 2003).

Analysis of HCV replication has been hampered by the lack of convenient animal model and efficient cell culture systems. As a result, compounds against HCV have been screened using either surrogate viruses such as bovine viral diarrhea virus (BVDV) (Bukhtiyarova et al., 2001), biochemical targets such as the NS3 protease-helicase and/or the NS5B RdRp (Sarisky, 2004), and hepatoma cell line Huh7 expressing the subgenomic replicon (Lohmann et al., 1999; Blight et al., 2000; Guo et al., 2001; Ikeda et al., 2002). Subgenomic replicons are increasingly used to screen and characterize antivirals (Horscroft et al., 2005), although inhibitors identified using such cell-based screens still need to be tested against all viral proteins encoded by the replicon system to determine the mechanism of action. Thus, the HCV NS5B remains a target of choice for both nucleoside and nonnucleoside inhibitors. This chapter we will focus on the structural and functional aspects of the HCV RdRp.

Expression of NS5B

Expression of recombinant NS5B in insect and bacterial cells provided valuable reagents for the biochemical characterization. NS5B expressed using the baculovirus system can perform RNA-dependent RNA synthesis (Behrens et al., 1996; Lohmann et al., 1997). However, generation of soluble full length NS5B in bacterial cells proved unsuccessful in spite of a number of attempts (Yuan et al., 1997). A hydrophobic profile of the NS5B revealed that the C-terminal 21 amino acid residues is highly hydrophobic and is predicted to insert into membrane. In fact, membrane association of the RdRp is essential for the replication of HCV subgenomic replicons in cells (Moradpour et al., 2004). Deletion of this C-terminal tail of NS5B resulted in a soluble protein that had properties similar to that of protein expressed using insect cells, indicating that the C-terminal tail contributes minimally to nucleotide polymerization (Yamashita et al., 1998). Vo et al. (2004) have evidence suggesting that the C-terminal tail in the recombinant NS5B protein will increase interaction with RNA. However, it is unclear how a hydrophobic region can affect RNA binding.

Enzymatically active NS5B proteins fused to glutathione S-transferase or a histidine tag have been generated (Yamashita et al., 1998; Oh et al., 1999; Ferrari et al., 1999). However, NS5B with a N-terminal histidine tag was expressed at a lower level and also had lower activity compared to the C-terminally tagged protein (Lohmann et al., 1997; Ferrari et al., 1999). This phenomenon may be related to the results from Lohmann et al. (1997, and 1998), who found that changes in the N-terminal residues of NS5B reduced RdRp activity in vitro (Lohmann et al., 1997 and 1998) and that residue Cys14 of NS5B contributes to RNA binding (Bressanneli et al., 1999; O’Farrell et al., 2003).

Structural Features of HCV NS5B

Similar to other known RdRps, the HCV NS5B also contains six conserved motifs designated A-F. Comparison of the HCV NS5B sequence with the poliovirus RdRp and the [var phi]6 RNA polymerase, two other model RdRps, is shown in Fig. 1. The three-dimensional crystal structure of HCV NS5B has been determined independently by several groups (Lesburg et al., 1999; Bressanelli et al., 1999; Ago et al., 1999). Using the right-hand analogy for polymerases (Joyce and Steitz, 1995), the HCV RdRp has discernable fingers, palm and thumb subdomains. An unusual feature of this polymerase is that, due to the extensive interactions between the finger and thumb subdomains, the HCV RdRp has an encircled active site (Fig. 2) (Lesburg et al., 1999; Bressanelli et al., 1999; Ago et al., 1999). These contacts restrict the flexibility of the subdomains and possibly constrain flexibility between the subdomains. The HIV reverse transcriptase and other DdRps are known to undergo transition from an open or to closed conformation upon template binding and polymerization (Doublie et al., 1999; Huang et al., 1998). Structural analysis of HCV NS5B (J4 strain) revealed that de novo initiation is the probable mode of RNA synthesis, and limited structural changes take place upon nucleotide binding (O’Farrell et al., 2003). Structural studies with RdRp from the HCV genotype 2a indicate the presence of two conformations of the protein even in the absence of template RNA, where the key difference between the two forms is the relative orientation of the thumb domain in relation to fingers and palm domains (Biswal et al., 2005). Since both conformations lacked RNA, whether the template RNA will induce the same structural change(s) remains to be determined.

Fig. 1. A schematic of the HCV RdRp depicting the locations of the motifs and domains.

Fig. 1

A schematic of the HCV RdRp depicting the locations of the motifs and domains. The sequence alignments of the six recognizable motifs within RdRps from HCV, the double-stranded RNA phage [var phi]6 and poliovirus (more...)

Fig. 2. The structure of the HCV RdRp.

Fig. 2

The structure of the HCV RdRp. The figure is from Lesburg et al. (1999), reprinted with the permission of the publisher and modified to denote the positions of the thumb, finger and palm domains, and the (more...)

Another unusual feature of NS5B is a β-hairpin loop that protrudes into the active site located at the base of the palm subdomain (Fig. 2). This 12 amino acid loop was suggested to interfere with binding to double stranded RNA due to steric hindrance (Hong et al., 2001). The poliovirus RdRp lacks a similar β-loop, a feature that may be related to the poliovirus RdRp normally directing genome replication with a protein-nucleotide primer (Paul et al., 1998). It was suggested that the β-loop could be involved in positioning the 3′ terminus of the viral RNA for correct initiation (Hong et al., 2001). Since the wild-type HCV RdRp is fully capable of primer-dependent RNA synthesis, additional factors are needed to prevent primer extension. Indeed, GTP can, along with structures within the RdRp, help prevent primer-extension (Ranjith-Kumar et al., 2003).

The C-terminal tail of NS5B also lines the RNA binding cleft in the active site. This region, which immediately precedes the C-terminal membrane anchorage domain, forms a hydrophobic pocket and interacts extensively with several important structural elements including the β-loop (Adachi et al., 2002; Leveque et al., 2003). Deletions of up to 55 residues of the C-terminal tail resulted in increased RdRp activity, suggesting a direct role for the C-terminal tail in RNA synthesis activity and providing evidence for regulation at the active site (Lohmann et al., 1997; Tomei et al., 2000; Ranjith-Kumar et al., 2002).

Catalytic Pocket

The active sites of HCV NS5B and HIV-1 reverse transcriptase are very similar and can be superimposed without significant steric clashes (Lesburg et al., 1999). The residues involved in nucleotidyl transfer are found in palm motifs A and C. Motif A harbors the metal binding residue D220 which is a part of conserved D-X4-D motif, while motif C has the conserved metal binding and nucleotidyl transfer residues D318 and D319 (Fig. 3). D225 within motif A forms a H-bond with the ribose 2′-hydroxyl group of the NTP and is thought to discriminate against the use of dNTPs. Crystal soaking experiments with HCV NS5B with NTPs revealed several residues in the catalytic pocket that contact the triphosphates of NTP (Fig. 3). These include R158, S367, R386, T390 and R394. Unfortunately, it was not possible to identify the base-interacting residues. The role of these residues in RdRp activity is discussed in more detail later in this chapter.

Fig. 3. The catalytic NTP binding pocket in the HCV RdRp.

Fig. 3

The catalytic NTP binding pocket in the HCV RdRp. The structure is from Bressanelli et al. (2002) and is reprinted with the permission of the publisher. Left panel: a detailed view of the NTP binding site (more...)

Soaked NS5B crystals with GTP also revealed a low affinity GTP binding site on the surface of the protein (Fig. 4; Bressanelli et al., 2002). This second site, which is apparently specific for GTP, lies between the fingers and thumb subdomains, approximately 30 Å away from the catalytic pocket. By virtue of its position and the requirement of higher GTP concentrations to saturate the pocket, it was suggested that it might play a regulatory role in RNA synthesis (Bressanelli et al., 2002). To date, any allosteric properties of this low affinity site in response to GTP has not been determined.

Fig. 4. A low affinity rGTP binding site in the HCV RdRp.

Fig. 4

A low affinity rGTP binding site in the HCV RdRp. The structure is from Bressanelli et al. (2002) and reprinted with the permission of the publisher. Left panel: a view of the back of the HCV RdRp emphasizing (more...)

Activities of NS5B

Recombinant NS5B was sufficient to synthesize full length HCV RNA in vitro (Hwang et al., 1997; Yamashita et al., 1998; Behrens et al., 1996; Lohmann et al., 1997; Ferrari et al., 1999). This observation indicates that NS5B can also unwind stable secondary and tertiary RNA structures. As covered in Chapter 2 of this book, the 5′ and 3′ untranslated regions (UTRs) of the HCV genome contain highly ordered and complex RNA structures , which are highly conserved and contain cis-acting elements for viral RNA replication. Recently it was shown that a pseudoknot structure is formed between the 3′ end of the HCV genome and a novel RNA element in the NS5B coding sequence (Friebe et al., 2005). The 3′ terminal 150 nt of the HCV RNA contain signals that are essential for RdRp binding and replication of viral RNA (Yi and Lemon, 2003a and b; Reigadas et al., 2001; Cheng et al., 1999). In vitro HCV NS5B was found to replicate the 3′ terminal region of the (−)-strand RNA more efficiently than the 3′ terminal region of (+)-strand RNA (Reigadas et al., 2001). Analysis of the promoter elements in 3′ terminus of (−)-strand revealed that complementary strand of second stem-loop of the internal ribosome entry sequence (IRES) binds NS5B and acts as a positive element for RNA synthesis (Kashiwagi et al., 2002). However, the complementary strand of the first stem-loop of the IRES worked as a negative regulator of RNA synthesis (Kashiwagi et al., 2002).

Though some genome specific recognition was observed, recombinant NS5B largely lacked specificity for binding to HCV RNA (Lohmann et al., 1997). Several groups used different RNAs to analyze RdRp activity. In general, the HCV NS5B preferred a template with a stable 5′ secondary structure(s) and a single stranded sequence that contained at least one 3′ cytidylate (Kao et al., 2000). This observation was further supported when high-affinity RNA ligands to HCV NS5B were isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure (Vo et al., 2003). The high affinity RNA was found to have three stem-loop structures.

Our lab uses short RNAs to study de novo initiation of RNA synthesis by the HCV NS5B because products from these templates can be identified with single-nucleotide resolution. The prototype RNA, LE19, was derived from BVDV. LE19 is predicted by mfold to form a stem-loop with five intramolecular base pairs and with singlestranded sequences of three nucleotides at both the 5′ and 3′ ends (Fig. 5). The 3′ sequence contains a cytidylate that can be used as an initiation nucleotide. Two LE19 molecules also form a heterodimer which can be extended to form a 32-nt primer extension product. In addition, NS5B can add nontemplated nucleotides to the RNA, a process known as terminal nucleotidyl transferase (TNTase). Lastly, HCV RdRp can generate recombinant RNA product from two or more non-covalently linked templates, a process known as template switch. All these activities can be studied using LE19 in a single reaction (Fig. 5). The different activities of NS5B and their potential importance in viral replication are discussed in detail below.

Fig. 5. An RNA template that can be used to examine several activities of the HCV RdRp in one reaction.

Fig. 5

An RNA template that can be used to examine several activities of the HCV RdRp in one reaction. A) Schematics of the various activities of the HCV RdRp using LE19. LE19 exists in an equilibrium between monomeric (more...)

De novo Synthesis and Primer Extension

Initiation of RNA synthesis in infected cells likely starts de novo, by use of a one-nucleotide primer (Bressanelli et al., 2002; Ferrari et al., 1999). This process is well studied in DNA-dependent RNA polymerase, which uses two NTP binding sites in the catalytic pocket. The first site is called the I site and specifically recognizes the initiating NTP (NTPi). The second site, the I+1 site, is less specific and recognizes the NTP complementary to the second template nucleotide. The flaviviral RdRps can also accommodate two NTPs, with the I site preferentially binding to GTP (Ferrari et al., 1999; Kao et al., 1999; Lohmann et al., 1999; O’Farrell et al., 2003; Oh et al., 1999; Luo et al., 2000; Ranjith-Kumar et al., 2002; Sun et al., 2000; Zhong et al., 2000; Zhong et al., 2000). The catalytic aspartates coordinate divalent metals that are in position to help form a phosphodiester bond between the NTPi and the second NTP (Ferrari et al., 1999; O’Farrell et al., 2003).

While GTP is generally accepted as the NTPi for RNA synthesis by the HCV RdRp in vitro, the 3′ terminal residue of many HCV isolates is a uridylate, suggesting that ATP may be used to initiate (−)-strand RNA synthesis. Recently, a subgenomic replicon that was passaged in vitro was demonstrated to switch from using GTP to ATP as the NTPi for both (+)- and (−)-strand RNA replication (Cai et al., 2004). The exact identity of the NTPi is specific for a purine triphosphate, but can be somewhat flexible, as it is for DNA-dependent RNA polymerases (Kuzmine et al., 2003 and references within).

Polymerases require divalent metal ions for activity. RNA synthesis by NS5B is increased by 4–20 fold when Mn2+ is present in the reaction in comparison to a reaction with only Mg2+ (Zhong et al., 2000; Ferrari et al., 1999; Luo et al., 2000; Ranjith-Kumar et al., 2002). However, other divalent metal ions such as Co2+, Cu2+, Ni2+ and Zn2+ did not support RdRp activity (Luo et al., 2000; Ranjith-Kumar et al., 2002). Recently it was shown that iron binds specifically to the Mg2+ binding site of NS5B and can inhibit RNA synthesis (Fillebeen et al., 2005). Mn2+ appears to more specifically contribute to de novo initiation by lowering the KM for GTP by about 30-fold (Ranjith-Kumar et al., 2002). While the concentration of Mn2+ used in vitro is far higher than concentrations present in the cell, physiologically relevant Mn2+ levels can increase de novo initiation with GTP (Ranjith-Kumar et al., 2002). Analysis of proteins with C-terminal deletions revealed that the C-terminus of the HCV RdRp plays a role in Mn2+ induced de novo initiation and can contribute to the suppression of primer extension (Ranjith-Kumar et al., 2002). Spectroscopy examining the intrinsic tryptophan and tyrosine fluorescence of the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of divalent metals (Ranjith-Kumar et al., 2002; Bougie et al., 2003).

Even though de novo initiation seems to be the preferred mechanism of initiation, it has always been puzzling that primer-extension activity was far more robust than de novo initiation in vitro. This begs the question of whether the two mechanisms will affect each other. Deletion of the β-loop led to an increase in primer extension activity (Hong et al., 2001). However, this deletion was only part of the requirement for the suppression of primer extension since additional characterizations revealed that the NTPi-binding site, higher concentrations of the initiation GTP, and the C-terminal tail that lines the catalytic pocket all participate to suppress primer extension (Ranjith-Kumar et al., 2003). Thus, the features and requirements for de novo initiation by the HCV RdRp significantly prevent primer extension.

Mutational Analysis

Site directed mutagenesis has been employed to study the role of specific residues in RdRp activities (Lohmann et al., 1997, Qin et al., 2001; Labonte et al., 2002; Ranjith-Kumar et al., 2002, 2003 and 2004). Mutation of the conserved catalytic residues D318 and D220 resulted in inactive proteins. Lohmann et al. (1997) investigated the effects of mutations of some conserved residues in motifs A, B, C and D. Most of the mutations on the conserved residues in motifs A and B led to inactive protein and inactive subgenomic replicons in cell culture (Cheney et al., 2002). However, mutations of the conserved residues G317, and D319 in motif C were tolerated somewhat. Interestingly, substitution of R345 with lysine in motif D enhanced the enzymatic activity (Lohmann et al., 1997). A similar increase in activity was observed when K151 was mutated to glutamate (Labonte et al., 2002). Qin et al., (2001) generated a series of clustered and point mutations and studied their effects on RdRp activity and template binding. The residues that affected RdRp activity included E18, Y191, C274, Y276 and H502. Y276 was also found to be important for interaction with the template/primer.

The structures of the HCV RdRp and nucleotides identified a number of interacting residues at D225, R48, R158, R386, R394, and S367 that interact with the initiation GTP (Bressanelli et al., 2002; Fig. 3). In this structure, it is not clear whether the GTP is binding to the I site or the I+1 site. NTPi binding to the I site is base-specific while binding of the second NTP to the I+1 site should be directed by the template (Ranjith-Kumar et al., 2002). Because the RdRp-GTP structure was determined without a template, it is likely that the residues identified recognized the NTPi. Alanine substitutions of these residues were analyzed for effects on de novo initiation, primer extension, TNTase, and template switch (Ranjith-Kumar et al., 2004). Although all mutations retained the capability for primer extension, alanine substitutions at R48, R158, R386, R394, and D225 decreased de novo initiation, and two or more mutations in combination abolished de novo initiation (Ranjith-Kumar et al., 2004). It is likely that these mutations affected the stability of the initiation complex, since many of the defects were rescued when the reactions were supplemented with Mn2+. We note that several of the mutant enzymes were selectively affected for de novo initiation and/or terminal nucleotide addition, indicating that the residues in the active site can contribute differentially to the known activities of the HCV RdRp. Furthermore, while the prototype enzyme had a KM for GTP of 3.5 μM, all mutations except one negatively affected the KM for GTP by 3 to 7 fold, demonstrating that the affected residues are functionally required to interact with the initiation nucleotide. Lastly, mutations in D225 are dramatically affected in template switch, suggesting that this residue of the NTPi pocket also participates in the elongation complex.

Terminal Nucleotidyl Transferase (TNTase) Activity

TNTase activity could be a significant concern in biochemical screens where the products of the HCV RdRp are not visualized by denaturing gel electrophoresis. This activity was observed by some (Behrens et al., 1996; Ishii et al., 1999; Ranjith-Kumar et al., 2001), but not by others, leading some to suggest that it should be attributed to cellular contaminants rather than the HCV RdRp (Lohmann et al., 1997, Oh et al., 1999; Johnson et al., 2000; Zhong et al., 2000). Ranjith-Kumar et al. (2001; 2003) demonstrated that the HCV NS5B does have TNTase activity. First, mutation of the conserved GDD motif inactivated both polymerase and TNTase activities. Second, a HCV NS5B specific inhibitor inhibited both activities. Third, proteins purified from eukaryotic and prokaryotic expression systems both showed TNTase activity. Fourth, several mutations in the residues in the HCV RdRp that affected de novo initiation also affected TNTase activity, implicating the NTPi pocket in TNTase activity. Consistent with this last claim, Mn2+, which increases de novo initiation, also increases TNTase activity (Ranjith-Kumar, 2004). Perhaps one reason for the discrepant observations of TNTase activity from different laboratories is that the amount of TNTase depends on the template sequence and the NTPs used (Ranjith-Kumar et al., 2001).

Template Switch

RNA recombination contributes to genetic diversity and pathogenesis of RNA viruses (Nagy and Simon, 1997; Jarvis and Kirkegaard, 1991). HCV NS5B can generate RNA products that are larger than the size of the template RNA by a process wherein the RdRp ternary complex does not terminate RNA synthesis from a template, but will bind to a second template and continue RNA synthesis. Template switch by the related BVDV RdRp and replicase complexes from plant viruses have been characterized and require the template initiation cytidylate as well as the NTPi (Kim et al., 2001).

Several of the mutations in the NTPi pocket of the HCV RdRp affected template switch (Ranjith-Kumar et al., 2004). A defect in the template switch is to be expected with many of the NTPi mutants since fewer ternary complexes are available. However, mutant D225A, which was capable of robust de novo initiation from the first template, was debilitated for template switch in comparison to the wild-type RdRp (Ranjith-Kumar et al., 2004). We hypothesize that D225, which recognizes the ribose 2′ hydroxyl of the NTPi, also plays an additional role either in recognition of the second template or in the release of the nascent RNA

Interaction with Other HCV Nonstructural Proteins

HCV replication uses a replicase complex that is associated with the endoplasmic reticulum (ER) (Hwang et al., 1997). The complex is thought to contain both host and virally encoded proteins, although detailed information concerning the composition of the replicase remains to be determined. A growing number of cellular proteins that could affect HCV replication activity have been identified. For example, it was recently shown that NS5B is phosphorylated by a protein kinase C-related kinase, PRK2, and this phosphorylation is involved in regulating HCV replication (Kim et al., 2004). (For general reviews of cellular factors involved in (+)-strand RNA virus infection, please see Lai et al., 1998, and Kushner et al., 2003).

HCV nonstructural proteins are co-localized with NS5B within the ER membrane and likely modulate NS5B activities (Behrens et al., 1996; Brass et al., 2002; Egger et al., 2002; Hwang et al., 1997; Wolk et al., 2000). One case is that the HCV NS5B can form oligomers in vitro and may catalyze RNA synthesis in a cooperative manner (Wang et al., 2002; Qin et al., 2002). While the C-terminal tail is not involved in this process, it was shown that two amino acids, E18 and H502, are very critical for oligomerization (Qin et al., 2002). While NS5B oligomerization can be demonstrated by several independent assays, its role in the infected cell remains to be determined.

Other interactions between HCV NS5B and other HCV nonstructural proteins have already been reported with low-resolution assays, such as protein pull-down experiments, co-immunolocalization, and yeast two-hybrid experiments. These results indicate that NS5B likely binds to NS3, and that NS3 will interact with NS4A and possibly NS4B and NS5B (Piccininni et al., 2002). NS5A may interact with NS2, NS3, NS4A, NS4B, NS5B and with itself (Dimitrova et al., 2003, Shirota et al., 2002). The NS5A protein is important for HCV replication and mutation that can increase the efficiency of subgenomic replicon replication can be mapped to it (Blight et al., 2000; Krieger et al., 2001). By using GST pull down and coimmunoprecipitation assays, NS5A was shown to directly interact with NS5B and modulate its activity (Shirotta et al., 2002).

NS3 is a multifunctional protein possessing protease, helicase, and NTPase activities (Borowski et al., 2002). Recently, the NS5B protein was shown to bind to NS3 through the protease domain and increase the helicase activity of NS3 by approximately five fold (Zhang et al., 2005). These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, full-length NS3 reduced RNA synthesis by the NS5B RdRp in vitro, possibly due to the NTPase activity of NS3 degrading NTPs nonspecifically. Whether this effect is relevant in vivo is unclear, and additional regulations of NS5B activity in the context of the HCV replicase remain to be characterized.

A major hurdle in understanding the HCV replication complex is that the replicase is not only present in relatively low abundance, but enriched replicase can only perform elongative RNA synthesis from the template RNA that copurified with the enzymatic activity (Hardy et al., 2003; Lai et al., 2003, Ali et al., 2002). These features have made the replicase a poor reagent to understand the requirements for HCV replication in vitro. Nonetheless, the recent demonstration of the ability to produce HCV elongation-competent replicase is an important first step in its characterization and could provide a useful reagent to characterize drugs identified in cell-based screens.

Conclusion and Future Trends

Although the initial effort to characterize the HCV RdRp was in response to a need to develop biochemical targets against HCV, recent research by the virology and structural biology communities has developed a strong basic understanding of the mechanism of action of a viral RNA-dependent RNA synthesis. This information should also prove useful in understanding the mechanism of action of antivirals targeted against HCV replication. Despite the progress made, much remains to be done. One bottom line is that we still do not have an effective drug targeting the HCV RdRp. In terms of future biochemical analyses, we need to: 1) gain a deeper understanding of the mechanism of polymerization and the conformational changes as the polymerase goes from initiation to elongative synthesis to the termination of RNA synthesis. Toward this effort, a true ternary complex at high resolution will be critical; and 2) define the effects of other viral and cellular subunits that participate in RNA-dependent RNA replication. Since HCV replication is a process intimately associated with the cellular membranes, understanding HCV replication will benefit from an infusion of expertise from membrane biologists and biochemists, and scientists trained to study the mechanism of complex enzymes, including those who have analyzed nucleic acid synthesis from other template-dependent polymerases. It is such multidisciplinary approach that could ultimately yield significant benefits to society against a disease with enormous worldwide impact.

Acknowledgements

We thank our colleagues at Texas A and M University for helpful discussions and Y. Kim for editing this manuscript. The Kao lab is supported by the National Science Foundation MCB grant 0332259.

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