A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase
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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 3Ј 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 3Ј 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 3Ј 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 known about this class of polymerases until recently. human medical problem with an estimated prevalence of Structural studies of unliganded poliovirus 3Dpol (9) and 170 million cases globally. Up to four million individuals HCV NS5B (4, 13) have revealed unique features and may be infected in the United States alone (1). In most provided useful information for the comparative analysis instances, the virus establishes a chronic and insidious of RdRps in relation to other classes of polymerases. infection that persists for decades. This usually results in This will in turn help to advance our understanding of recurrent and progressively worsening liver inflamma- viral replication at the molecular level for positive- tion, which often leads to more severe disease states stranded RNA viruses. such as cirrhosis and hepatocellular carcinoma. A Unique -Hairpin in the Thumb Subdomain of HCV HCV, a member of the Flaviviridae family, is a positive- NS5B Polymerase. One novel structural component stranded RNA virus. Its life cycle consists of several unique to HCV polymerase is a -hairpin found in the important and interrelated processes that occur primarily thumb subdomain, which protrudes toward the active in the cytoplasm of the host cells (19). Among these site located at the base of the palm subdomain (yellow intricate processes, RNA replication is the centerpiece of ribbon, Fig. 1A). The -hairpin consists of 12 amino acids the viral proliferation cycle. While many viral and/or host (LDCQIYGACYSI) with a tyrosine (Y) and glycine (G) pair factors are suspected to be involved, one certainty is that at the top of the hairpin (position and direction of the the virally encoded polymerase has a key role in the -hairpin is shown at the bottom of Fig. 1A). In stark entire replication process. The interplay between viral contrast, this -hairpin is absent in polioviral polymerase polymerase and its substrates (template, primer, and (3Dpol) (Fig. 1B). This structural difference suggests that incoming nucleotide) sets the initial and minimal frame- the -hairpin may serve a special function important for work for viral replication. For HCV, the nonstructural pro- HCV replication. Poliovirus, a well-studied virus, initiates tein 5B (NS5B) is the RNA-dependent RNA polymerase its genome replication using a protein primer (VPg, prod- (RdRp) required for genome replication (3, 7, 14). Al- uct of 3B) (18). Contrary to the inability of HCV NS5B to though the first RdRp was discovered more than 20 years bind dsRNA substrates productively as reported previ- ago in poliovirus infected cells, relatively little was ously (21), polioviral 3Dpol can efficiently assemble an elongation-competent complex with a symmetrical du- plex RNA substrate (sym/sub), suggesting that the 3Dpol 1 To whom correspondence and reprint requests should be ad- apoenzyme can accommodate preannealed double- dressed at ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa stranded template/primer (2). This observation coincides Mesa, CA 92626. Fax: (714) 668-3141. E-mail: [email protected]. with the lack of an analogous -hairpin in 3Dpol (Fig. 1B) 0042-6822/01 $35.00 Copyright © 2001 by Academic Press 6 All rights of reproduction in any form reserved. RAPID COMMUNICATION 7 FIG. 1. Structural comparison between HCV NS5B RdRp and poliovirus 3Dpol 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 3Dpol 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 3Dpol structure to be constructed. Based on the similar polypeptide fold in regions common to both crystallographic models, the missing portions of the 3Dpol 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 hin- shorten the -hairpin without disrupting the structural drance against docking the dsRNA (13). For HCV, assem- integrity and the catalytic activity of HCV NS5B. bly of a catalytically competent complex requires the Shortened -Hairpin Allows Internal Initiation from RNA template to be single-stranded at the 3Ј terminus Preannealed Duplex RNA. Guided by the crystal struc- (10, 21). Upon template binding to the polymerase, short ture of HCV NS5B polymerase (4), four amino acids from RNA primers (i.e., the initiating nucleotide, one to three each strand were deleted simultaneously to shorten the nucleotides in length) basepair with the terminal tem- -hairpin without potential global structural perturbation plate bases in the active site of the polymerase and that may affect the RdRp activity. The resulting deletion prime RNA synthesis from the 3Ј terminus of the template mutant, BL⌬8, retained two amino acids at the top of the (21). A structural model for the quaternary complex of -hairpin, which were changed from YG to GG to favor HCV polymerase/template/primer/nucleotide has been the formation of the observed type IЈ -turn between the proposed, which suggests that the unique -hairpin two -strands (from LDCQIYGACYSItoLGGI). This dele- plays an important role in positioning the 3Ј terminus of tion mutant as well as its parent wild-type NS5B protein viral RNA properly in the polymerase active site for cor- were produced in bacterial cells and purified as de- rect initiation of RNA replication (21). To test this hypoth- scribed previously (9, 13). The purity of the wild-type and esis, a deletion mutant was constructed to significantly BL⌬8 NS5B is above 95% and comparable to that of the 8 RAPID COMMUNICATION 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 3Dpol (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 accom- modation of the nascent dsRNA (a product of elongation FIG. 2. A -hairpin deletion mutant allows productive binding to dsRNA substrate to initiate RNA synthesis. (A) Expression and purification of HCV during replication) by HCV polymerase must be accom- NS5B and poliovirus 3Dpol. NS5B cDNA was isolated from a subgenomic panied by a conformational change from initiation to Ј replicon clone (pFKI389/NS3-3 /wt) kindly provided by Dr. Ralf Barten- elongation in the polymerase active site, perhaps by schlager (Johannes-Gutenberg University, Mainz, Germany). The region moving the -hairpin away from the active site. encoding the C-terminal 21 amino acids was removed to improve the ⌬ HCV NS5B initiates RNA synthesis from the initiating solubility of NS5B as described previously (7). The BL 8 deletion mutant Ј was created from the C-terminally deleted NS5B by using the Quick- nucleotide (de novo) or a dinucleotide at the 3 terminus Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).