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• W2045740959 abstract "RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis. RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis. West Nile virus (WNV), 1The abbreviations used are: WNV, West Nile virus; TR, terminal region; UTR, untranslated region; CYC, cyclization; nt, nucleotide; RdRP, RNA-dependent RNA polymerase; mutCYC, mutant cyclization motif; PIPES, 1,4-piperazinediethanesulfonic acid. a mosquito-borne member of Flaviviridae family that consists of more than 70 human pathogens, was first isolated in 1937 from the blood of a female patient in the West Nile district of Uganda (1Smithburn K.C. Hugh T.P. Burke A.W. Paul J.H. Am. J. Trop. Med. Hyg. 1940; 20: 471-492Crossref Google Scholar). Since then, the virus has been isolated in humans, birds, and bird-feeding mosquitoes, Culex univittatus. Serological studies revealed a broad distribution of the virus in Africa, West Asia including India, and the Middle East, but only occasionally isolated in Europe, and is thought to be spread by migratory birds (Ref. 2Rappole J.H. Derrickson S.R. Hubalek Z. Emerg. Infect. Dis. 2000; 6: 319-328Crossref PubMed Scopus (468) Google Scholar, for reviews, see Refs. 3Meek J. Curr. Opin. Pediatr. 2002; 14: 72-77Crossref PubMed Scopus (21) Google Scholar and 4Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar). WNV belongs to the Japanese encephalitis serocomplex group, which includes St. Louis encephalitis, Murray Valley encephalitis, Kunjin virus, and Japanese encephalitis virus (5Westaway E.G. J. Immunol. 1968; 100: 569-580PubMed Google Scholar, 6Calisher C.H. Karabatsos N. Dalrymple J.M. Shope R.E. Porterfield J.S. Westaway E.G. Brandt W.E. J. Gen. Virol. 1989; 70: 37-43Crossref PubMed Scopus (700) Google Scholar). WNV was not previously isolated in the Western hemisphere. The first outbreak of WNV infections occurred in New York in 1999 (7Asnis D.S. Conetta R. Teixeira A.A. Waldman G. Sampson B.A. Clin. Infect. Dis. 2000; 30: 413-418Crossref PubMed Scopus (289) Google Scholar, 8Nash D. Mostashari F. Fine A. Miller J. O'Leary D. Murray K. Huang A. Rosenberg A. Greenberg A. Sherman M. Wong S. Layton M. N. Engl. J. Med. 2001; 344: 1807-1814Crossref PubMed Scopus (1072) Google Scholar, 9Campbell G.L. Marfin A.A. Lanciotti R.S. Gubler D.J. Lancet Infect. Dis. 2002; 2: 519-529Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar, 10Roehrig J.T. Layton M. Smith P. Campbell G.L. Nasci R. Lanciotti R.S. Curr. Top. Microbiol. Immunol. 2002; 267: 223-240PubMed Google Scholar) in which 62 people were confirmed to be infected, seven of which were fatal. Since then, transmission of WNV is spreading rapidly throughout the United States; 12 states along the eastern seaboard in 2000 to 25 states and the District of Columbia by 2001 (3Meek J. Curr. Opin. Pediatr. 2002; 14: 72-77Crossref PubMed Scopus (21) Google Scholar). WNV, like other flaviviruses, contain a positive (+)-strand RNA of ∼11 kb in length that is capped at the 5′-end and nonpolyadenylated at the 3′-end. WNV RNA contains conserved sequence elements within the 5′- and 3′-terminal regions (TR), which include two self-complementary cyclization motifs (5′-CYC and 3′-CYC) of 9 nt in length (11Hahn C.S. Hahn Y.S. Rice C.M. Lee E. Dalgarno L. Strauss E.G. Strauss J.H. J. Mol. Biol. 1987; 198: 33-41Crossref PubMed Scopus (295) Google Scholar). There is a highly conserved stem-loop structure within the 3′-terminal 96 nt of 3′-UTR of flaviviral RNAs and a less conserved stem-loop structure within 5′-UTR (12Brinton M.A. Fernandez A.V. Dispoto J.H. Virology. 1986; 153: 113-121Crossref PubMed Scopus (203) Google Scholar, 13Brinton M.A. Dispoto J.H. Virology. 1988; 162: 290-299Crossref PubMed Scopus (164) Google Scholar, 14Mohan P.M. Padmanabhan R. Gene (Amst.). 1991; 108: 185-191Crossref PubMed Scopus (40) Google Scholar) (for reviews, see Refs. 4Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar, 15Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1612) Google Scholar, and 16Rice C.M. Fields N.B. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven, Philadelphia1996: 931-959Google Scholar). In addition, there is a potential pseudoknot tertiary structure within the 3′-terminal stem-loop structure of WNV RNA (17Shi P.Y. Brinton M.A. Veal J.M. Zhong Y.Y. Wilson W.D. Biochemistry. 1996; 35: 4222-4230Crossref PubMed Scopus (115) Google Scholar). Several host proteins have been shown to bind to the 5′- and 3′-UTR although their functional role in viral RNA replication has not been clearly established (18Blackwell J.L. Brinton M.A. J. Virol. 1995; 69: 5650-5658Crossref PubMed Google Scholar, 19Shi P.Y. Li W. Brinton M.A. J. Virol. 1996; 70: 6278-6287Crossref PubMed Google Scholar, 20De Nova-Ocampo M. Villegas-Sepulveda N. del Angel R.M. Virology. 2002; 295: 337-347Crossref PubMed Scopus (157) Google Scholar, 21Yocupicio-Monroy R.M. Medina F. Reyes-del Valle J. del Angel R.M. J. Virol. 2003; 77: 3067-3076Crossref PubMed Scopus (49) Google Scholar) (for a review, see Ref. 22Brinton M.A. Ann. N. Y. Acad. Sci. 2001; 951: 207-219Crossref PubMed Scopus (74) Google Scholar). By mutational analysis, the 3′ stem-loop region within the 3′-UTR was shown to be important for viral replication in vivo (23Men R. Bray M. Clark D. Chanock R.M. Lai C.J. J. Virol. 1996; 70: 3930-3937Crossref PubMed Google Scholar, 24Zeng L. Falgout B. Markoff L. J. Virol. 1998; 72: 7510-7522Crossref PubMed Google Scholar) and in vitro (25You S. Falgout B. Markoff L. Padmanabhan R. J. Biol. Chem. 2001; 276: 15581-15591Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The flaviviral RNA genome encodes a single polyprotein precursor that were processed co-translationally and post-translationally into three structural proteins (capsid, C; precursor membrane, prM; its processed form, membrane protein, M; and the envelope, E) and at least seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (for reviews, see Refs. 4Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar, 15Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1612) Google Scholar, and 16Rice C.M. Fields N.B. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven, Philadelphia1996: 931-959Google Scholar). NS3 is a multifunctional protein; the N-terminal region of NS3 contains the serine protease domain and it requires NS2B for cleavage at the junctions of 2A–2B, 2B–3, 3–4A, and 4B–5. NS3 contains conserved motifs found in RNA helicases of the DEXH family (for reviews, see Refs. 4Brinton M.A. Annu. Rev. Microbiol. 2002; 56: 371-402Crossref PubMed Scopus (305) Google Scholar, 15Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1612) Google Scholar, and 16Rice C.M. Fields N.B. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippincott-Raven, Philadelphia1996: 931-959Google Scholar). Purified NS3 was shown to possess NTPase and RNA helicase activities (26Borowski P. Niebuhr A. Mueller O. Bretner M. Felczak K. Kulikowski T. Schmitz H. J. Virol. 2001; 75: 3220-3229Crossref PubMed Scopus (74) Google Scholar, 27Cui T. Sugrue R.J. Xu Q. Lee A.K. Chan Y.C. Fu J. Virology. 1998; 246: 409-417Crossref PubMed Scopus (94) Google Scholar, 28Kim D.W. Gwack Y. Han J.H. Choe J. Virus Res. 1997; 49: 17-25Crossref PubMed Scopus (32) Google Scholar, 29Kuo M.D. Chin C. Hsu S.L. Shiao J.Y. Wang T.M. Lin J.H. J. Gen. Virol. 1996; 77: 2077-2084Crossref PubMed Scopus (51) Google Scholar, 30Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar, 31Warrener P. Tamura J.K. Collett M.S. J. Virol. 1993; 67: 989-996Crossref PubMed Google Scholar, 32Wengler G. Wengler G. Virology. 1991; 184: 707-715Crossref PubMed Scopus (162) Google Scholar) as well as 5′-RNA triphosphatase activity (33Wengler G. Wengler G. Virology. 1993; 197: 265-273Crossref PubMed Scopus (175) Google Scholar, 34Bartelma G. Padmanabhan R. Virology. 2002; 299: 122-132Crossref PubMed Scopus (118) Google Scholar), the first enzyme required in 5′-cap synthesis. NS5 is the largest of the flaviviral nonstructural proteins. Flaviviral NS5 contains conserved motifs found in several 5′-RNA methyltransferases at its N-terminal region (35Koonin E.V. J. Gen. Virol. 1993; 74: 733-740Crossref PubMed Scopus (203) Google Scholar, 36Rozanov M.N. Koonin E.V. Gorbalenya A.E. J. Gen. Virol. 1992; 73: 2129-2134Crossref PubMed Scopus (329) Google Scholar). The crystal structure of the N-terminal region consisting of 296 amino acid residues of DEN2 NS5 containing the guanylyltransferase/methyltransferase domain was recently reported (37Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (489) Google Scholar). This domain can catalyze the transfer of methyl group from the S-adenosylmethionine to form the 2′-O-methyladenine but no 7-methylguanosine was formed. The C-terminal region of the flaviviral NS5 contains conserved motifs consistent with those of RNA-dependent RNA polymerases (RdRP) encoded by several positive-strand RNA viruses (38Poch O. Sauvaget I. Delarue M. Tordo N. EMBO J. 1989; 8: 3867-3874Crossref PubMed Scopus (981) Google Scholar, 39O'Reilly E.K. Kao C.C. Virology. 1998; 252: 287-303Crossref PubMed Scopus (264) Google Scholar). In this study, we expressed the WNV NS5 (from the EG101 strain) with an N-terminal His tag in Escherichia coli and purified the protein using metal affinity chromatography and characterized the biochemical properties of the polymerase in RNA synthesis. An in vitro viral RdRP assay system for DEN2 was reported that was dependent on addition of exogenous subgenomic viral RNA (+)-strand templates containing conserved sequence motifs within the 5′- and 3′-TR and purified polymerase in the presence of four nucleoside triphosphatase (40Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Two RNA products were formed, a template-sized product and a hairpin product, twice the size of the template RNA. The template-sized RNA (1×) was shown to be double-stranded in nature by its resistance to RNase A digestion under high ionic strength conditions, and it was formed as a result of de novo synthesis from the input (+)-strand RNA (40Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). The hairpin RNA (2×) was formed by 3′-end elongation of the template RNA by a “copy-back” mechanism. RNase A digestion of the hairpin RNA also gave rise to a template-sized product upon digestion of the single-stranded “fold-back” region. In this study, we describe the first in vitro RdRP assay for WNV that utilizes purified NS5 and exogenous subgenomic WNV RNA templates of either (+)- or (–)-strand polarity; the 5′-end of template RNAs was either triphosphorylated or had a 5′-cap structure. We show that whereas the uncapped (+)- strand RNA template produced both hairpin RNA and the template-sized de novo synthesized RNA, addition of 5′-cap on the subgenomic (+)-strand RNA template reduced the formation of hairpin RNA by 48% without affecting the synthesis of the de novo product RNA. The newly synthesized RNA from the (+)-strand template RNA was of (–)-strand polarity as determined by RNase H mapping. Moreover, we show that the WNV NS5 could also utilize uncapped or 5′-capped subgenomic WNV (–)-strand templates to produce predominantly de novo product that are of (+)-strand polarity. Finally, mutational analysis of the 5′- and 3′-CYC motifs in both subgenomic WNV (+)- and (–)-strand templates showed that these motifs play a critical role in (–)-strand synthesis from (+)-strand RNA templates but do not seem to affect the (+)-strand synthesis from (–)- strand templates. The sequences corresponding to the WNV NS5 were PCR amplified from the plasmid containing the full-length WNV EG101 strain genome (NCBI number AF260968) using the following primers: the forward primer, 5′-TCCCCCGGGTGGGCAAAGGGACGCACCTTG-3′ and the reverse primer, 5′-TCCCCCGGGTTACAGTACTGTGTCCTCAACC-3′. The product from this PCR was cloned into the pGEM-T easy vector (Promega) and the sequence was verified. The positive clone was digested with XmaI (underlined) and the fragment was cloned into pQE30 vector (Qiagen), giving rise to pQE30-WNV NS5. For the bacterial expression of WNV NS5, E. coli Top10 F′ cells (Invitrogen) were transformed with pQE30-WNV NS5 plasmid. Transformed E. coli cells were grown at 37 °C in LB medium containing 100 μg/ml ampicillin and 0.5% (w/v) glucose to an A600 nm of 0.6–0.8. Cells were collected by centrifugation (5000 × g) to remove the glucose from the medium, the pellet was resuspended in LB medium containing 100 μg/ml ampicillin and 1 mm isopropyl-1-thio-β-d-galactopyranoside (Sigma), and incubated for 48 h at 18 °C. Bacteria were then collected by centrifugation and stored at –80 °C. For purification of WNV NS5, bacteria were resuspended in a buffer containing 100 mm Tris-HCl, pH 7.5, 300 mm NaCl, 1% Triton X-100, 10 mm 2-mercaptoethanol, and 20% glycerol and were lysed by using a French press. The soluble fraction was loaded onto a Talon column (Clontech). WNV NS5 proteins were eluted with 500 mm imidazole. The peak fractions containing the WNV NS5 protein were pooled and dialyzed against 50 mm Tris-HCl, pH 7.5, 300 mm NaCl, and 40% glycerol. The purified protein was stored at –20 °C. cDNA Constructs Encoding Subgenomic RNA Containing the 5′- and 3′-TR of WNV Genome—A 281-nt DNA fragment from the 5′-TR that includes the 5′-untranslated region (UTR) (96 nt) and the 5′ cyclization motif (CYC, which corresponds to 137–144 nt of the viral genome) was generated by PCR using WNV EG101 plasmid as a template and the forward primer, 5′-TAATACGACTCACTATAGAGTAGTTCGCCTGTGTGAGCTGAC-3′ (T7 promoter (underlined) was fused to 1–24 nt of the viral genome) and the reverse primer, 5′-CACTGCTCGGGTCGGAGCAAT-3′ (complementary to 271–291 nt of the viral genome containing the authentic AvaI site (underlined)). An amplified PCR product, WNV5′TR281nt, was cloned into pGEM-T easy vector (pGEM-T easy-WNV5′TR). The 3′-TR (770 nt) that includes the 3′-UTR (631 nt) was amplified using WNV EG101 plasmid as a template and the forward primer, 5′-CACAAGAACCCGAGCCACG-3′ (corresponding to 10251–10269 nt of the viral genome containing the authentic AvaI site (underlined)) and the reverse primer, 5′-GCTCTAGAAGATCCTGTGTTCTCGCACCA-3′ (complementary to 11009–11029 nt fused with the XbaI site (underlined)). The plasmid DNA, pGEM-T easy-WNV3′TR, was obtained by cloning of 3′TR770nt into the pGEM-T easy vector. The sequences of the 5′- and 3′-TR cDNAs in the pGEM-T easy plasmids were verified. The 5′- and 3′-TR encoding fragments were obtained by digestion of the pGEM-T easy plasmids with AvaI and EcoRI (in the vector) and AvaI and XbaI, respectively, and cloned into EcoRI- and XbaI-digested pSP64 vector (Promega) to yield the plasmid, pSP64-WNV1051nt encoding the WNV subgenomic RNA (Fig. 2). Plasmids Containing Mutant Cyclization Motif (mutCYC)—pSP64-WNV1051nt containing mutCYC motif was obtained using the site-directed mutagenesis kit (Stratagene). The primers used in the mutagenesis reaction were the following. For the 5′ mutCYC, the 5′-CCGGCAAGAGCCGGGCTGCCTGCAGGCTAAAACGCGGAATGCCC-3′ and its complement as primers, and for the 3′ mutCYC, the 5′-CACCACAACAAAACAGCCTGCAGGCACCTGGGATAGACTAGGAG-3′ and its complement as primers were used for PCR. The underlined sequence indicates the replacement of the wild type CYC sequence (wtCYC), TCAATATG, with the mutCYC sequence, CCTGCAGG. The plasmid containing both 5′- and 3′-mutCYC motifs was obtained by PCR-based site-directed mutagenesis using primers containing 3′ mutCYC sequences and the plasmid containing the 5′ mut-CYC motif as the template (see above). PCR Products Encoding Subgenomic WNV (+)-Strand RNA1051nt (wt and mutCYC)—To generate the subgenomic WNV (+)RNA1051nt containing wild type as well as mutCYC motifs, PCR was performed using the appropriate plasmid as the template (Fig. 2) and two primers for PCR as follows: the forward primer, 5′-AGCTATGACCATGATTACGAATTC-3′ (pSP64-EcoRI primer) that corresponds to the sequences upstream of the T7 promoter and the reverse primer, 5′-AGATCCTGTGTTCTCGCACCAC-3′ (WNV-3′ end primer) that anneals with the 3′ end region of the WNV 3′-UTR. PCR Products Encoding Subgenomic Wild Type and CYC Mutant WNV (–)-Strand RNA1051nt—The first PCR was carried out with the same plasmid construct as above as a template and two primers: the forward primer, 5′-TAATACGACTCACTATAGAGATCCTGTGTTCTCGCACCAC-3′ that contains T7 promoter (underlined) fused with the 5′-end of WNV (–)-strand, and the reverse primer, 5′-AGTAGTTCGCCTGTGTGAGCTGAC-3′ (WNV-5′UTR (+)-strand primer). A second PCR was carried out with the forward primer containing the T7 promoter, 5′-TTACGAATTCGAGCTCGCCCTAATACGACTCACTATAG-3′ and the same reverse primer consisting of the WNV-5′UTR (+)-strand sequence used for the first PCR. PCR Product Encoding the WNV (+)-Strand 5′TR230nt RNA—PCR was performed with the pSP64-WNV1051nt as the template and two primers: pSP64 (EcoRI) primer as the forward primer and the reverse primer, 5′-CGTATTGGTCCCTTGCCGTCGATC-3′ that corresponds to the sequence complementary to 207–230 nt of the WNV genome. PCR Product Encoding the WNV (+)-Strand 3′-UTR631nt RNA—To generate the WNV(+) 3′-UTR631nt RNA, the first PCR was carried out with pSP64-WNV1051nt as the template and two primers: the forward primer, 5′-TAATACGACTCACTATAGATACTTTATTAATTGTAAATAGAC-3′ that contains the T7 promoter (underlined) fused with the 5′ terminal sequence of the 3′-UTR, and the sequence complementary to the WNV-3′ end as the reverse primer. The second PCR was performed using the same forward primer containing the T7 promoter used in the second PCR described above and the reverse primer consisting of the sequence complementary to the 3′-end of WNV (–)-strand was the same as that used in the first PCR. To generate WNV (–)-strand 3′-TR230nt RNA, the first PCR was carried out with pSP64-WNV1051nt as the template and the two following primers: the forward primer, 5′-TAATACGACTCACTATAGCGTATTGGTCCCTTGCCGTCGATC-3′ that contains the T7 promoter (underlined) fused with the sequence complementary to 207–230 nt of the viral genome, and the reverse primer sequence from the beginning of the WNV (+)-strand 5′-UTR. The second PCR was performed as described the above. All PCR were performed with Vent polymerase (New England Biolabs) using the appropriate template and primers. The PCR products were purified using the QIAquick gel extraction kit (Qiagen), and was used in the in vitro transcription reaction. In vitro transcription was performed with Ampliscribe™ T7 Transcription kit (Epicenter Technologies) as described by the manufacturer using the T7 promoter containing PCR products prepared as described above or using linearized plasmid DNAs (pTM1 vector plasmid digested with BamHI or pSP64 digested with AflIII). In the case of PCR product or linearized plasmid containing SP6 promoter, the Ampliscribe™ SP6 Transcription kit (Epicenter Technologies) was used as described by the manufacturer. 5′-Capped RNAs were prepared using AmpliCap™ T7 High Yield Message Maker kit (Epicenter Technologies) as described by the manufacturer. The RNA transcripts were purified as described previously (41You S. Padmanabhan R. J. Biol. Chem. 1999; 274: 33714-33722Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and then used in the RdRP assays. RNA templates with blocked 3′-OH groups were prepared using 3′-dATP (TriLink BioTechnologies) and poly(A) polymerase (Ambion) as described by Luo et al. (42Luo 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). The in vitro RdRP assays were carried out as described previously (40Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) except when indicated. Briefly, the reaction mixture (50 μm) contained 50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 5 mm MgCl2, template RNA (0.3 μg), 500 μm each of ATP, GTP, and UTP, 10 μm unlabeled CTP, and 10 μCi of [α-32P]CTP along with 270 ng of purified WNV NS5 (2.6 pmol). The incubation was carried out at 25 °C, which favored de novo synthesis of RNA over 3′-end elongation (40Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). When the specificity of the RdRP was analyzed using various nonspecific RNA templates, the incubation temperature of the RdRP reaction was 30 °C at which the total RNA synthesis was higher than at 25 °C. Following extraction with acid phenol/chloroform, the reaction mixture was passed through a P30 gel filtration column (Bio-Rad) to remove the unincorporated nucleotides. RNA was then analyzed by formaldehydeagarose gel electrophoresis and visualized by autoradiography. When indicated, the labeled bands were excised from dried gels and quantified by liquid scintillation counting and density analysis using the imagej program, a free software product available at the National Institutes of Health website. 2rsb.info.nih.gov/ij. The amount of template RNA, NS5, and incubation time for the RdRP assays were chosen from the linear range of RNA synthesis. The hairpin and the de novo products produced in the RdRP assays were either analyzed directly by formaldehyde-agarose gel electrophoresis or (in the case of wild type and mutCYC (+)-strand RNA templates in the RdRP assays) were first analyzed by native polyacrylamide gel electrophoresis (PAGE; 5%) followed by autoradiography. The bands containing the RNA products were cut out, eluted from the gel (elution buffer: 0.5 m ammonium acetate, 1 mm EDTA and 0.1% SDS), and precipitated with ethanol. The RNA products were further analyzed by formaldehyde-agarose gel, followed by autoradiography as described previously (41You S. Padmanabhan R. J. Biol. Chem. 1999; 274: 33714-33722Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). To analyze the RdRP products, RNase A digestion was performed as described previously (41You S. Padmanabhan R. J. Biol. Chem. 1999; 274: 33714-33722Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Briefly, RdRP products were distributed equally and treated with or without RNase A (Sigma; 200 ng) in 20 μl of 2× SSC at 30 °C for 30 min. The reactions were stopped by adding 30 μl of TES stop buffer (10 mm Tris-HCl, pH 8.0, 50 mm EDTA, and 0.2% SDS), followed by acid phenol/chloroform extraction and ethanol precipitation in the presence of 10 μg of yeast tRNA (Ambion). The RNase A-treated products were analyzed on formaldehyde-agarose gel followed by autoradiography. RNase H mapping was carried out essentially as reported previously (42Luo 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, 43Behrens S.E. Tomei L. De Francesco R. EMBO J. 1996; 15: 12-22Crossref PubMed Scopus (648) Google Scholar). Following the RdRP assay using either WNV (+)RNA1051nt or WNV (–)RNA1051nt template, RNase A treatment was carried out only for RdRP products synthesized from the WNV (+)RNA1051nt template to digest the single-stranded loop region of the 2× hairpin product. After RNase A treatment, the reaction mixture was digested with proteinase K (Sigma; 40 μg) followed by extraction with acid phenol/chloroform and precipitation with ethanol. The RdRP products were then mixed with 1.5 μg of either WNV (+)RNA1051nt or WNV (–)RNA1051nt that was complementary to the template used in the RdRP reaction, denatured at 95 °C for 5 min, and reannealed in a hybridization buffer (80% formamide, 40 mm PIPES, pH 6.8, 1 mm EDTA, 400 mm NaCl) at 42 °C overnight. RNA samples were then precipitated with ethanol and resuspended with 20 μl of RNase H mapping buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 10 units of RNasin (Promega), 4 units of RNase H (Thermostable; Epicenter Technologies), 200 μm oligodeoxynucleotide of either (+)- or (–)-strand polarity). The reaction mixture was incubated at 45 °C for 1 h. The reaction was terminated by addition of 2× stop buffer (100 mm Tris-HCl, pH 8.0, 300 mm NaCl, 0.5% SDS). RNA was extracted with acid phenol/chloroform, then precipitated with ethanol, and analyzed by formaldehyde-agarose gel. Purification of Full-length WNV NS5—We found that E. coli TOP10F′ cells transformed by WNV NS5 yielded higher amounts of NS5 protein in the soluble form compared with E. coli XL1-BLUE, which was used for DEN2 NS5. Moreover, addition of glucose (0.5%) to the LB growth medium during the initial phase of E. coli growth was necessary as observed for E. coli XL1-BLUE transformed by DEN2 NS5 plasmid presumably because of toxicity of the leaky expression of the NS5 protein from the lac promoter (40Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) (data not shown). After the growth of E. coli cells reached A600 nm of 0.6–0.8, glucose was removed and expression of WNV NS5 was induced by the addition of isopropyl-β-d-thiogalactopyranoside and the bacterial culture was incubated as described under “Experimental Procedures.” The NS5 protein, subsequent to elution from the metal affinity (Talon™ resin) column and dialysis, was analyzed by SDS-PAGE (8%) and Coomassie Blue staining (Fig. 1). The NS5 protein purified in this manner had some minor contaminants and its purity was estimated to be 80% by density analysis. The purified WNV NS5 protein was free of any RNase contamination as judged from the results of incubation with the subgenomic WNV (+)RNA1051nt template at 37 °C for 2 h followed by analysis by electrophoresis on a 4% acrylamide, 8 m urea PAGE gel and staining with ethidium bromide. No contamination of any RNase was detected (data not shown). Therefore, this WNV NS5 protein was used for subsequent experiments (described below). RdRP Assay Using WNV Subgenomic RNA Templates— First, we determined whether the purified WNV NS5 was enzymatically active in RNA synthesis on the WNV (+)RNA1051nt as the template used in the RdRP assay. This template RNA contains the authentic regulatory elements within 5′- and 3′-TRs including the two self-complementary 9-nucleotide long CYC motifs located within the 5′- and 3′-conserved sequence elements reported previously (11Hahn C.S. Hahn Y.S. Rice C.M. Lee E. Dalgarno L. Strauss E.G. Strauss J.H. J. Mol. Biol. 1987; 198: 33-41Crossref PubMed Scopus (295) Google Scholar) (see Fig. 2B). RdRP assay was carried out in a reaction mixture containing the four NTPs in which the α-32P-labeled CTP was included along with the template RNA and the purified NS5 polymerase. The RNA products synthesized in the reaction were analyzed by a formaldehyde-agarose gel followed by autoradiography. In this in vitro a" @default.
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• W2045740959 date "2004-03-01" @default.
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• W2045740959 title "Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli" @default.
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• W2045740959 doi "https://doi.org/10.1074/jbc.m310839200" @default.
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