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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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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.


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 φ6 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 φ6 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.


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.


  1. Adachi T, Ago H, Habuka N, Okuda K, Komatsu M, Ikeda S, Yatsunami K. The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase. Biochim Biophys Acta. 2002;1601:38–48. [PubMed: 12429501]
  2. Ago H, Adachi T, Yoshida A, Yamamoto M, Habuka N, Yatsunami K, Miyano M. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure Fold Des. 1999;7:1417–1426. [PubMed: 10574802]
  3. Ali N, Tardif KD, Siddiqui A. Cell-free replication of the hepatitis C virus subgenomic replicon. J Virol. 2002;76:12001–12007. [PMC free article: PMC136887] [PubMed: 12414942]
  4. Beaulieu PL, Tsantrizos YS. Inhibitors of the HCV NS5B polymerase: new hope for the treatment of hepatitis C infections. Curr Opin Investig Drugs. 2004;5:838–50. [PubMed: 15600240]
  5. Behrens S-E, Tomei L, De Francesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 1996;15:12–22. [PMC free article: PMC449913] [PubMed: 8598194]
  6. Biswal BK, Cherney MM, Wang M, Chan L, Yannopoulos CG, Bilimoria D, Nicolas O, Bedard J, James MN. Crystal structures of the RNA dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J. Biol. Chem. 2005;280:18202–18210. [PubMed: 15746101]
  7. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science. 2002;290:1972–1974. [PubMed: 11110665]
  8. Borowski P, Schalinski S, Schmitz H. Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. Antiviral Res. 2002;55:397–412. [PubMed: 12206878]
  9. Bougie I, Charpentier S, Bisaillon M. Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase. J Biol Chem. 2003;278:3868–3875. [PubMed: 12458224]
  10. Brass V, Bieck E, Montserret R, Wolk B, Hellings JA, Blum HE, Penin F, Moradpour D. An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem. 2002;277:8130–8139. [PubMed: 11744739]
  11. Bressanelli S, Tomei I, Roussel A, Incitti I, Vitale RL, Mathieu M, DeFrancesco R. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci USA. 1999;96:13034–13039. [PMC free article: PMC23895] [PubMed: 10557268]
  12. Bressanelli S, Tomei L, Rey FA, DeFrancesco R. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol. 2002;76:3482–3492. [PMC free article: PMC136026] [PubMed: 11884572]
  13. Buck KW. Comparison of the replication of positive-strand RNA viruses of plants and animals. Adv Virus Res. 1996;47:159–251. [PubMed: 8895833]
  14. Bukhtiyarova M, Rizzo CJ, Kettner CA, Korant BD, Scarnati HT, King RW. Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate. Antivir Chem Chemother. 2001;12:367–373. [PubMed: 12018682]
  15. Cai Z, Liang TJ, Luo G. Effects of mutations of the initiation nucleotides on hepatitis C virus RNA replication in the cell. J Virol. 2004;78:3633–3643. [PMC free article: PMC371060] [PubMed: 15016884]
  16. Cheney IW, Naim S, Lai VC, Dempsey S, Bellows D, Walker MP, Shim JH, Horscroft N, Hong Z, Zhong W. Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture. Virology. 2002;297:298–306. [PubMed: 12083828]
  17. Cheng JC, Chang MF, Chang SC. Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3′ end of the viral RNA. J Virol. 1999;73:7044–7049. [PMC free article: PMC112794] [PubMed: 10400807]
  18. Dimitrova M, Imbert I, Kieny MP, Schuster C. Protein-protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 2003;77:5401–5414. [PMC free article: PMC153952] [PubMed: 12692242]
  19. Doublie S, Sawaya MR, Ellenberger T. An open and closed case for all polymerases. Structure Fold Des. 1999;7:R31–5. [PubMed: 10368292]
  20. Ferrari E, Wright-Minogue J, Fang JWS, Baroudy BM, Lau JYN, Hong Z. Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. J. Virol. 1999;73:1649–1654. [PMC free article: PMC103993] [PubMed: 9882374]
  21. Fillebeen C, Rivas-Estilla AM, Bisaillon M, Ponka P, Muckenthaler M, Hentze MW, Koromilas AE, Pantopoulos K. Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C Virus. J Biol Chem. 2005;280:9049–9057. [PubMed: 15637067]
  22. Friebe P, Boudet J, Simorre JP, Bartenschlager R. Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J Virol. 2005;79:380–392. [PMC free article: PMC538730] [PubMed: 15596831]
  23. Guo JT, Bichko VV, Seeger C. Effect of alpha interferon on the hepatitis C virus replicon. J Virol. 2001;75:8516–8523. [PMC free article: PMC115097] [PubMed: 11507197]
  24. Hardy RW, Marcotrigiano J, Blight KJ, Majors JE, Rice CM. Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J. Virol. 2003;77:2029–2037. [PMC free article: PMC140877] [PubMed: 12525637]
  25. Hong Z, Cameron CE, Walker MP, Castro C, Yao N, Lau JY, Zhong W. A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase. Virology. 2001;285:6–11. [PubMed: 11414800]
  26. Horscroft N, Lai VC, Cheney W, Yao N, Wu JZ, Hong Z, Zhong W. Replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis C virus. Antivir Chem Chemother. 2005;16:1–12. [PubMed: 15739617]
  27. Huang H, Chopra R, Verdine GL, Harrison SC. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science. 1998;282:1669–1675. [PubMed: 9831551]
  28. Hwang SB, Park KJ, Kim YS, Sung YC, Lai MM. Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology. 1997;227:439–446. [PubMed: 9018143]
  29. Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol. 2002;76:2997–3006. [PMC free article: PMC135991] [PubMed: 11861865]
  30. Ishii K, Tanaka Y, Yap CC, Aizaki H, Matsuura Y, Miyamura T. Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding. Hepatology. 1999;29:1227–1235. [PubMed: 10094969]
  31. Jarvis TC, Kirkegaard K. The polymerase in its labyrinth: mechanisms and implications of RNA recombination. Trends Genet. 1991;7:186–191. [PubMed: 1712518]
  32. Johnson RB, Sun XL, Hockman MA, Villarreal EC, Wakulchik M, Wang QM. Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase. Arch Biochem Biophys. 2000;377:129–134. [PubMed: 10775451]
  33. Joyce CM, Steitz TA. Polymerase structures and function: variations on a theme? J Bacteriol. 1995;177:6321–6329. [PMC free article: PMC177480] [PubMed: 7592405]
  34. Kao CC, Del Vecchio AM, Zhong W. De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase. Virology. 1999;253:1–7. [PubMed: 9887312]
  35. Kao CC, Yang X, Kline A, Wang QM, Barket D, Heinz BA. Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase. J Virol. 2000;74:11121–11128. [PMC free article: PMC113194] [PubMed: 11070008]
  36. Kashiwagi T, Hara K, Kohara M, Iwahashi J, Hamada N, Honda-Yoshino H, Toyoda T. Promoter/origin structure of the complementary strand of hepatitis C virus genome. J Biol Chem. 2002;277:28700–28705. [PubMed: 12039953]
  37. Kim MJ, Kao C. Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: implications for RNA-RNA recombination. Proc Natl Acad Sci U S A. 2001;98:4972–4977. [PMC free article: PMC33148] [PubMed: 11309487]
  38. Kim SJ, Kim JH, Kim YG, Lim HS, Oh JW. Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. J Biol Chem. 2004;279:50031–50041. [PubMed: 15364941]
  39. Krieger N, Lohmann V, Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol. 2001;75:4614–4624. [PMC free article: PMC114214] [PubMed: 11312331]
  40. Kushner DB, Lindenbach BD, Grdzelishvili VZ, Noueiry AO, Paul SM, Ahlquist P. Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc Natl Acad Sci U S A. 2003;100:15764–15769. [PMC free article: PMC307642] [PubMed: 14671320]
  41. Kuzmine I, Gottlieb PA, Martin CT. Binding of the priming nucleotide in the initiation of transcription by T7 RNA polymerase. J Biol Chem. 2003;278:2819–1823. [PubMed: 12427761]
  42. Labonte P, Axelrod V, Agarwal A, Aulabaugh A, Amin A, Mak P. Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. J Biol Chem. 2002;277:38838–38846. [PubMed: 12145289]
  43. Lai MM. Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology. 1998;244:1–12. [PubMed: 9581772]
  44. Lai VC, Dempsey S, Lau JY, Hong Z, Zhong W. In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus. J. Virol. 2003;77:2295–2300. [PMC free article: PMC140981] [PubMed: 12525668]
  45. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 1999;6:937–943. [PubMed: 10504728]
  46. Leveque VJ, Johnson RB, Parsons S, Ren J, Xie C, Zhang F, Wang QM. Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis. J Virol. 2003;77:9020–9028. [PMC free article: PMC167210] [PubMed: 12885918]
  47. Lohmann V, Korner F, Herian U, Bartenschlager R. Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol. 1997;71:8416–8428. [PMC free article: PMC192304] [PubMed: 9343198]
  48. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110–113. [PubMed: 10390360]
  49. Lohmann V, Roos A, Korner F, Koch JO, Bartenschlager R. Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus. Virology. 1998;249:108–118. [PubMed: 9740782]
  50. Luo G, Hamatake RK, Mathis DM, Racela J, Rigat KL, Lemm J, Colonno RJ. De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J. Virol. 2000;74:851–863. [PMC free article: PMC111606] [PubMed: 10623748]
  51. Moradpour D, Brass V, Bieck E, Friebe P, Gosert R, Blum HE, Bartenschlager R, Penin F, Lohmann V. Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication. J Virol. 2004;78:13278–13284. [PMC free article: PMC524999] [PubMed: 15542678]
  52. Nagy PD, Simon AE. New insights into the mechanisms of RNA recombination. Virology. 1997;235:1–9. [PubMed: 9300032]
  53. O’Farrell D, Trowbridge R, Rowlands D, Jager J. Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): structural evidence for nucleotide import and denovo initiation. J. Mol. Biol. 2003;326:1025–1035. [PubMed: 12589751]
  54. Oh JW, Ito T, Lai MM. A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J. Virol. 1999;73:7694–7702. [PMC free article: PMC104296] [PubMed: 10438859]
  55. Paul AV, van Boom JH, Filippov D, Wimmer E. Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature. 1998;393:280–284. [PubMed: 9607767]
  56. Piccininni S, Varaklioti A, Nardelli M, Dave B, Raney KD, McCarthy JE. Modulation of the hepatitis C virus RNA-dependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 2002;277:45670–45679. [PubMed: 12235135]
  57. Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989;8:3867–3874. [PMC free article: PMC402075] [PubMed: 2555175]
  58. Qin W, Luo H, Nomura T, Hayashi N, Yamashita T, Murakami S. Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase. J Biol Chem. 2002;277:2132–2137. [PubMed: 11673460]
  59. Qin W, Yamashita T, Shirota Y, Lin Y, Wei W, Murakami S. Mutational analysis of the structure and functions of hepatitis C virus RNA-dependent RNA polymerase. Hepatology. 2001;33:728–737. [PubMed: 11230755]
  60. Ranjith-Kumar CT, Gajewski L, Gutshall R, Maley R, Sarisky R, Kao C. Viral RNA-dependent RNA polymerase has terminal transferase activity: implications for viral RNA synthesis. J Virol. 2001;75:8615–8623. [PMC free article: PMC115107] [PubMed: 11507207]
  61. Ranjith-Kumar CT, Gutshall L, Kim MJ, Sarisky RT, Kao CC. Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases. J Virol. 2002;76:12526–12536. [PMC free article: PMC136677] [PubMed: 12438578]
  62. Ranjith-Kumar CT, Gutshall L, Sarisky RT, Kao CC. Multiple interactions within the hepatitis C virus RNA polymerase repress primer-dependent RNA synthesis. J Mol Biol. 2003;330:675–685. [PubMed: 12850139]
  63. Ranjith-Kumar CT, Kim YC, Gutshall L, Silverman C, Khandekar S, Sarisky RT, Kao CC. Mechanism of de novo initiation by the hepatitis C virus RNA-dependent RNA polymerase: Role of divalent metals. J. Virol. 2002;76:12513–12525. [PMC free article: PMC136676] [PubMed: 12438577]
  64. Ranjith-Kumar CT, Sarisky RT, Gutshall L, Thomson M, Kao CC. De novo initiation pocket mutations have multiple effects on hepatitis C virus RNA-dependent RNA polymerase activities. J. Virol. 2004;78:12207–12217. [PMC free article: PMC525054] [PubMed: 15507607]
  65. Reigadas S, Ventura M, Sarih-Cottin L, Castroviejo M, Litvak S, Astier-Gin T. HCV RNA-dependent RNA polymerase replicates in vitro the 3′ terminal region of the minus-strand viral RNA more efficiently than the 3′ terminal region of the plus RNA. Eur. J. Biochem. 2001;268:5857–67. [PubMed: 11722573]
  66. Sarisky RT. Non-nucleoside inhibitors of the HCV polymerase. J Antimicro Chemotherapy. 2004;54:14–16. [PubMed: 15190019]
  67. Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T, Kobayashi K, Murakami S. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRp) NS5B and modulates RNA-dependent RNA polymerase activity. J. Biol. Chem. 2002;277:11149–11155. [PubMed: 11801599]
  68. Sun XL, Johnson RB, Hockman MA, Wang QM. De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase. Biochem Biophys Res Commun. 2000;268:798–803. [PubMed: 10679285]
  69. Tomei L, Vitale RL, Incitti I, Serafini S, Altamura S, Vitelli A, De Francesco R. Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence. J Gen Virol. 2000;81:759–767. [PubMed: 10675414]
  70. Vo NV, Oh JW, Lai MM. Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates. Virology. 2003;307:301–316. [PubMed: 12667800]
  71. Vo NV, Tuler JR, Lai MM. Enzymatic characterization of the full-length and C-terminally truncated hepatitis C virus RNA polymerases: function of the last 21 amino acids of the C terminus in template binding and RNA synthesis. Biochemistry. 2004;43:10579–10591. [PubMed: 15301555]
  72. Wang QM, Hockman MA, Staschke K, Johnson RB, Case KA, Lu J, Parson S, Zhang F, Rathnachalam R, Kirkegaard K, Colacino J. Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J Virol. 2002;76:3865–3872. [PMC free article: PMC136118] [PubMed: 11907226]
  73. Wolk B, Sansonno D, Krausslich HG, Dammacco F, Rice CM, Blum HE, Moradpour D. Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J Virol. 2000;74:2293–2304. [PMC free article: PMC111711] [PubMed: 10666260]
  74. Wu J, Hong Z. Targeting NS5B RNA-dependent RNA polymerase for anti-HCV chemotherapy. Curr Drug Targets Infect Disord. 2003;3:207–19. [PubMed: 14529354]
  75. Yamashita T, Kaneko S, Shirota Y, Qin W, Nomura T, Kobayashi K, Murakami S. RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. J Biol Chem. 1998;273:15479–15486. [PubMed: 9624134]
  76. Yi M, Lemon SM. 3′ nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol. 2003a;77:3557–3568. [PMC free article: PMC149512] [PubMed: 12610131]
  77. Yi M, Lemon SM. Structure-function analysis of the 3′ stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA. 2003b;9:331–45. [PMC free article: PMC1370400] [PubMed: 12592007]
  78. Yuan ZH, Kumar U, Thomas HC, Wen YM, Monjardino J. Expression, purification, and partial characterization of HCV RNA polymerase. Biochem Biophys Res Comm. 1997;232:231–235. [PubMed: 9125138]
  79. Zhang C, Cai Z, Kim YC, Ranjith Kumar CT, Yuan F, Shi PY, Kao CC, Luo G. Stimulation of hepatitis C virus (HCV) NS3 helicase activity by the NS3 protease domain and by the HCV RNA-dependent RNA polymerase. J Virol. 2005;79:8687–8697. [PMC free article: PMC1168731] [PubMed: 15994762]
  80. Zhong W, Ferrari E, Lesburg CA, Maag D, Gosh A, Cameron C, Lau J, Hong Z. Template-primer requirements and single-nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase. J Virol. 2000;74:9134–9143. [PMC free article: PMC102112] [PubMed: 10982360]
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