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Template Requirement and Initiation Site Selection by Hepatitis C Virus Polymerase on a Minimal Viral RNA Template

2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês

10.1074/jbc.m908781199

1083-351X

Jong‐Won Oh, Gwo‐Tarng Sheu, Michael M. C. Lai,

HIV Research and Treatment

RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3′-end. We have previously shown that NS5B can utilize the 3′-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3′-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3′-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3′-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3′-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3′-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3′-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3′-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells. RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3′-end. We have previously shown that NS5B can utilize the 3′-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3′-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3′-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3′-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3′-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3′-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3′-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3′-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells. hepatitis C virus nucleotide(s) RNA-dependent RNA polymerase untranslated region electrophoretic mobility shift assay nitrilotriacetic acid Hepatitis C virus (HCV)1is the etiological agent of non-A, non-B hepatitis, often causing liver diseases including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1.Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6238) Google Scholar, 2.Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.-L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1084) Google Scholar, 3.Alter H.J. Blood. 1995; 85: 1681-1695Crossref PubMed Google Scholar, 4.Houghton M. Fields B.N. Knipe D.M. Howley P.M. Virology. Lippincott-Raven Publishers, Philadelphia1996: 1035-1058Google Scholar). HCV has a positive-sense, single-stranded RNA genome of approximately 9700 nucleotides (nt) in length, which is terminated with a stretch (98 nt) of highly conserved sequence, termed the X region (5.Kolykhalov A.A. Agapov E.V. Blight K.J. Mihalik K. Feinstone S.M. Rice C.M. Science. 1997; 277: 570-574Crossref PubMed Scopus (623) Google Scholar, 6.Yanagi M. Purcell R.H. Emerson S.U. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar, 7.Yanagi M. Claire M.S. Shapiro M. Emerson S.U. Purcell R.H. Bukh J. Virology. 1998; 244: 161-172Crossref PubMed Scopus (199) Google Scholar, 8.Tanaka T. Kato N. Cho M.-J. Shimotohno K. Biochem. Biophys. Res. Commun. 1995; 215: 744-749Crossref PubMed Scopus (247) Google Scholar, 9.Tanaka T. Kato M. Cho M.-J. Sugiyama K. Shimotohno K. J. Virol. 1996; 70: 3307-3312Crossref PubMed Google Scholar, 10.Kolykhalov A.A. Feinstone S.M. Rice C.M. J. Virol. 1996; 70: 3363-3371Crossref PubMed Google Scholar, 11.Yamada N. Tanihara K. Takada A. Yorihuzi T. Tsutsumi M. Shimomura H. Tsuji T. Date T. Virology. 1996; 223: 255-261Crossref PubMed Scopus (111) Google Scholar). The X region folds into a stable secondary structure consisting of three stem-loop domains (12.Blight K.J. Rice C.M. J. Virol. 1997; 71: 7345-7352Crossref PubMed Google Scholar, 13.Ito T. Lai M.M.C. J. Virol. 1997; 71: 8698-8706Crossref PubMed Google Scholar). Upstream of the X region is a stretch of poly(U-U/C)-rich sequences of variable length and highly variable sequences of about 30–40 nt (5.Kolykhalov A.A. Agapov E.V. Blight K.J. Mihalik K. Feinstone S.M. Rice C.M. Science. 1997; 277: 570-574Crossref PubMed Scopus (623) Google Scholar, 6.Yanagi M. Purcell R.H. Emerson S.U. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar, 7.Yanagi M. Claire M.S. Shapiro M. Emerson S.U. Purcell R.H. Bukh J. Virology. 1998; 244: 161-172Crossref PubMed Scopus (199) Google Scholar, 8.Tanaka T. Kato N. Cho M.-J. Shimotohno K. Biochem. Biophys. Res. Commun. 1995; 215: 744-749Crossref PubMed Scopus (247) Google Scholar, 9.Tanaka T. Kato M. Cho M.-J. Sugiyama K. Shimotohno K. J. Virol. 1996; 70: 3307-3312Crossref PubMed Google Scholar, 10.Kolykhalov A.A. Feinstone S.M. Rice C.M. J. Virol. 1996; 70: 3363-3371Crossref PubMed Google Scholar, 11.Yamada N. Tanihara K. Takada A. Yorihuzi T. Tsutsumi M. Shimomura H. Tsuji T. Date T. Virology. 1996; 223: 255-261Crossref PubMed Scopus (111) Google Scholar). Infectivity assays showed that the X region and U-U/C-rich sequences are required for viral infectivity, but the variable sequences are not (14.Yanagi M. Claire M.S. Emerson S.U. Purcell R.H. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2291-2295Crossref PubMed Scopus (192) Google Scholar). As implicated by sequence conservation among all HCV genotypes, the structure and/or sequence of the X region of HCV is important for minus-strand RNA synthesis and translational regulation (15.Ito T. Tahara S.M. Lai M.M.C. J. Virol. 1998; 72: 8789-8796Crossref PubMed Google Scholar, 16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). The replication of HCV RNA is mediated by NS5B, which is an RNA-dependent RNA polymerase (RdRp) (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar, 17.Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (645) Google Scholar, 18.Lohmann V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar, 19.Yamashita T. Kaneko S. Shirota Y. Qin W. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 1998; 273: 15479-15486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20.Ferrari E. Wright-Minogue J. Fang J.W.S. Baroudy B.M. Lau J.Y.N. Hong Z. J. Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar). The initial step of viral RNA replication is recognition of the 3′-end of RNA template by RdRp, which may occur directly or indirectly with the help of cellular proteins (21.Lai M.M.C. Virology. 1998; 244: 1-12Crossref PubMed Scopus (238) Google Scholar, 22.Strauss J.H. Strauss E.G. Science. 1999; 283: 802-804Crossref PubMed Scopus (32) Google Scholar). For example, Qβ bacteriophage replicase recognizes the replicable RNA templates with the help of cellular factors, including ribosomal protein S1 and translation elongation factor Tu (23.Blumenthal T. Carmichael G.G. Annu. Rev. Biochem. 1979; 48: 525-548Crossref PubMed Scopus (309) Google Scholar, 24.Barrera I. Schuppli D. Sogo J.M. Weber H. J. Mol. Biol. 1993; 232: 512-521Crossref PubMed Scopus (96) Google Scholar, 25.Schuppli D. Barrera I. Weber H. J. Mol. Biol. 1994; 243: 811-815Crossref PubMed Scopus (18) Google Scholar), which are also important for template recognition on certain in vitro selected RNA templates (26.Brown D. Gold L. Biochemistry. 1995; 34: 14765-14774Crossref PubMed Scopus (42) Google Scholar, 27.Brown D. Gold L. Biochemistry. 1995; 34: 14775-14782Crossref PubMed Scopus (42) Google Scholar, 28.Brown D. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11558-11562Crossref PubMed Scopus (63) Google Scholar). Qβ replicase contains two RNA-binding domains; one is the catalytic site, and the other is for sequence-specific recognition of template RNA. However, template specificity is conferred by the host factors. Encephalomyocarditis virus polymerase recognizes the 3′-untranslated region (UTR) of viral RNA only when it contains a poly(A) tail (29.Cui T. Sanker S. Porter A.G. J. Biol. Chem. 1993; 268: 26093-26098Abstract Full Text PDF PubMed Google Scholar, 30.Cui T. Porter A.G. Nucl. Acid Res. 1995; 23: 377-382Crossref PubMed Scopus (40) Google Scholar). The influenza virus polymerase PB1 subunit also specifically binds, via three separate RNA-binding domains, to the 5′- and 3′-arms of either viral or complementary RNA panhandles (31.Gonzalez S. Ortin J. EMBO J. 1999; 18: 3767-3775Crossref PubMed Scopus (97) Google Scholar). In contrast, poliovirus polymerase appears to bind the viral RNA genome through a nonspecific cooperative binding mechanism (32.Pata J.D. Schultz S.C. Kirkegaard K. RNA. 1995; 1: 466-477PubMed Google Scholar). HCV RdRp has an RNA binding activity and preferentially binds poly(U) and poly(G) over poly(C) and poly(A) homopolymeric RNA (18.Lohmann V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar). However, no specific binding of HCV polymerase to the 3′-end of HCV viral RNA has been reported, although the X region is important for infectivity in vivo (14.Yanagi M. Claire M.S. Emerson S.U. Purcell R.H. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2291-2295Crossref PubMed Scopus (192) Google Scholar) and acts as a minimal RNA template in vitro(16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). The mechanism of initiation of RNA synthesis by RdRp for most RNA viruses is poorly understood. Brome mosaic virus RdRp appears to be able to recognize an internal promoter for subgenomic mRNA synthesis de novo (33.Adkins S. Siegel R.W. Sun J.-H. Kao C.C. RNA. 1997; 3: 634-647PubMed Google Scholar, 34.Siegel R.W. Bellon L. Beigelman L. Kao C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11613-11618Crossref PubMed Scopus (50) Google Scholar). However, it is not clear whether there is a direct interaction between the promoter and viral RdRp holoenzyme, since the purified RdRp complex contained two virus-encoded proteins, the polymerase and helicase, and a subunit of translation elongation factor eIF3 (35.Quadt R. Kao C.C. Browning K.S. Hershberger R.P. Ahlquist P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1498-1502Crossref PubMed Scopus (148) Google Scholar). Similarly, bovine diarrhea virus polymerase can also recognize the minimal 21-nt RNA template and synthesize complementary RNA in a primer-independent manner and start RNA synthesis preferentially from a cytidylate at the most 3′-end of the template (36.Kao C.C. Del Vecchio A.M. Zhong W. Virology. 1999; 253: 1-7Crossref PubMed Scopus (103) Google Scholar). For HCV polymerase, RNA synthesis has been shown to depend on exogenous or snap-back RNA primers in vitro (17.Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (645) Google Scholar, 18.Lohmann V. Korner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar, 19.Yamashita T. Kaneko S. Shirota Y. Qin W. Nomura T. Kobayashi K. Murakami S. J. Biol. Chem. 1998; 273: 15479-15486Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20.Ferrari E. Wright-Minogue J. Fang J.W.S. Baroudy B.M. Lau J.Y.N. Hong Z. J. Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar). However, we recently reported that an HCV NS5B expressed and purified fromEscherichia coli is able to initiate RNA synthesis de novo using the full-length HCV genome or the 3′-end of the HCV genome in both senses as templates without requirement of a primer (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). Furthermore, we demonstrated that NS5B can utilize the 98-nt, X region RNA at the 3′-end of plus-strand HCV genomic RNA as a minimal template. The upstream sequences including the variable sequence region and U-U/C-rich tract were found to enhance the efficiency of RNA synthesis. For the minus-strand template, a minimum of 239 nt at the 3′-end of minus-strand HCV RNA was required for efficient RNA synthesisin vitro. Thus, our recombinant HCV polymerase by itself appears to be able to recognize HCV RNA and utilize the X region as a minimal RNA template. We used this minimal RNA template to further elucidate the mechanism of HCV RNA synthesis. Our results show that HCV polymerase can bind to the X region directly, but its binding is enhanced by an upstream U-U/C-rich tract and part of the NS5B-coding region. Furthermore, we identified the RNA initiation site and determined the sequence requirement for initiation of RNA synthesis. These results shed further light on the mechanism of HCV RNA replication. Recombinant HCV RdRp NS5B enzyme was expressed in E. coli BL21 transformed with plasmid pThNS5B and purified using a Ni2+-nitrilotriacetic acid (NTA)-agarose column (Qiagen) as described previously (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). The NS5B-containing fractions were collected and dialyzed against buffer A (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 10% glycerol) and applied to a heparin-Sepharose CL-4B column (Amersham Pharmacia Biotech) equilibrated in the same buffer. The column was washed with buffer A and step-eluted with 100 mm to 1m NaCl. The peak fractions containing pure NS5B were pooled, and the salt concentration of the pooled fractions was adjusted to 100 mm NaCl with buffer A without NaCl. The protein was further purified by passing it through an SP-Sepharose column (Amersham Pharmacia Biotech) that had been equilibrated with buffer A. The adsorbed protein was then eluted with a 10-ml linear gradient of NaCl from 100 mm to 1 m. Fractions (1 ml) were collected, and small aliquots of the peak fractions were stored at −80 °C after dialyzing against buffer A containing 20% glycerol. Protein concentrations were determined by Bio-Rad protein assay, and the purity of protein was estimated by densitometric analysis of a silver-stained SDS-polyacrylamide gel. NS5B proteins were subjected to a SDS-10% polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The membrane was probed with rabbit anti-His6 antibody (Santa Cruz Biotechnology, Inc.), and proteins were detected by using goat anti-rabbit IgG conjugated with peroxidase (American Qualex) and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Polymerase activity assays were carried out as described previously (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). Briefly, 200 ng of RNA template was incubated with 2 pmol of NS5B enzyme in a 25-μl reaction containing 50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 100 mm potassium glutamate, 5 mm MgCl2, 1 mm dithiothreitol, 20 μg/ml actinomycin D (Sigma), 20 units of RNase inhibitor (Promega), 10% glycerol, 0.5 mmeach ATP, CTP, GTP, and 5 μm UTP with 10 μCi of [α-32P]UTP (3000 Ci/mmol; NEN Life Science Products) for 1 h at 25 °C. After reactions, the products were extracted with acidic phenol emulsion (phenol, chloroform (Ambion), 10% SDS, 0.5 m EDTA (1:1:0.2:0.04)), precipitated with 2.5 volumes of 5 m ammonium acetate/isopropyl alcohol (1:5), denatured in a denaturing buffer containing 95% formamide with 10 mm EDTA and 0.025% each xylene cyanol and bromphenol blue, and then resolved on an 8 m urea, 6% polyacrylamide gel (14 × 17 cm). After electrophoresis, gels were stained with ethidium bromide to localize positions of template RNAs. For better resolution, products were also analyzed on a 5% denaturing sequencing gel (38 × 43 cm). The dried gels were exposed to x-ray film, and the amount of 32P incorporated into the newly synthesized RNA was quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Wild-type and mutant X region RNAs were synthesized by in vitro transcription using polymerase chain reaction-amplified DNA templates fused to bacteriophage T7 RNA polymerase promoter as described previously (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). The full-length 3′-UTR of HCV containing a long U-U/C-rich tract (81 nt) (named HCV-3′(+) Full) was amplified by polymerase chain reaction using the infectious genotype 1b HCV cDNA (7.Yanagi M. Claire M.S. Shapiro M. Emerson S.U. Purcell R.H. Bukh J. Virology. 1998; 244: 161-172Crossref PubMed Scopus (199) Google Scholar) as a template. The 3′-UTR of HCV containing a short U-rich sequence (13 nt) (named HCV-3′(X)) was amplified from an HCV Korean isolate of 1b genotype (15.Ito T. Tahara S.M. Lai M.M.C. J. Virol. 1998; 72: 8789-8796Crossref PubMed Google Scholar, 16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). The FCR RNA, which contains the HCV-3′(+) Full plus the neighboring NS5B-coding region (nt 9300–9364 of the infectious genotype 1b HCV RNA) and the CR RNA, which contains C-terminal portion of NS5B-coding region only (nt 9300–9364), were amplified in a similar way. The gel-purified polymerase chain reaction-amplified DNA templates were used directly for in vitro transcription using T7 RNA polymerase. After transcription with T7 RNA polymerase, DNA templates were digested by RQ1 DNase (Promega) for 15 min at 37 °C. Then in vitrotranscribed RNAs were purified using a Sephadex G-25 spin column, extracted with acidic phenol/chloroform, and then precipitated with ethanol. RNA concentrations were estimated by measuring the absorbance at 260 nm. For 5′-end 32P-labeling of X region and FCR RNAs, in vitro transcripts were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals) and phosphorylated with T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (6000 Ci/mmol; NEN Life Science Products). After heat inactivation of the enzyme, free nucleotides were removed using a Sephadex G-25 spin column, and the labeled RNA was purified by electrophoresis on a 6% polyacrylamide gel containing 8 m urea in 1× TBE buffer (90 mmTris base, 90 mm boric acid, 1 mm EDTA, pH 8.2). The labeled RNAs were eluted in 2 ml of a buffer consisting of 0.5 m ammonium acetate, 1 mm EDTA (pH 7.5), and 0.5% SDS. The 5′-end-labeled RNAs were then precipitated with ethanol and resuspended in double distilled H2O to a final concentration of 1.2 μm (specific activity: 4.4 × 104 cpm/pmol for X RNA and 3.7 × 104cpm/pmol for FCR RNA). 32P-Labeled RNA was incubated with 20 μl of Na2CO3/NaHCO3 buffer (pH 10) for 20 min at 90 °C. The partially hydrolyzed RNA was mixed with an equal volume of a denaturing buffer containing 95% formamide with 10 mm EDTA and 0.025% each of xylene cyanol and bromphenol blue and loaded onto a denaturing polyacrylamide gel. 32P-Labeled 3′-UTR of HCV RNA genome was synthesized by in vitro transcription using the HCV-3′(X) or HCV-3′(+) Full DNA template as described previously (13.Ito T. Lai M.M.C. J. Virol. 1997; 71: 8698-8706Crossref PubMed Google Scholar). UV cross-linking experiments were carried out in 96-well microtiter plates in a buffer containing 25 mm HEPES-KOH, pH 7.8, 150 mm NaCl, 5% glycerol, 1 mm EDTA, and 2 mm dithiothreitol. Purified NS5B (1.5 pmol) was preincubated with 10 μg of yeast tRNA in a 20-μl binding buffer at 30 °C for 10 min. Then 32P-labeled RNA probe (2.5 × 105 cpm) was added to a final concentration of 4 pmol/ml, and the mixture was further incubated for 10 min. UV cross-linking was conducted as described previously (13.Ito T. Lai M.M.C. J. Virol. 1997; 71: 8698-8706Crossref PubMed Google Scholar). For competition assays, the indicated amounts of unlabeled RNAs or homopolymeric RNAs (Amersham Pharmacia Biotech) were preincubated with NS5B prior to the addition of probe. After UV cross-linking, RNAs were digested with 2 mg/ml RNase A at 37 °C for 30 min, and the UV-cross-linked products were subjected to SDS-polyacrylamide gel electrophoresis. The dried gels were exposed to x-ray films for autoradiography. The amounts of free and bound RNA were analyzed using a PhosphorImager (Molecular Dynamics). The purified NS5B diluted to appropriate concentrations in buffer A were mixed with the radiolabeled RNA in the same buffer and incubated for 15 min at 25 °C. RNA-protein complexes were formed in a 10-μl reaction mixture in the same buffer as that for the RdRp assay, with the exception that rNTPs and actinomycin D were omitted. The 5′-end32P-labeled RNA (50 fmol) was incubated with 2.5 pmol of NS5B, unless otherwise specified, or increasing amounts of NS5B (0.5, 0.7, 0.9, 1, 2, 3, 4, and 5 pmol) for 15 min on ice and then for an additional 15 min at 25 °C. After reactions, 2 μl of loading buffer (50% glycerol, 0.01% each xylene cyanol and bromphenol blue) was added, and the samples were loaded directly onto 1-mm-thick nondenaturing 4% polyacrylamide (59:1 acrylamide/bisacrylamide) gels. Gels were prerun at 5 V/cm for 30 min and run at room temperature in 0.5× TBE. For competition assays, increasing amounts of the unlabeled RNAs were preincubated with NS5B for 15 min on ice, and incubation was continued for 15 min at 25 °C with the labeled RNA. The unlabeled competitor HCV RNAs or homopolymeric RNAs were used in the competition assays. The 5′-end 32P-labeled X RNA (50 fmol) was preincubated with 3 pmol of NS5B in buffer A on ice for 15 min and then at 25 °C for 15 min. After reactions, RNAs were digested with RNase T1 (Ambion; 0.1 units/reaction) for 15 min at 25 °C in 10 μl of RdRp assay buffer (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 100 mm potassium glutamate, 5 mm MgCl2, 1 mm dithiothreitol, 10% glycerol). Samples were mixed with an equal volume of the denaturing buffer, boiled for 2 min, and analyzed by electrophoresis on a 5% denaturing sequencing gel. We have previously expressed and purified an enzymatically active, full-length recombinant HCV NS5B by Ni2+-NTA column chromatography (16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar). This preparation contained trace amounts of E. coliproteins of 75 and 110 kDa, which were detectable by silver staining but not by Coomassie Blue staining (see Ref. 16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar; data not shown). To study RNA binding and enzymatic properties of NS5B, we first performed further purification of NS5B. The pooled NS5B fractions eluted with 250–350 mm imidazole from Ni2+-NTA column were loaded on a heparin-Sepharose CL-4B column. The bound NS5B was eluted as a broad peak by NaCl gradient (Fig.1 A) and was detected by anti-His6 antibody in Western blotting (Fig.1 B). The main peak fractions (∼400–600 mmNaCl-eluted fraction) were pooled and loaded onto an SP-Sepharose column after adjusting the NaCl concentration to 100 mm. The NS5B was eluted with a buffer containing 500–700 mmNaCl. The eluate with the highest purity, as visualized by silver staining of a SDS-polyacrylamide gel, was found to be over 98% pure (Fig. 1 C). No contaminating proteins could be detected. The highly purified NS5B protein (65 kDa) was used for all the experiments described in this study. To investigate whether NS5B can directly bind to the 3′-UTR of HCV RNA, we first performed UV cross-linking studies. We used a full-length 3′-UTR RNA (HCV-3′(X)), which consists of a stretch of variable sequence, a short U-rich tract (13 nt), and the X region (15.Ito T. Tahara S.M. Lai M.M.C. J. Virol. 1998; 72: 8789-8796Crossref PubMed Google Scholar). The purified recombinant NS5B was incubated with 32P-labeled HCV 3′-UTR, and the complex was covalently cross-linked by UV irradiation. As shown in Fig. 2 A, a 65-kDa protein was labeled. To assess the specificity of this cross-linking, increasing amounts of various unlabeled RNA were used for competition studies. We found that the unlabeled homologous 3′-UTR RNA competed very effectively with the labeled RNA for binding (lanes 2–5). We next used homopolymeric RNAs for competition to assess the nucleotide preference for NS5B binding. Among the homopolymeric RNAs used (Fig. 2 B), poly(U) competed most efficiently (lanes 5–7), followed by poly(G) (lanes 11–13). In contrast, poly(C) and poly(A) were poor competitors (lanes 2–4 andlanes 8–10, respectively). This order of competition agrees well with the previous direct filter binding assays of NS5B using homopolymeric RNAs (17.Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (645) Google Scholar). However, the 98-nt X region alone did not significantly compete for binding; only in the presence of a 500-fold molar excess of unlabeled RNA was some inhibition of NS5B binding observed (Fig. 2 C, lane 5). In direct UV cross-linking studies using 32P-labeled 98-nt X RNA, a very weak NS5B band was detected only after a very long exposure (data not shown). These results indicate that NS5B binds weakly to the X region. We have also tested the 3′-UTR (HCV-3′(+) Full) from an infectious HCV genotype 1b RNA (14.Yanagi M. Claire M.S. Emerson S.U. Purcell R.H. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2291-2295Crossref PubMed Scopus (192) Google Scholar), which contains a longer (81-nt U/U-C) pyrimidine-rich tract; comparable binding activity was detected (data not shown). These results indicate that NS5B binds to the HCV 3′-UTR mainly through interaction with the U-rich sequence. It also weakly interacts with the X region. Since UV cross-linking studies showed a weak but still detectable interaction between NS5B and the X region (data not shown), we took another approach, electrophoretic mobility shift assay (EMSA), to characterize the interaction. We first estimated the binding affinity of NS5B to X RNA. The NS5B proteins at various concentrations were incubated with a fixed amount of radiolabeled X RNA, and the RNA-protein complex was resolved on a nondenaturing polyacrylamide gel. As shown in Fig. 3 A, an RNA-NS5B complex was retarded at the loading wells when increasing amounts of NS5B were used. The percentage of probe bound to NS5B was quantified using a PhosphorImager and graphically illustrated in Fig.3 B. The apparent dissociation constant was estimated to be about 170 nm. The binding affinity of HCV NS5B to this RNA is lower than those of influenza virus polymerase subunit PB1 to the viral complementary RNA (70 nm) (31.Gonzalez S. Ortin J. EMBO J. 1999; 18: 3767-3775Crossref PubMed Scopus (97) Google Scholar) or Qβ replicase toin vitro selected RNA templates (20–30 nm) (26.Brown D. Gold L. Biochemistry. 1995; 34: 14765-14774Crossref PubMed Scopus (42) Google Scholar, 27.Brown D. Gold L. Biochemistry. 1995; 34: 14775-14782Crossref PubMed Scopus (42) Google Scholar, 28.Brown D. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11558-11562Crossref PubMed Scopus (63) Google Scholar), but it is slightly higher than that of Qβ replicase to unselected RNA ligand pools (>200 nm). Competition assays indicated that the NS5B-X RNA interaction can be disrupted efficiently with a 5–10-fold excess of unlabeled X RNA (Fig.4 A) and also very efficiently inhibited by poly(U) (Fig. 4 B). Other homopolymers were less effective in competition, with the following order of binding efficiency: poly(U) ≫ poly(C) ≥ poly(G) > poly(A). These results were similar to the competition data observed with the full-length 3′-UTR by UV cross-linking, except that poly(C) was a slightly better competitor than poly(G) for the X RNA probe in EMSA. To further confirm that the binding of NS5B to X RNA was specific, we used several X RNA mutants that contain various degrees of deletion of stem I and/or II for competition assays. These mutants have deletions of 40, 46, and 57 nt from the 3′-end (−40X, −46X, and −57X, respectively), and none of them can serve as RNA templates (see Ref. 16.Oh J.-W. Ito T. Lai M.M.C. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar; see also Fig.7 B, left panel, lanes 9–11). The results showed that all of them competed poorly as compared with the 98-nt X RNA for binding to NS5B (Fig.4 C). These results suggest that NS5B binding to the X region is necessary for polymerase activity. Nevertheless, −40X RNA competed slightly better than the −46X and −57X RNAs at 10-fold molar excess, suggesting that −40X RNA contains part of the NS5B-binding sequence.Figure 4Specificity of interaction of HCV NS5B with X RNA. A, EMSA was carried out in the absence (−) and in the presence of a 1–100-fold molar excess of unlabeled X RNA. Increasing am