FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022198259> ?p ?o ?g. }

• W2022198259 endingPage "28705" @default.
• W2022198259 startingPage "28700" @default.
• W2022198259 abstract "Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3′ termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp. Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3′ termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp. hepatitis C virus untranslated region internal ribosome entry site RNA-dependent RNA polymerase dithiothreitol complementary strand IRES Hepatitis C virus (HCV)1 has a positive-stranded RNA genome and belongs to the family Flaviviridae (1van Regenmortel M.H.V. Fauquet C.M.F. Bishop D.H.L. Carstens E.B. Estes M.K. Lemon S.M. Maniloff J. Mayo M.A. McGeoch D.J. Pringle C.R. Wickner R.B. Virus Taxonomy, Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego2000: 872-878Google Scholar). HCV is a major causative agent of post-transfusion sporadic non-A and non-B hepatitis worldwide (2Alter M.J.H. Hepatology. 1997; 26: 62-65Crossref PubMed Scopus (910) Google Scholar). The HCV RNA genome is about 9.6 kb and has a long open reading frame encoding a polyprotein of ∼3,010 amino acids (3Bartenschlager R. Antiviral Chem. Chemother. 1997; 8: 281-301Crossref Scopus (62) Google Scholar, 4Lindenbach B.D. Rice C.M. Fields B.N. Knipe D.M. Howly P.M. Flaviviridae: The Viruses and Their Replication. 4th Ed. Lippincott-Raven Publishers, Philadelphia, NY2001: 991-1042Google Scholar), which is processed into at least 10 polypeptides (NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) by host and viral proteases (5Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar, 6Matsuura Y. Miyamura T. Semin. Virol. 1993; 4: 297-304Crossref Scopus (56) Google Scholar, 7Tomei L. Failla C. Santolini E. De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar). The 5′-untranslated region (UTR) contains the highly conserved internal ribosome entry site (IRES) of 341 nucleotides (8Fukushi S. Katayama K. Kurihara C. Ishiyama N. Hoshino F.B. Ando T. Oya A. Biochem. Biophys. Res. Commun. 1994; 199: 425-432Crossref PubMed Scopus (78) Google Scholar, 9Kohara T.K. Iizuka N. Kohara M. Nomoto A. J. Virol. 1992; 66: 1476-1483Crossref PubMed Google Scholar, 10Lemon S.H. Honda M. Semin. Virol. 1997; 8: 274-288Crossref Scopus (71) Google Scholar). The 3′-UTR contains a polypyrimidine “U/C” tract, a variable region, and a highly conserved 98-base X region (11Tanaka T. Kato N. Cho M.-J. Sugiyama K. Shimotohno K. J. Virol. 1996; 70: 3307-3312Crossref PubMed Google Scholar,12Tanaka T. Kato N. Cho M.-J. Shimotohno K. Biochem. Biophys. Res. Commun. 1995; 215: 744-749Crossref PubMed Scopus (248) Google Scholar). The X region and polypyrimidine U/C tract are required for viral infectivity (13Yanagi M. St Claire M. Emerson S.U. Purcell R.H. Bukh J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2291-2295Crossref PubMed Scopus (193) Google Scholar). HCV RNA-dependent RNA polymerase (RdRp) binds the X region and is important for minus strand RNA synthesis (14Cheng J.-C. Chang M.-F. Chang S.C. J. Virol. 1999; 73: 7044-7049Crossref PubMed Google Scholar, 15Kolykhalov A.A. Mihalik K. Feinstone S.M. Rice C.M. J. Virol. 2000; 74: 2046-2051Crossref PubMed Scopus (565) Google Scholar, 16Oh J.W. Ito T. Lai M.M. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar, 17Oh J.W. Sheu G.T. Lai M.M. J. Biol. Chem. 2000; 275: 17710-17717Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). NS5B shows RdRp activity in vitro (16Oh J.W. Ito T. Lai M.M. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar, 18Behrens S. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 19Hagedorn C.H. van Beers E.H. De Staaercke C. Curr. Top. Microbiol. Immunol. 2000; 242: 225-260PubMed Google Scholar, 20Lesburg C.A. Radfar R. Weber P.C. Curr. Opin. Investig. Drugs. 2000; 1: 289-296PubMed Google Scholar, 21Lohmann V. Köner F. Herian U. Bartenschlager R. J. Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar, 22Lohmann V. Roos A. Köner F. Herian U. Bartenschlager R. J. Viral Hep. 2000; 7: 167-174Crossref PubMed Scopus (66) Google Scholar, 23Lohmann V. Roos A. Köner F. Koch J.O. Bartenschlager R. Virology. 1998; 249: 108-118Crossref PubMed Scopus (128) Google Scholar, 24Sun X.-L. Johnson R.B. Hockman M.A. Wang Q.M. Biochem. Biophys. Res. Commun. 2000; 268: 798-803Crossref PubMed Scopus (65) Google Scholar). NS5B has a highly hydrophobic 21-amino acid sequence in its C terminus, and when it is removed NS5B becomes soluble (25Ferrari 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, 26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar, 27Yamashita 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 (223) Google Scholar). HCV RdRp exhibits de novo and copy-back initiation activities (18Behrens S. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 24Sun X.-L. Johnson R.B. Hockman M.A. Wang Q.M. Biochem. Biophys. Res. Commun. 2000; 268: 798-803Crossref PubMed Scopus (65) Google Scholar, 28Luo G. Hamatake R.K. Mathis D.M. Racela J. Rigat K.L. Lemm J. Colonno R.J. J. Virol. 2000; 74: 851-863Crossref PubMed Scopus (254) Google Scholar, 29Zhong W. Uss A.S. Ferrari E.J. Lau Y.N. Hong Z. J. Virol. 2000; 74: 2017-2022Crossref PubMed Scopus (166) Google Scholar). It can utilize single-stranded RNA as a template but not double-stranded RNA (30Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y.N. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 31Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K.B. Cameron C.E. Lau J.Y.N. Hong Z. J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar). It prefers a cytidine at the 3′ terminus of the template, andde novo initiation in vitro by HCV RdRp was selectively activated by a high GTP concentration (22Lohmann V. Roos A. Köner F. Herian U. Bartenschlager R. J. Viral Hep. 2000; 7: 167-174Crossref PubMed Scopus (66) Google Scholar, 23Lohmann V. Roos A. Köner F. Koch J.O. Bartenschlager R. Virology. 1998; 249: 108-118Crossref PubMed Scopus (128) Google Scholar, 26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar, 30Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y.N. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 32Kao C.C. Yang X. Kline A. Wang Q.M. Barket D. Heinz B.A. J. Virol. 2000; 74: 11121-11128Crossref PubMed Scopus (115) Google Scholar). It can utilize both viral and non-viral RNA templates although a specific promoter has not been identified (18Behrens S. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar, 22Lohmann V. Roos A. Köner F. Herian U. Bartenschlager R. J. Viral Hep. 2000; 7: 167-174Crossref PubMed Scopus (66) Google Scholar, 24Sun X.-L. Johnson R.B. Hockman M.A. Wang Q.M. Biochem. Biophys. Res. Commun. 2000; 268: 798-803Crossref PubMed Scopus (65) Google Scholar). NS5B bound to the poly(U) stretch (18Behrens S. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar) and the upstream conserved stem-loop structures at the 3′ end of the genome (14Cheng J.-C. Chang M.-F. Chang S.C. J. Virol. 1999; 73: 7044-7049Crossref PubMed Google Scholar). We have recently found that the complementary sequence of IRES showed very strong template activity (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar, 34Reigadas S. Ventura M. Sarih-Cottin L. Castroviejo M. Litvak S. Astier-Gin T. Eur. J. Biochem. 2001; 268: 5857-5867Crossref PubMed Scopus (69) Google Scholar). In this study, we analyzed the promoter/origin structure of the complementary sequence of IRES, and we determined the role of the complementary strand of the first and second stem-loops of IRES in RNA synthesis in vitro. This domain perfectly overlaps with those identified in the HCV replicon system in vivo(35Friebe P. Lohnmann V. Krieger N. Bartenschlager R. J. Virol. 2000; 75: 12047-12057Crossref Scopus (287) Google Scholar). HCV NS5B protein truncated by 21 C-terminal amino acids with a His6 tag was expressed inSpodoptera frugiperda (Sf)-21AE cells and purified as described previously (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar). Purified HCV RdRp was stored at −25 °C in the presence of 50% glycerol. RNA templates designed from the complementary sequences of HCV IRES (cIRES) were synthesized with a MEGAscript T7 RNA polymerase kit (Ambion) (Fig. 1). The DNA templates were produced by PCR using the primer pairs listed in TableI. The sequence UGGC was added to the 3′ terminus of all the templates. The DNA templates were removed by digestion with DNase I after in vitro transcription, and all transcripts were purified by 6% PAGE, 7 m urea for use as the templates of in vitro transcription by HCV RdRp, followed by successive phenol-chloroform extraction and ethanol precipitation. These RNA templates were resuspended in RNase-free water and stored at −80 °C until used.Table ISequences of the primers used in this studyView Large Image Figure ViewerDownload (PPT)The T7 polymerase promoter sequence is shown in bold (TK-1 and TK-2). The mutated nucleotides are shown in italics (TK-10 to TK-12). An extra GCCA sequence was added to the 3′ terminus of the RNA templates (TK-4 to TK-13). Open table in a new tab The T7 polymerase promoter sequence is shown in bold (TK-1 and TK-2). The mutated nucleotides are shown in italics (TK-10 to TK-12). An extra GCCA sequence was added to the 3′ terminus of the RNA templates (TK-4 to TK-13). Unless otherwise indicated, HCV RdRp activity was measured in 50 μl of standard transcription buffer, TxG(+) (20 mm Tris/HCl (pH 8.0), 100 mm KCl, 2.5 mm MnCl2, 50 μm ATP, 50 μm CTP, 5 μm UTP, 0.5 mm GTP, 0.185 Mbq of [α-32P]UTP, 10 pmol of RNA template, 25 μg/ml actinomycin D, 5 units of human placental RNase inhibitor (Nacalai Tesque, Japan), 1 mm DTT, and 10 pmol of NS5B). For the single-round transcription assay, the reaction mixture without nucleotides was preincubated with 50 or 500 μm GTP at 29 °C for 30 min. Then 0.2 mg/ml heparin (Wako Chemicals, Japan) was added to the mixture, followed by ATP, CTP, and UTP, respectively, and the reaction mixture was further incubated at 29 °C for an additional 90 min. The reaction was stopped by extraction of 150 μl of Sepasol RNA II (Nacalai Tesque, Japan) and 40 μl of chloroform. [32P]UMP-labeled RNA was precipitated with the equal volume of 2-propanol. The radiolabeled RNA was washed with 70% ethanol, dried, and resuspended in formamide dye loading buffer and analyzed by electrophoresis on a 6% PAGE containing 7 murea. The radioactivity of the transcribed RNA was measured with a BAS-2000 image analyzer (Fuji Film), and the amount of transcribed RNA was calculated from the amount of UMP in the transcripts. Each value was calculated from the average of at least three independent assays. cIRES, SL234–1D, SL234, SL34, SL4, and SL0 were transcribed in vitro in 40 mm Tris/HCl (pH 7.6), 10 mm DTT, 0.5 mm each of ATP, CTP, and GTP, 50 mm UTP, 50 μCi of UTP (Amersham Biosciences), 10 μg of DNA template, 20 units of human placental RNase inhibitor, and 50 units of T7 RNA polymerase (Toyobo, Japan) at 37 °C for 4 h. The transcribed RNA was purified by electrophoresis on a 6% PAGE containing 7 m urea. [32P]UMP-labeled RNA templates (20,000 cpm) were incubated in 20 μl of 20 mmTris/HCl (pH 8.0), 100 mm KCl, 2.5 mmMnCl2, 5 units of human placental RNase inhibitor, 1 mm DTT, 2 mg/ml yeast tRNA (Roche Molecular Biochemicals), and 3.2 pmol NS5B with or without 0.5 mm GTP on ice for 1 h. Then 4 μl of DNA sample buffer (0.002% bromphenol blue, 0.002% xylene cyanol, and 50% glycerol) was added, and the mixture was analyzed by electrophoresis on 0.8% agarose gel (Nacalai Tesque, Japan) in TAE buffer (40 mm Tris/HCl, 20 mmacetic acid, and 1 mm EDTA). After the electrophoresis the gel was dried and analyzed with the BAS-2000 image analyzer. For the competition assay, 1, 10, 100, and 1000 pmol of non-labeled cIRES, SL234–1D, SL234, SL34, SL4, SL0, 3NTR, and XREG (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar) were incubated in the binding reaction mixture with [32P]UMP-labeled cIRES (20,000 cpm). For the detection of the HCV RdRp protein, the agarose gel was stained with 0.2% Coomassie Brilliant Blue R-250 (Wako Chemicals, Japan) for 1 h and destained with 50% methanol and 5% acetic acid. First of all, the concentration of heparin for the single-round transcription was determined (Fig.2 A). HCV RdRp and cIRES template (10 pmol each) were treated with 3.1, 6.25, 12.5, 25, 50, 100, and 200 μg/ml of heparin after preincubation with 50 μm (G(−)) or 0.5 mm GTP (G(+)) in 20 mm Tris/HCl (pH 8.0), 100 mm KCl, 2.5 mm MnCl2, 25 μg/ml actinomycin D, and 5 units of human placental RNase inhibitor at 29 °C for 30 min. Then 50 μm ATP, 50 μm CTP, 5 μm UTP, and 0.185 Mbq of α-[32P]UTP were added and incubated for an additional 90 min. The RdRp activity with more than 0.2 mg/ml heparin was about 20% that without heparin and did not drop below 20% even when more heparin was added. Next, the time course with 0.2 mg/ml heparin was examined (Fig. 1 B). Under these conditions, the accumulation of transcribed RNA continued for 90 min and reached a plateau. Thus, 0.2 mg/ml heparin was used for the single-round transcription assay of HCV RdRp. First, the activity of deletion mutant templates for stem-loop structures was measured by the single-round transcription assay because the initiation activity could be accurately measured with this assay (Figs. 1 and 3). Under de novo initiation by 0.5 mm GTP, the template activity of SL234–1D, SL234, SL34, SL4, and SL0 was 74.8, 88.2, 34.2, 27.9, and 9.7% that of cIRES, respectively (Fig. 3 D). There was no significant difference among cIRES, SL234–1D, and SL234. However, there was a big decrease in activity between SL234 and SL34. Without the initiation, far less RNA was synthesized de novo, and there was little difference among them. Next, in order to analyze the effect of the stem-loop structures and sequences between SL234 and SL34 on de novo transcription more precisely, single-round transcriptions using SL34-S, SL1234–1S, SL1234–1LD, and SL34-SS were performed (Figs. 1 and 5 A). Again, the sequence UGGC was added to the 3′ terminus of each template. The amount of RNA synthesized from 10 pmol of cIRES, SL234–1D, SL234, SL34, SL4, SL0, SL34-S, SL1234–1S, SL1234–1LD, and SL34-SS was 8.06, 6.03, 7.11, 2.76, 2.25, 0.78, 3.90, 4.49, 9.22, and 5.16 fmol, respectively. Several bands smaller than the template were found among the transcripts from cIRES, SL234–1D, SL234, and SL34 (Fig. 3,A and B). Additional transcripts were also found in SL34-S, SL1234–1S, SL1234–1LD, and SL34-SS (Fig. 5 A). However, no additional bands were found in SL4 and SL0. From the pattern and size of the transcripts, we concluded that they were produced by early termination. From the size of the two major additional transcripts estimated from PAGE (Fig. 3, *1 and*2), transcription terminated in bulge *1 and*2 of cSL3, respectively (Fig. 1). Until this experiment, we only calculated the transcripts of template size, excluding those derived from early termination. To compare the effect of cSL1 and cSL2, we designed templates without early termination as follows: SL12, SL2, SL12–1S, SL12–1LD, and SL0+SL1 (Fig. 1 and Fig. 5, B and C). From these templates few additional transcripts were obtained. The transcripts of SL2, SL12–1S, and SL12–1LD were 165.5, 258.4, and 91.7% the level of SL12 (Fig. 5 B). When cSL1 was added to SL0 (SL0+SL1), the transcripts were 69.0% the level without cSL1 (SL0) (Fig. 1 C). The same deletion mutant templates were examined for binding to HCV RdRp by gel shift assay (Fig. 4). The relative binding ratio was calculated after correcting for the number of UMP in the probes. The relative binding ratio with 0.5 mm GTP was 100, 74, 83, 46, 58, and 60% whereas that without GTP was 178, 89, 79, 89, 58, and 60%, for cIRES, SL-234–1D, SL234, SL34, SL4, and SL0, respectively. For the competition assay, HCV RdRp and radiolabeled cIRES were incubated with 1, 10, 100, and 1000 pmol of unlabeled cIRES, SL234, SL34, SL34-S, SL4, SL0, 3NTR, and XREG, respectively. One pmol of cIRES and SL234–1D inhibited the binding of cIRES with HCV RdRp. One pmol of 3NTR almost inhibited the binding as well. Ten pmol of SL234, SL34, SL34-S, and XREG inhibited the gel shift with cIRES. From these results, cSL2 is concluded important for binding with HCV RdRp especially in 0.5 mm GTP. The HCV RNA genome contains conserved 5′- and 3′-UTRs (8Fukushi S. Katayama K. Kurihara C. Ishiyama N. Hoshino F.B. Ando T. Oya A. Biochem. Biophys. Res. Commun. 1994; 199: 425-432Crossref PubMed Scopus (78) Google Scholar, 9Kohara T.K. Iizuka N. Kohara M. Nomoto A. J. Virol. 1992; 66: 1476-1483Crossref PubMed Google Scholar, 10Lemon S.H. Honda M. Semin. Virol. 1997; 8: 274-288Crossref Scopus (71) Google Scholar, 11Tanaka T. Kato N. Cho M.-J. Sugiyama K. Shimotohno K. J. Virol. 1996; 70: 3307-3312Crossref PubMed Google Scholar, 12Tanaka T. Kato N. Cho M.-J. Shimotohno K. Biochem. Biophys. Res. Commun. 1995; 215: 744-749Crossref PubMed Scopus (248) Google Scholar). As in the case of Flaviviridae family viruses, the 3′-terminal X-region is expected to play an important role in the synthesis of the minus strand, and the complementary strand of IRES is expected to serve as the origin for plus strand synthesis in genome replication (4Lindenbach B.D. Rice C.M. Fields B.N. Knipe D.M. Howly P.M. Flaviviridae: The Viruses and Their Replication. 4th Ed. Lippincott-Raven Publishers, Philadelphia, NY2001: 991-1042Google Scholar). The complementary strand of IRES may also work as a promoter of transcription. Mutations in IRES also affected the replication of genomic RNA (35Friebe P. Lohnmann V. Krieger N. Bartenschlager R. J. Virol. 2000; 75: 12047-12057Crossref Scopus (287) Google Scholar). The reason for this may be that the mutation in the 3′ terminus of the complementary strand (cIRES) affected the replication and transcription. Both termini of the viral genomic RNA have stem-loop secondary structures. The 3′ termini of both the plus and minus strands of genomic RNA are able to serve as templates for RdRp in vitro (16Oh J.W. Ito T. Lai M.M. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar) The complementary sequences of IRES had the highest template activity for de novo RNA synthesisin vitro (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar, 34Reigadas S. Ventura M. Sarih-Cottin L. Castroviejo M. Litvak S. Astier-Gin T. Eur. J. Biochem. 2001; 268: 5857-5867Crossref PubMed Scopus (69) Google Scholar). However, activity for the de novo synthesis of RNA in vitro by RdRp in the X region was very weak (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar). Therefore, we determined the promoter/origin structure of the complementary strand of IRES. In the multiround transcription system, the products from short templates were sometimes larger than those from long ones. To compare the initiation activity, we established a de novosingle-round transcription system using 0.2 mg/ml heparin to treat HCV RdRp and templates followed by preincubation with 0.5 mmGTP (Fig. 2). Because HCV RdRp prefers a cytidine at the 3′ terminus and interacts with GTP (28Luo G. Hamatake R.K. Mathis D.M. Racela J. Rigat K.L. Lemm J. Colonno R.J. J. Virol. 2000; 74: 851-863Crossref PubMed Scopus (254) Google Scholar, 30Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y.N. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 32Kao C.C. Yang X. Kline A. Wang Q.M. Barket D. Heinz B.A. J. Virol. 2000; 74: 11121-11128Crossref PubMed Scopus (115) Google Scholar, 33Lohmann V. Overton H. Bartenschlager R. J. Biol. Chem. 1999; 274: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), all the templates are designed to have UGGC at the 3′ terminus (Figs. 1 and5). We have temporally used the secondary structure of cIRES as a mirror image of that of IRES in Fig. 1until it is determined experimentally (36Honda M. Beard M.R. Ping L.H. Lemon S.M. J. Virol. 1999; 73: 1165-1174Crossref PubMed Google Scholar, 37Lukavsky P.J. Otto G.A. Lancaster A.M. Sarnow P. Puglisi J.D. Nat. Struct. Biol. 2000; 7: 1105-1110Crossref PubMed Scopus (198) Google Scholar). Because the transcription activity following the treatment with 0.5 mm GTP decreased markedly when cSL2 was deleted, cSL2 was important for de novo initiation (Fig. 3 D). However, the activity of SL1234–1S was also half of that of cIRES. Because the T m of the UA stem was 12 °C, a stem structure might not form in the reaction at 29 °C. A comparison of the activity of cIRES, SL1234–1D, SL1234–1S, and SL1234–1LD indicated the complicated secondary structure of the template, although cSL2 and the stem of cSL1 may affect the structure of the promoter/origin. The sequence between cSL2 and cSL3 could also affect the activity. The seven Gs, which exist between cSL2 and cSL3, did not affect the activity. From a comparison of the activity of SL32-S, SL34-SS, and SL34, the length of the single stranded sequence at the 3′ terminus was confirmed important as reported previously (26Kashiwagi T. Hara K. Kohara M. Kohara K. Iwahashi J. Hamada N. Honda H. Toyoda T. Biochem. Biophys. Res. Commun. 2002; 250: 1188-1194Crossref Scopus (17) Google Scholar). In this series of experiments, we calculated the product amount from only the template-sized bands. Additional products were transcribed from the templates containing cSL3 (Figs. 2 and 5). We measured the size of the products of template size and smaller (Fig. 3, *1 and *2). By comparing their size with that of the templates, the products *1 and *2 were identified as early termination products from the templates. The positions of possible termination sites are mapped in the bulges of cSL3 (Fig. 1). There is a triple helical structure in IRES corresponding to bulge *1, and a complex stem-loop in the stem structure of stem-loop 3 corresponding to bulge *2. We predict a strong secondary structure for these sequences in cIRES. The results and the prediction of secondary structure by mFold (bioinfo.math.rpi.edu/∼zukerm/) (38Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3232) Google Scholar, 39Zuker M. Mathews D.H. Turner D.H. Barciszewski J. Clark B.F.C. RNA Biochemistry and Bio/Technology. Kluwer Academic Publishers, Norwell, MA1999: 11-43Google Scholar), which predicted too many to be shown here, suggest a complicated secondary structure of cIRES. The results obtained with deletion mutants of stem-loop structures of cIRES were difficult to interpret. The cIRES sequence may make complicated stem-loop structures which interact with each other. Therefore, we decided to design a simpler template. Because we could not predict the secondary structure of cIRES but wanted to elucidate the role of cSL1 and cSL2 in the template activity, we constructed templates carrying only cSL1 and cSL2 (SL2) to exclude early termination (Fig. 5 B). In this experiment, the activity of SL12–1S was more than twice that of SL12 but that of SL12–1LD was similar to the activity of SL2. Because theT m of the UA stem in Fig. 1 D was 12 °C, a stem structure might not form in the reaction at 29 °C. The stem structure of cSL1 could inhibit the activity. The sequence of the cSL1 loop did not affect the activity. Because the results from the templates carrying mutant sequences of cSL1 were not conclusive, we made additional templates carrying simple structures. When cSL1 was added to SL0 (SL0+SL1), the amount of product decreased, confirming that cSL1 was a negative element (Fig. 5 C). cSL1 was always identified as shown in Fig. 1 when the secondary structure of the templates was predicted by mFold (data not shown). When cSL1 exists, the length of the 3′ single-stranded RNA is only four nucleotides, too short to initiate the reaction because at least a five-nucleotide single-stranded RNA is required for initiation (31Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K.B. Cameron C.E. Lau J.Y.N. Hong Z. J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar, 40Ago H. Adachi T. Yoshida A. Yamamoto M. Habuka N. Yatsunami K. Miyano M. Structure. 2000; 7: 1417-1426Abstract Full Text Full Text PDF Scopus (392) Google Scholar, 41Bressanelli S. Tomei L. Roussel A. Vitale R.L. Mathieu M. De Francesco R. Rey F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 96: 13034-13039Crossref Scopus (547) Google Scholar, 42Butcher S.J. Grimes J.M. Makeyev E.V. Bamford D.H. Stuart D.I. Nature. 2001; 410: 235-240Crossref PubMed Scopus (438) Google Scholar, 43Lesburg C.A. Cable M.B. Ferrari E. Hong Z. Mannarino A.F. Weber P.C. Nat. Struct. Biol. 1999; 6: 937-943Crossref PubMed Scopus (703) Google Scholar). We demonstrated that HCV RdRp bound to the 3′ terminus of the complementary strand RNA as well as that of the positive strand RNA (14Cheng J.-C. Chang M.-F. Chang S.C. J. Virol. 1999; 73: 7044-7049Crossref PubMed Google Scholar). Gel shift assay indicates that cSL2 is also important for the binding of HCV RdRp especially with 0.5 mm GTP (Fig.4 A). Without 0.5 mm GTP, the template-RdRp interaction was enhanced, and SL34 also bound to RdRp effectively. The RdRp binding activity of cSL2 was similar to that of the poly(U/C) tract in the 3′ terminus of HCV genome RNA (Fig. 4 B). RdRp could also bind to other stems or sequences although the binding was weaker than that of cIRES, SL234–1D, and SL234. Considering the results of transcription and RdRp binding, cSL2 would be a positive element of the promoter/origin structure because it binds specifically to RdRp with 0.5 mm GTP. 0.5 mm GTP may give specificity to the binding of RdRp to cSL2. Although we did not show the results, copy-back products became apparent without 0.5 mm GTP preincubation even in single-round transcription. The mechanism of switching from de novo initiation with 0.5 mm GTP preincubation to copy-back initiation remains to be resolved. Fig. 6 shows the proposed scheme of initiation from the 3′ terminus of the complementary strand of IRES. HCV RdRp binds to cSL2. HCV RdRp can start with single-stranded 3′ termini (17Oh J.W. Sheu G.T. Lai M.M. J. Biol. Chem. 2000; 275: 17710-17717Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 31Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K.B. Cameron C.E. Lau J.Y.N. Hong Z. J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar). However, the 3′ single-stranded sequences of cIRES are only four nucleotides long and are too short to reach the active site of RdRp because of the β-hairpin structure (30Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y.N. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 31Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K.B. Cameron C.E. Lau J.Y.N. Hong Z. J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar, 42Butcher S.J. Grimes J.M. Makeyev E.V. Bamford D.H. Stuart D.I. Nature. 2001; 410: 235-240Crossref PubMed Scopus (438) Google Scholar), so they cannot initiate transcription efficiently. NS3 helicase binds to cSL1 (44Banerjee R. Dasgupta A. J. Virol. 2001; 75: 1708-1721Crossref PubMed Scopus (70) Google Scholar) and may relax the stem-loop structure to bring the 3′ terminus to the RdRp active site so that RdRp interacts with GTP. NS3 interacts with RdRp (45Ishido S. Fujita T. Hotta H. Biochem. Biophys. Res. Commun. 1998; 244: 35-40Crossref PubMed Scopus (123) Google Scholar). In both cases, NS3 and HCV RdRp are expected to form a transcription and replication initiation complex. Recently, HS5A was found to bind RdRp and modulate its activity (46Shirota Y. Luo H. Quin W. Kaneko S. Yamashita T. Kobayashi K. Murakami S. J. Biol. Chem. 2002; 277: 2132-2137Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). We have demonstrated that cSL2 played an important role in the initiation of transcription and replication of the HCV genome by interacting with RdRp. High concentrations of GTP may give specificity to the interaction between RdRp and template. The HCV initiation complex needs to be reconstitutedin vitro using purified RdRp and NS3." @default.
• W2022198259 created "2016-06-24" @default.
• W2022198259 creator A5022430910 @default.
• W2022198259 creator A5031399834 @default.
• W2022198259 creator A5036481822 @default.
• W2022198259 creator A5056055237 @default.
• W2022198259 creator A5062643792 @default.
• W2022198259 creator A5078428570 @default.
• W2022198259 creator A5087740365 @default.
• W2022198259 date "2002-08-01" @default.
• W2022198259 modified "2023-10-14" @default.
• W2022198259 title "Promoter/Origin Structure of the Complementary Strand of Hepatitis C Virus Genome" @default.
• W2022198259 cites W1520082092 @default.
• W2022198259 cites W1531200550 @default.
• W2022198259 cites W1542390893 @default.
• W2022198259 cites W1576685899 @default.
• W2022198259 cites W1600974081 @default.
• W2022198259 cites W1823300406 @default.
• W2022198259 cites W1914838920 @default.
• W2022198259 cites W1928472258 @default.
• W2022198259 cites W1932632320 @default.
• W2022198259 cites W1965505230 @default.
• W2022198259 cites W1965962980 @default.
• W2022198259 cites W1972459659 @default.
• W2022198259 cites W1988237823 @default.
• W2022198259 cites W1991378085 @default.
• W2022198259 cites W1992048113 @default.
• W2022198259 cites W1995000176 @default.
• W2022198259 cites W2008701068 @default.
• W2022198259 cites W2009818318 @default.
• W2022198259 cites W2019579401 @default.
• W2022198259 cites W2021451720 @default.
• W2022198259 cites W2037829048 @default.
• W2022198259 cites W2045378360 @default.
• W2022198259 cites W2049146226 @default.
• W2022198259 cites W2051767229 @default.
• W2022198259 cites W2054700330 @default.
• W2022198259 cites W2054896136 @default.
• W2022198259 cites W2066931983 @default.
• W2022198259 cites W2089154590 @default.
• W2022198259 cites W2090326661 @default.
• W2022198259 cites W2098368373 @default.
• W2022198259 cites W2098897538 @default.
• W2022198259 cites W2106870343 @default.
• W2022198259 cites W2132114909 @default.
• W2022198259 cites W2139624902 @default.
• W2022198259 cites W2140518449 @default.
• W2022198259 cites W2144881432 @default.
• W2022198259 cites W2157280530 @default.
• W2022198259 cites W2157549311 @default.
• W2022198259 cites W2161945006 @default.
• W2022198259 cites W2167360353 @default.
• W2022198259 cites W2199569371 @default.
• W2022198259 doi "https://doi.org/10.1074/jbc.m201251200" @default.
• W2022198259 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12039953" @default.
• W2022198259 hasPublicationYear "2002" @default.
• W2022198259 type Work @default.
• W2022198259 sameAs 2022198259 @default.
• W2022198259 citedByCount "17" @default.
• W2022198259 countsByYear W20221982592012 @default.
• W2022198259 crossrefType "journal-article" @default.
• W2022198259 hasAuthorship W2022198259A5022430910 @default.
• W2022198259 hasAuthorship W2022198259A5031399834 @default.
• W2022198259 hasAuthorship W2022198259A5036481822 @default.
• W2022198259 hasAuthorship W2022198259A5056055237 @default.
• W2022198259 hasAuthorship W2022198259A5062643792 @default.
• W2022198259 hasAuthorship W2022198259A5078428570 @default.
• W2022198259 hasAuthorship W2022198259A5087740365 @default.
• W2022198259 hasBestOaLocation W20221982591 @default.
• W2022198259 hasConcept C104317684 @default.
• W2022198259 hasConcept C141231307 @default.
• W2022198259 hasConcept C159047783 @default.
• W2022198259 hasConcept C185592680 @default.
• W2022198259 hasConcept C2522874641 @default.
• W2022198259 hasConcept C54355233 @default.
• W2022198259 hasConcept C70721500 @default.
• W2022198259 hasConcept C86803240 @default.
• W2022198259 hasConceptScore W2022198259C104317684 @default.
• W2022198259 hasConceptScore W2022198259C141231307 @default.
• W2022198259 hasConceptScore W2022198259C159047783 @default.
• W2022198259 hasConceptScore W2022198259C185592680 @default.
• W2022198259 hasConceptScore W2022198259C2522874641 @default.
• W2022198259 hasConceptScore W2022198259C54355233 @default.
• W2022198259 hasConceptScore W2022198259C70721500 @default.
• W2022198259 hasConceptScore W2022198259C86803240 @default.
• W2022198259 hasIssue "32" @default.
• W2022198259 hasLocation W20221982591 @default.
• W2022198259 hasOpenAccess W2022198259 @default.
• W2022198259 hasPrimaryLocation W20221982591 @default.
• W2022198259 hasRelatedWork W1874513932 @default.
• W2022198259 hasRelatedWork W2016551366 @default.
• W2022198259 hasRelatedWork W2086525401 @default.
• W2022198259 hasRelatedWork W2094230406 @default.
• W2022198259 hasRelatedWork W2111970140 @default.
• W2022198259 hasRelatedWork W2123163123 @default.
• W2022198259 hasRelatedWork W2138564085 @default.
• W2022198259 hasRelatedWork W2147145791 @default.
• W2022198259 hasRelatedWork W2188759708 @default.

• next