RAPID COMMUNICATION. A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase. Zhi Hong,*,1

Virology 285, 6 11 (2001) doi:10.1006/viro.2001.0948, available online at http://www.idealibrary.com on RAPID COMMUNICATION A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase Zhi Hong,*,1 Craig E. Cameron, Michelle P. Walker,* Christian Castro, Nanhua Yao,* Johnson Y. N. Lau,* and Weidong Zhong* *ICN Pharmaceuticals, 3300 Hyland Avenue, Costa Mesa, California 92626; and Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802 Received January 31, 2001; returned to author for revision February 20, 2001; accepted April 10, 2001 Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique -hairpin in the thumb subdomain which protrudes toward the active site. We report here that this -hairpin plays an important role in positioning the 3 terminus of the viral RNA genome for correct initiation of replication. The presence of this -hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsrna) molecules and allows only the single-stranded 3 terminus of an RNA template to bind productively to the active site. We propose that this -hairpin may serve as a gate which prevents the 3 terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a -hairpin deletion mutant that utilizes dsrna substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses. 2001 Academic Press Infection by hepatitis C virus (HCV) is a significant human medical problem with an estimated prevalence of 170 million cases globally. Up to four million individuals may be infected in the United States alone (1). In most instances, the virus establishes a chronic and insidious infection that persists for decades. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma. HCV, a member of the Flaviviridae family, is a positivestranded RNA virus. Its life cycle consists of several important and interrelated processes that occur primarily in the cytoplasm of the host cells (19). Among these intricate processes, RNA replication is the centerpiece of the viral proliferation cycle. While many viral and/or host factors are suspected to be involved, one certainty is that the virally encoded polymerase has a key role in the entire replication process. The interplay between viral polymerase and its substrates (template, primer, and incoming nucleotide) sets the initial and minimal framework for viral replication. For HCV, the nonstructural protein 5B (NS5B) is the RNA-dependent RNA polymerase (RdRp) required for genome replication (3, 7, 14). Although the first RdRp was discovered more than 20 years ago in poliovirus infected cells, relatively little was 1 To whom correspondence and reprint requests should be addressed at ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626. Fax: (714) 668-3141. E-mail: zhihong@icnpharm.com. known about this class of polymerases until recently. Structural studies of unliganded poliovirus 3D pol (9) and HCV NS5B (4, 13) have revealed unique features and provided useful information for the comparative analysis of RdRps in relation to other classes of polymerases. This will in turn help to advance our understanding of viral replication at the molecular level for positivestranded RNA viruses. A Unique -Hairpin in the Thumb Subdomain of HCV NS5B Polymerase. One novel structural component unique to HCV polymerase is a -hairpin found in the thumb subdomain, which protrudes toward the active site located at the base of the palm subdomain (yellow ribbon, Fig. 1A). The -hairpin consists of 12 amino acids (LDCQIYGACYSI) with a tyrosine (Y) and glycine (G) pair at the top of the hairpin (position and direction of the -hairpin is shown at the bottom of Fig. 1A). In stark contrast, this -hairpin is absent in polioviral polymerase (3D pol ) (Fig. 1B). This structural difference suggests that the -hairpin may serve a special function important for HCV replication. Poliovirus, a well-studied virus, initiates its genome replication using a protein primer (VPg, product of 3B) (18). Contrary to the inability of HCV NS5B to bind dsrna substrates productively as reported previously (21), polioviral 3D pol can efficiently assemble an elongation-competent complex with a symmetrical duplex RNA substrate (sym/sub), suggesting that the 3D pol apoenzyme can accommodate preannealed doublestranded template/primer (2). This observation coincides with the lack of an analogous -hairpin in 3D pol (Fig. 1B) 0042-6822/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved. 6

RAPID COMMUNICATION 7 FIG. 1. Structural comparison between HCV NS5B RdRp and poliovirus 3D pol RdRp. Molecular surface structures were constructed for both polymerases (turquoise color for HCV and blue color for poliovirus) and oriented with the palm at the base, the fingers to the left, and the thumb subdomain to the right. The essential GDD motifs (polymerase motif C; part of the active site) are colored red. In HCV NS5B, the -hairpin comprising residues 443 through 454 is colored yellow and protrudes toward the active site (4). The sequence of the -hairpin is shown at the bottom of (A). In both structures, the unique molecular shape of the RdRps with the fully encircled NTP binding site is clear. This encircled active site likely exists in 3D pol because the extreme aminoterminal residues in the crystal structure contact the thumb subdomain, just as in HCV NS5B, before proceeding to form the fingers subdomain. This figure was produced using the program INSIGHT II (Molecular Simulation Inc., San Diego, CA). In the crystal structure of poliovirus 3Dpol (9), approximately 30% of the protein residues were unobserved. The availability of HCV NS5B RdRp structure (4) allows a model of the complete poliovirus 3D pol structure to be constructed. Based on the similar polypeptide fold in regions common to both crystallographic models, the missing portions of the 3D pol structure were simply transferred from NS5B and the correct side chains were assigned with the most common rotamer. The junctions between crystallographically observed and inferred residues were adjusted according to local similarity to a database of polypeptide fragments derived from high-resolution crystal structures. which, in HCV NS5B polymerase, imposes steric hindrance against docking the dsrna (13). For HCV, assembly of a catalytically competent complex requires the RNA template to be single-stranded at the 3 terminus (10, 21). Upon template binding to the polymerase, short RNA primers (i.e., the initiating nucleotide, one to three nucleotides in length) basepair with the terminal template bases in the active site of the polymerase and prime RNA synthesis from the 3 terminus of the template (21). A structural model for the quaternary complex of HCV polymerase/template/primer/nucleotide has been proposed, which suggests that the unique -hairpin plays an important role in positioning the 3 terminus of viral RNA properly in the polymerase active site for correct initiation of RNA replication (21). To test this hypothesis, a deletion mutant was constructed to significantly shorten the -hairpin without disrupting the structural integrity and the catalytic activity of HCV NS5B. Shortened -Hairpin Allows Internal Initiation from Preannealed Duplex RNA. Guided by the crystal structure of HCV NS5B polymerase (4), four amino acids from each strand were deleted simultaneously to shorten the -hairpin without potential global structural perturbation that may affect the RdRp activity. The resulting deletion mutant, BL 8, retained two amino acids at the top of the -hairpin, which were changed from YG to GG to favor the formation of the observed type I -turn between the two -strands (from LDCQIYGACYSItoLGGI). This deletion mutant as well as its parent wild-type NS5B protein were produced in bacterial cells and purified as described previously (9, 13). The purity of the wild-type and BL 8 NS5B is above 95% and comparable to that of the

8 RAPID COMMUNICATION FIG. 2. A -hairpin deletion mutant allows productive binding to dsrna substrate to initiate RNA synthesis. (A) Expression and purification of HCV NS5B and poliovirus 3D pol. NS5B cdna was isolated from a subgenomic replicon clone (pfki 389 /NS3-3 /wt) kindly provided by Dr. Ralf Bartenschlager (Johannes-Gutenberg University, Mainz, Germany). The region encoding the C-terminal 21 amino acids was removed to improve the solubility of NS5B as described previously (7). The BL 8 deletion mutant was created from the C-terminally deleted NS5B by using the Quick- Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The poliovirus 3D pol was produced as described (8). The purity of each polymerase was above 95%. (B) Use of duplex RNA substrate. Single-nucleotide incorporation assay was performed using end-labeled sym/sub RNA as the template/primer pair. AMP incorporation was compared among wild-type NS5B (lane 2), -hairpin deletion mutant BL 8 (lane 3), poliovirus 3D pol (lane 4). No template control is in lane 1. A measure of 2 M of each enzyme was employed, respectively. poliovirus 3D pol (Fig. 2A). The BL 8 mutant was first tested for its ability to utilize the dsrna substrate, sym/ sub. The wild-type NS5B, as shown previously (5, 21), was unable to utilize sym/sub or preannealed dsrna efficiently to direct RNA synthesis (Fig. 2B, lane 2). In contrast, the BL 8 mutant was able to utilize sym/sub as efficiently as poliovirus 3D pol (Fig. 2B, compare lanes 3 and 4) under the same assay conditions (described in Fig. 3 legend). Interestingly, the overall RdRp activity of BL 8 was increased approximately fivefold compared to the wild-type NS5B (Fig. 4). These results support the notion that the -hairpin prevents HCV polymerase from binding to a double-stranded template/primer duplex in a productive manner, while polioviral polymerase lacking the -hairpin binds to dsrna and efficiently extends the labeled primer. This observation argues that the accommodation of the nascent dsrna (a product of elongation during replication) by HCV polymerase must be accompanied by a conformational change from initiation to elongation in the polymerase active site, perhaps by moving the -hairpin away from the active site. HCV NS5B initiates RNA synthesis from the initiating nucleotide (de novo) or a dinucleotide at the 3 terminus of the template (21). The dinucleotide-mediated terminal initiation assay was selected to characterize the -hairpin deletion mutant. The initiating dinucleotide, believed to be the product of abortive initiation, was shown to prime RNA synthesis efficiently by HCV NS5B (21), a phenomenon that was also demonstrated in bacteriophage T7 RNA polymerase-mediated RNA synthesis (6). Terminal initiation is in accordance with the quaternary structural model wherein the 3 terminus of the RNA template sterically clashes with the tip of the -hairpin loop and is unable to slip through the active site (21). FIG. 3. The -hairpin deletion mutant initiates RNA synthesis internally. (A) Internal initiation from a dinucleotide primer. TER and INT template RNAs were employed to distinguish terminal vs internal initiation from the primer 33 pgpg. RdRp activities of wild-type NS5B (lanes 2 and 3), deletion mutant BL 8 (lanes 4 and 5) were compared. in lane 1 represents a control without the template RNA. P represents the dinucleotide pgpg and P 1 is the trinucleotide product as a result of single nucleotide addition. (B) Internal initiation from a preannealed primer. A preannealed template and primer (P, an end-labeled primer 33 pcgggcc) were tested for single-nucleotide (AMP) incorporation. Lane 1, no template control ; lane 2, wild-type NS5B; lane 3, BL 8 mutant; lane 4, poliovirus 3D pol. The standard nucleotide incorporation reaction was carried out in a volume of 20 l containing 50 mm HEPES (ph 7.3), 10 mm -mercaptoethanol, 50 mm NaCl, 5 mm MgCl 2,2 M of end-labeled sym/sub RNA, or 10 M of TER or INT template RNA and 10 M of end-labeled dinucleotide, 2 M of polymerase (HCV NS5B or poliovirus 3D pol ), and 100 M of CTP or ATP (2, 21). The product was separated on a 23 25% PAGE/6 M urea/1 TBE gel and exposed to X-ray film. Individual bands were quantified using a Phosphorimager (Typhoon 8600, Molecular Dynamics, Sunnyvale, CA). All synthetic RNA templates and primers were chemically synthesized (Oligos, Etc., Wilsonville, OR).

RAPID COMMUNICATION 9 FIG. 4. RdRp activity of the wild-type and the BL 8 mutant NS5Bs on in vitro transcribed HCV RNA. (A) Structure of the minigenome RNA used for RdRp assay. Numbers refer to nucleotide positions within the HCV subgenomic replicon (15). An internal deletion between two KpnI sites at positions 1684 and 7477 reduced the size of the minigenome RNA to about 2.1 kb. (B) Comparison of the RNA products from the HCV minigenome RNA template. RdRp reactions were initiated by adding 1 g of each protein: lanes 2 7, wild-type NS5B; lanes 8 13, BL 8 NS5B. Reactions were terminated by the addition of SDS-containing buffer and proteinase K at 10 (lanes 3 and 8), 20 (lanes 4 and 9), 30 (lanes 5 and 10), 50 (lanes 2, 6, 11, and 13), or 70 (lanes 7 and 12) min after the reactions started. Control reactions contained no nucleotides (lanes 2 and 13) for 50 min. Samples were analyzed by denaturing guanidiniumisothiocyanate-1% agarose gel electrophoresis. Minigenome monomer size marker was obtained by in vitro transcription of the HCV minigenome in the presence of -[ 33 P]UTP (lane 1). (C) Comparison of the RNA products from the homopolymeric RNA template/primer, poly(a)/ oligo(dt), using increasing enzyme concentrations. Reactions were initiated by the addition of 0.5 (lanes 1 and 4), 1.0 (lanes 2 and 5), or 2.0 (lanes 3 and 6) g of enzyme and terminated after 45 min at 30 C by the addition of phenol/chloroform. Indeed, our results (Fig. 3A) demonstrate that HCV NS5B is unable to utilize the INT template (lacking the terminal dicytidylate bases) to initiate RNA synthesis from the diguanylate primer ( 33 pgpg; lane 4), whereas the enzyme utilized the TER template with the terminal dicytidylate bases very efficiently (lane 5). Clearly, the wild-type HCV NS5B could not initiate nucleotide incorporation from the internal dicytidylate (CC) template bases. However, the -hairpin deletion mutant BL 8 could utilize the INT template to initiate RNA synthesis (Fig. 3A, lanes 2 and 3), indicating that the shortened -hairpin allowed internal initiation of RNA synthesis by permitting the 3 terminus of the template to slip through the active site. Poliovirus 3D pol, which lacks the -hairpin, allowed internal initiation (Fig. 3B, lane 4) from a primer (P) preannealed to the middle of a template, an activity that could be duplicated by the BL 8 mutant but not by the wildtype NS5B polymerase (Fig. 3B, compare lanes 2 and 3). The above results suggest that the apo form of the HCV polymerase is unable to initiate efficient RNA synthesis internally due to the presence of the -hairpin, whereas poliovirus, although capable of internal initiation, has acquired a discrete mechanism to ensure terminal initiation from the poly(a) stretch at the end of the viral genome (18). The BL 8 Mutant NS5B Produces Predominantly Truncated RNA Products. To compare the RdRp activity of the wild-type and the BL 8 mutant NS5Bs on a natural HCV template, an HCV minigenome was constructed that contains authentic 5 and 3 termini and portions of the coding sequence (pminigenome) (Fig. 4A) (15). In vitro transcribed minigenome RNAs were used as templates in a standard RdRp assay. Products larger than the input RNA, particularly of dimer size, were apparent for both wild-type and BL 8 mutant NS5Bs (Fig. 4B, lanes 3 7 for the wild-type NS5B; lanes 8 12 for the BL 8 mutant NS5B). This is consistent with the observation that NS5B is capable of initiating RNA synthesis via the copy-back mechanism at the 3 terminus (3, 14). Also in agreement with previous reports (14, 17), the near dimer-size hairpin product appeared 15 20 min after the start of the reaction, corresponding to an incorporation rate of approximately 100 140 nucleotides per minute. Interestingly, the BL 8 mutant produced RNA products varying in size, which appeared as a smear by denaturing agarose gel electrophoresis (Fig. 4B, lanes 8 12). Many of these heterogeneous RNA products could occur by internal initiation as a result of the -hairpin deletion, consistent with the notion that the -hairpin is necessary to ensure terminal initiation. The overall RNA synthesis activity of the BL 8 mutant was at least five times greater than that of the wild-type NS5B (Fig. 4B, compare lanes 8 12 with lanes 3 7). In concert, enhanced RdRp activity by the BL 8 mutant was observed when using the homopolymeric RNA template/primer, poly(a)/oligo(dt), in a standard RdRp assay (Fig. 4C). Much of the enhanced RdRp activity resulted from initiation from internally annealed primers, leading to smaller than template-sized products (Fig. 4C, compare lanes 1 3 with lanes 4 6). Again, these results support the proposed function of the -hairpin. Identification of a Conserved Arginine Critical for De Novo Initiation from the 3 Terminus. A structural model was constructed to further define the molecular requirements at the active site for efficient assembly of the template RNA and the short initiating dinucleotide (21). Based on this model, a conserved arginine (R 386 ) was

10 RAPID COMMUNICATION FIG. 5. A conserved arginine critical for de novo initiation. A critical arginine at position 386 was identified to play a role in stabilizing the initiating nucleotide(s). (A) Effect on RNA synthesis from the dinucleotide primer, pgpg. Lane 1, no template control ; lane 2, wild-type NS5B; lane 3, NS5B with R 386 Q mutation; lane 4, BL 8 mutant; lane 5, BL 8 with R 386 Q mutation. P represents the end-labeled primer and P 1 represents the extended primer. (B) Effect on RNA synthesis from the preannealed primer, sym/sub. The lane assignment is the same as (A). identified and proposed to play a role in stabilizing the initiating nucleotide (for de novo synthesis) or the dinucleotide (for pgpg-mediated synthesis), which would otherwise be unable to basepair efficiently with the template base(s). Interestingly, our previous mutational analysis had demonstrated that the equivalent Arg residue (R 518 ) in bovine viral diarrhea virus (BVDV) NS5B was important for de novo RNA synthesis (12). Indeed, sitedirected mutagenesis revealed that R 386 was also important for the dinucleotide-initiated RNA synthesis, since mutation to glutamine (R 386 Q) significantly reduced the ability to utilize the dinucleotide for both the wild-type and BL 8 mutant NS5B (Fig. 5A, compare lanes 2 vs 3 and 4 vs 5). However, this mutation had little effect on RNA synthesis from a preannealed duplex RNA (sym/ sub) catalyzed by the deletion mutant BL 8 (Fig. 5B, lanes 4 and 5). These observations support the hypothesis that R 386 plays a critical role in stabilizing the interaction between the 3 terminus of the RNA template and the initiating nucleotide(s). We believe that the -hairpin and R 386 coordinate RNA synthesis from an initiating nucleotide (de novo) or the initiating dinucleotide. The dinucleotide is probably an abortive transcript of de novo initiation that can prime efficient RNA synthesis when annealed to a bound template in the active site. Concluding Remarks. Terminal initiation is a critical first step for many positive-stranded RNA virus replications. In the absence of repetitive sequence motifs, strict terminal initiation ensures the integrity of a viral genome. For poliovirus, a prototype virus representing the Picornaviridae family, precise terminal initiation is achieved by a protein primer which assembles the replication complex at the 3 poly(a) region and initiates RNA synthesis via a uridylylated protein primer (VPg) (18). This uridylylation is highly specific as it requires specifically 3D pol activity, UTP, and poly(a), which coincides with the observation that the 3 terminal poly(a) region of the poliovirus genome is the origin of RNA replication (20). HCV, an RNA virus in the Flaviviridae family, appears to have adopted a different strategy for de novo RNA replication (16, 22). This study shows that an intrinsic structural element is employed by HCV for precise terminal initiation of the viral genome replication. A recent study demonstrated that BVDV NS5B polymerase specifically recognizes the terminal template base(s) for de novo RNA synthesis (11), providing evidence that terminal initiation of RNA replication is also required by this related virus. Thus, the identification of a novel and intrinsic structural element (such as the -hairpin in HCV NS5B) for terminal initiation may have a broader impact on our understanding of viral replication, as it may represent a replication strategy employed by related RNA viruses. ACKNOWLEDGMENTS C.E.C. is a recipient of a Howard Temin Award (CA75118) and is supported in part by NIAID Grant AI47350. We thank Ralf Bartenschlager for providing the valuable subgenomic HCV replicon DNA. We also acknowledge the technical assistance of Shannon Dempsey. REFERENCES 1. Alter, M. J., Kruszon-Moran, D., Nainan, O. V., McQuillan, G. M., Gao, F., Moyer, L. A., Kaslow, R. A., and Margolis, H. S. (1999). The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N. Engl. J. Med. 341, 556 562. 2. Arnold, J. J., and Cameron, C. E. (2000). Poliovirus RNA-dependent RNA polymerase (3D pol ): Assembly of stable, elongation-competent complexes by using a symmetrical primer/template substrate (sym/sub). J. Biol. Chem. 275(8), 5329 5336. 3. Behrens, S.-E., Tomei, L., and De Francesco, R. (1996). Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15(1), 12 22. 4. Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu,

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