A Novel in Vitro Replication System for Dengue Virus
1999; Elsevier BV; Volume: 274; Issue: 47 Linguagem: Inglês
10.1074/jbc.274.47.33714
ISSN1083-351X
Autores Tópico(s)Plant Virus Research Studies
ResumoPositive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5′- and 3′-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5′- and 3′-TR present in the same template molecule or when the 5′-TR RNA was added in trans to the 3′-untranslated region (UTR) RNA. However, the 3′-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3′-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5′- and 3′-TR of the viral RNA that modulates the 3′-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction. Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5′- and 3′-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5′- and 3′-TR present in the same template molecule or when the 5′-TR RNA was added in trans to the 3′-untranslated region (UTR) RNA. However, the 3′-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3′-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5′- and 3′-TR of the viral RNA that modulates the 3′-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction. RNA-dependent RNA polymerase(s) untranslated region nucleotide(s) terminal region(s) polyacrylamide gel electrophoresis polymerase chain reaction conserved sequence stem-loop wild type The mosquito-borne dengue viruses, members of positive strand RNA viruses of Flavivirus family, are human pathogens that cause dengue fever, dengue hemorrhagic fever/dengue shock syndrome (for a review see Refs. 1Monath T.P. Proc. Natl. Acad. Sci. U. S. 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The nucleoside triphosphatase and RNA helicase activities of DEN2 NS3 have recently been demonstrated by expression and purification of recombinant NS3 in Escherichia coli (23Li H. Clum S. You S. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar). The RNA helicase activity is thought to be involved in unwinding of a double-stranded RNA replicative intermediate formed during replication of the flavivirus genome (8Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1613) Google Scholar, 24Westaway E.G. Adv. Virus Res. 1987; 33: 45-90Crossref PubMed Scopus (125) Google Scholar). NS5, the largest of the flaviviral proteins, contains conserved motifs found in many viral RNA-dependent RNA polymerases (RdRP)1 (25Poch O. Sauvaget I. Delarue M. Tordo N. EMBO J. 1989; 8: 3867-3874Crossref PubMed Scopus (981) Google Scholar, 26O'Reilly E.K. Kao C.C. 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In flavivirus-infected cells, three RNA species have been detected: a genomic RNA of 40–44 S; a double-stranded, RNase-resistant, replicative form of 20–22 S; and a partially RNase-sensitive, replicative intermediate of 20–28 S RNA (37Chu P.W. Westaway E.G. Virology. 1985; 140: 68-79Crossref PubMed Scopus (149) Google Scholar, 38Grun J.B. Brinton M.A. J. Virol. 1986; 60: 1113-1124Crossref PubMed Google Scholar, 39Chu P.W. Westaway E.G. Virology. 1987; 157: 330-337Crossref PubMed Scopus (76) Google Scholar, 40Bartholomeusz A.I. Wright P.J. Arch. Virol. 1993; 128: 111-121Crossref PubMed Scopus (85) Google Scholar). The in vitro RdRP assays that have been developed to study flavivirus replication utilize membrane-bound complexes isolated from the infected cell lysates (37Chu P.W. Westaway E.G. Virology. 1985; 140: 68-79Crossref PubMed Scopus (149) Google Scholar, 38Grun J.B. Brinton M.A. J. Virol. 1986; 60: 1113-1124Crossref PubMed Google Scholar, 39Chu P.W. Westaway E.G. 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To study the mechanism of viral replication in molecular detail, it is crucial to develop an in vitro RdRP assay that can utilize exogenous RNA templates containing essential regulatory elements of the viral genome involved in viral replication. For initiation of (−)- and (+)-strand RNA synthesis, conserved RNA sequences with intrinsic stem-loop structures from the 3′ and 5′ regions of many viral genomes are thought to play an important role in RNA replication (41Esteban R. Fujimura T. Wickner R.B. EMBO J. 1989; 8: 947-954Crossref PubMed Scopus (69) Google Scholar, 42Nakhasi H.L. Cao X.Q. Rouault T.A. Liu T.Y. J. Virol. 1991; 65: 5961-5967Crossref PubMed Google Scholar, 43Pardigon N. Strauss J.H. J. Virol. 1992; 66: 1007-1015Crossref PubMed Google Scholar, 44Pogue G.P. Hall T.C. J. Virol. 1992; 66: 674-684Crossref PubMed Google Scholar, 45Rohll J.B. Percy N. Ley R. Evans D.J. Almond J.W. Barclay W.S. J. Virol. 1994; 68: 4384-4391Crossref PubMed Google Scholar, 46Rohll J.B. Moon D.H. 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In flavivirus genomes, such conserved elements have been noted previously; their functions in replication remain to be established. For example, the 3′-terminal 96 nucleotides of the mosquito-borne flaviviruses within the 3′-UTR form a conserved and stable stem-loop structure, although the primary sequence is not conserved (for a review see Ref.8Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1613) Google Scholar). Formation of such a stable secondary structure within the 3′-terminal 373 nt of the 3′-UTR was shown to exist in solution (56Mohan P.M. Padmanabhan R. Gene (Amst.). 1991; 108: 185-191Crossref PubMed Scopus (40) Google Scholar). A potential secondary structure with a lesser predicted stability near the 5′ termini of several flavivirus RNAs including DEN2 was reported (57Brinton M.A. Dispoto J.H. Virology. 1988; 162: 290-299Crossref PubMed Scopus (164) Google Scholar). There are also two short conserved sequences (CS1 and CS2) in 3′-UTR shared by all mosquito-borne flaviviruses; CS1, 26 nucleotides in length, is located 5′ to the stem-loop structure of the 3′-UTR (3′-CS1). A portion of the 3′-CS1 is complementary to a conserved element is located within the N-terminal coding region of the capsid protein, C, in the 5′-TR of the viral genome (5′-CS). It has been proposed that these complementary sequences might play a role in cyclization of the viral genome (Ref. 58Hahn 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 and for a review see Ref. 8Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1613) Google Scholar). However, cyclization of any of the flavivirus genome has not been observed to date. Thus the role of cyclization motifs in genome replication is unknown at present. In this study, we report the development of the first in vitro RdRP assay that utilizes cell-free extracts of DEN2-infected mosquito (C6/36) cells and exogenous subgenomic RNA templates containing 5′- and/or 3′-terminal regions of the viral genome. The results indicate that there is an interaction between 5′- and 3′-terminal regions of the viral RNA that is required for RNA synthesisin vitro. This interaction is modulated by the complementary cyclization motifs. RNA synthesis occurs by the 3′-end elongation of the template RNA to yield a predominantly double-stranded RNA hairpin with a limited single-stranded loop region. The kinetics of the formation of the RNA hairpin product indicates that the template RNA is first modified yielding a RNase A-sensitive intermediate that is then converted to the hairpin product twice the size of the template RNA. This in vitro RdRP assay will be useful to study the sequence and protein requirements for RNA synthesis in vitro. Dengue virus type 2 (New Guinea Strain C) was propagated as described (59Yaegashi T. Vakharia V.N. Page K. Sasaguri Y. Feighny R. Padmanabhan R. Gene (Amst .). 1986; 46: 257-267Crossref PubMed Scopus (20) Google Scholar, 60Smith T.J. Brandt W.E. Swanson J.L. McCown J.M. Buescher E.L. J. Virol. 1970; 5: 524-532Crossref PubMed Google Scholar). To obtain the DEN2 viral replication complex, C6/36 cells were infected with DEN2 virus (multiplicity of infection = 5) in T-150 cm2 flasks for 36 h at 28 °C. Uninfected C6/36 cells were used as controls. Cells were harvested by centrifugation at 800 × g for 10 min, and the cell pellet was resuspended in 0.5 ml of TNMg buffer (10 mmTris-HCl, pH 8.0, 10 mm sodium acetate, 1.5 mmMgCl2) per T-150 cm2 flask. After passing the cell suspension 20 times through a syringe (one ml capacity), fitted with a 27-guage needle, the cell lysates were centrifuged at 800 × g for 10 min at 4 °C to fractionate the cytoplasmic and nuclear fractions as described (39Chu P.W. Westaway E.G. Virology. 1987; 157: 330-337Crossref PubMed Scopus (76) Google Scholar). The protein concentrations of the cytoplasmic extracts were determined as described (61Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar), and the extracts were stored in aliquots at −70 °C until used. An aliquot of the cytoplasmic extract was mixed with an equal volume of a 2× sample loading buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 0.7 m β-mercaptoethanol, 10% glycerol, 0.05% bromphenol blue). Proteins were separated by SDS-PAGE (10%) and transferred electrophoretically to a polyvinylidene difluoride membrane. The membrane was treated with nonfat dry milk (5%) for 2 h in Tris-buffered saline containing 0.1% Tween-20, pH 7.5, and then incubated overnight with polyclonal rabbit anti-NS3 or anti-NS5 antibodies (1:2000). The membranes were washed three times for 20 min each with this buffer and incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (1:20000) for 2 h at room temperature. Chemiluminescence detection of the immunoreactive bands were performed as described by the manufacturer (ECL system; Amersham Pharmacia Biotech). The p190-24 plasmid contains the 3′-terminal 373 nt from the DEN2 cDNA and 26 nt from the pGEM7Zf+ vector (TCAAGCTATGCATCCAACGCGTTGGG) under the control of SP6 promoter (56Mohan P.M. Padmanabhan R. Gene (Amst.). 1991; 108: 185-191Crossref PubMed Scopus (40) Google Scholar). When this plasmid is linearized withXbaI, the cDNA of (−)-strand polarity was extended by 4 additional nucleotides of the XbaI-5′ overhang. The RNA synthesized from this template strand would include the 4 additional nucleotides complementary to this overhang at its 3′-end. To generate 3′-UTR373nt RNA, which contains the wild type cyclization sequence but without these 4 additional nucleotides, two approaches were followed. Firstly, a PCR approach was followed using the two oligonucleotide primers: the upstream 5′-primer (A), 5′-TGACCATGATTACGCCAAGCTATTTAGGTG-3′ anneals a region upstream of the SP6 promoter and a part of the promoter (underlined); the downstream 3′ primer (B), 5′-AGAACCTGTTGATTCAACAGCACC-3′ is complementary to the 3′-end of the viral genome. The PCR fragment was purified and used for in vitro transcription (see below). In the second approach, the XbaI-linearized p190-24 plasmid (10 μg in each reaction) was treated with increasing amounts of S1 nuclease (0, 3, 10, 30, 75, and 150 units) in 100 μl of reaction mixtures containing 50 mm sodium acetate, pH 4.5, 28 mm NaCl, and 4.5 mm ZnSO4 at room temperature for 30′, extracted with phenol, and precipitated with ethanol. The S1 nuclease-treated plasmids were used for the in vitro transcription catalyzed by SP6 RNA polymerase. To ensure that the S1 nuclease precisely removed the XbaI overhang, the blunt ends were ligated by T4 DNA ligase and cloned. DNA sequencing was carried out using an automated DNA sequencer (Biotech Facility, University of Kansas Medical Center). A 230-nt DNA fragment from the 5′-terminal region that includes the 5′-UTR (96 nt), and the 5′-CS1 was generated by PCR using pMK4 as a template and the upstream primer (C), 5′-CGGAATTCGGATCGATCCCCCCTAATAC-3′ (containing the T7 promoter), and the downstream primer (D) 5′-CAGTTCCTGAGGTCCTCGTCCCTGCAG-3′ (complementary to 217–233 nt of the viral genome, which includes the 5′-CS1 element). The primers, C and D, contain the EcoRI andBsu36I sites (underlined), respectively. The PCR product was digested with EcoRI and Bsu36I and purified using a Qiagen cartridge. The 3′-terminal fragment (800 nt) that includes the 3′-UTR was derived from the plasmid clone pGEM-PCR1.3 (4Irie K. Mohan P.M. Sasaguri Y. Putnak R. Padmanabhan R. Gene (Amst.). 1989; 75: 197-211Crossref PubMed Scopus (151) Google Scholar) by digestion with Bsu36I (nt position 9885) and XbaI (nt position 10723). The pSP64 vector plasmid was digested at the multiple cloning site with EcoRI and XbaI. A three-fragment ligation and cloning yielded pSY-1 plasmid containing DEN2 cDNA sequences under the control of T7 promoter. To construct a plasmid encoding a shorter subgenomic RNA, the pSY-1 plasmid was partially digested with XmnI, followed by digestion withBsu36I. The overhang from the Bsu36I-cut plasmid was blunt-ended by treatment with E. coli Klenow DNA polymerase fragment and was cloned to yield the pSY-2 plasmid (720 nt) in which 348 nt from the pSY-1 was deleted but retaining all of 3′-UTR region. The pSY-1 and pSY-2 plasmids were used to produce the subgenomic RNAs of 1.0 kilobase and 720 nt, respectively, by in vitro transcription (Fig. 1). To construct a PCR fragment of 5′-TR/mutCYC (which contains substitution mutations within the cyclization sequence), a PCR-based mutagenesis protocol was followed. Two PCR products were generated with overlapping sequences using two sets of primers. PCR1 was obtained using the 5′-primer (E), containing the EcoRI site, 5′-AGCTATGACCATGATTACGAATTC-3′ that corresponds to the upstream region of the T7 promoter in the pSP64 vector and the 3′-primer (F), 5′-TTTCACAGAGAGAGAAGGCGTATTTCTCGCCTTT-3′. PCR2 was produced using the 5′ primer (G), 5′-GCCTTCTCTCTCTGTGAAACGCGAGAGAAACCG-3′, and the 3′ primer (H), 5′-TGAGGTCCTCGTCCTG-3′. The underlined complementary sequences in F and G primers represent the mutated cyclization element (mutCYC). The primer H shares identical sequences with pSY-1 plasmid in the vicinity of Bsu36I site (underlined). The two products, PCR1 and PCR2, produced using the primer sets E/F and G/H were purified, mixed, and used for a third PCR in the presence of the primer set E/H. This final PCR product was purified and used for in vitro transcription to generate 5′-TR/mutCYC RNA. To construct a PCR fragment containing the 3′-UTR373nt/mutCYC that contains mutations within the cyclization sequence complementary to that in 5′-TR/mutCYC), a PCR-based mutagenesis protocol was followed. PCR1 was obtained using the 5′-primer (A), the 3′-primer (I), 5′-CAGCGCTCTCTCTGTGTTTTTTGTTTTGGGGGGG-3′. PCR2 was produced using 5′ primer (J), 5′-AAACACAGAGAGAGCGCTGGGAAAGACCAGAGAT-3′ and the 3′-primer (B). The underlined complementary sequences in I and J primers represent mutated cyclization element (mutCYC). p190-24 plasmid was used as the template for PCR. The two products, PCR1 and PCR2, produced using the primer sets A/I and J/B were purified, mixed, and used for a third PCR in the presence of the primer set A/B. This final PCR product was purified and used for in vitro transcription to generate the 3′-UTR/mutCYC RNA. The RNA templates used in the in vitro RdRP assays are shown in Fig. 1. To synthesize RNA templates containing 3′-terminal 373 nt (3′-UTR373nt) or the subgenomic RNA templates, the plasmid constructs, p190.24 (56Mohan P.M. Padmanabhan R. Gene (Amst.). 1991; 108: 185-191Crossref PubMed Scopus (40) Google Scholar) and pSY-2, respectively, were linearized with XbaI. The supercoiled plasmids were used as templates for PCR. The PCR products or the XbaI-linearized plasmids were used in the in vitro transcription reaction catalyzed by either SP6 (for the 3′-UTR373nt RNA) or T7 RNA polymerase (for subgenomic RNAs) using conditions supplied by the manufacturer (Promega). Briefly, the in vitro transcription reactions were carried out at 37 °C for 3 h in reaction mixtures (100 μl) containing the 1× buffer (40 mmTris-HCl, pH 7.5, 6 mm MgCl2, 2 mmspermidine, 10 mm NaCl), 5 μg of the DNA templates, a mixture of four rNTPs (0.5 mm each), 100 units of RNasin, 10 mm dithiothreitol, and 5 units of T7 or SP6 RNA polymerase. Then the reaction mixtures were digested with 5 units of RNase-free DNase I (Promega) at 37 °C for 20 min to remove the DNA templates. The RNA transcripts were extracted with phenol at pH 4.5 and precipitated with ethanol. The quality of RNA products was analyzed electrophoretically and quantified spectrophotometically. Because of the 26 nt from the vector sequence at the 5′-end, the 3′-UTR373nt RNA is actually 399 nt in length but is referred to in this study as 3′-UTR373nt. Nonviral exogenous RNAs were prepared by linearizing three different vector plasmids (pSP64, pSP70, and pTM1) with six different restriction enzymes. RNAs were prepared by in vitro transcription and purified as described above. The in vitro RdRP assay was performed in 50-μl reaction mixtures containing 50 mm Hepes, pH 7.3, 3 mm magnesium acetate, 6 μm zinc acetate, 25 mm potassium acetate, 60 units of RNase inhibitor, 10 mm β-mercaptoethanol, 0.5 mm each ATP, GTP, and UTP, and 10 μm of CTP mixed with 10 μCi of [α-32P]CTP (800 Ci/mmol), 0.1 mg/ml actinomycin D, 10 μg of cytoplasmic extract from infected cells, and 5 μg of an exogenous RNA template. As a control, a parallel reaction mixture containing all the components except the cytoplasmic extract from the uninfected C6/36 cell lysate (10 μg) was included. Controls in which cytoplasmic extracts from infected or uninfected cells but without any exogenous RNA were also included. Reaction mixtures were incubated at 30 °C for 1.5 h. To carry out the kinetics of RdRP reaction, seven separate RdRP reactions that included DEN2-infected cytoplasmic extracts and the subgenomic RNA template were initiated. At time intervals of 0, 5, 10, 15, 20, 30, and 40 min, reactions were terminated by the addition of EDTA (10 mm). The samples were extracted with phenol, pH 4.5, and precipitated by the addition of 0.1 volume of 3 m sodium acetate, pH 5.2, and 2.5 volumes of 100% ethanol in the presence of 5 μg of yeast tRNA (Ambion). The RNA products were analyzed using either polyacrylamide, 7 murea or by formaldehyde-agarose gel electrophoresis, followed by autoradiography. The RdRP products separated on PAGE, 7 m urea gels were cut out and eluted in 400 μl of elution buffer (2 mammonium acetate, 1% SDS, 1 mm EDTA, and 25 μg/ml tRNA) either at 37 °C for overnight or at 65 °C for 4 h. After briefly spinning down gel slices, the supernatants were precipitated overnight with 1 ml of 100% ethanol at −20 °C. The eluted products were treated with or without RNase A (Sigma; 5 ng/μl) in 20 μl of either 2× SSC (300 mm NaCl and 30 mm sodium citrate, pH 7.2) or 0.1× SSC (15 mm NaCl and 1.5 mm sodium citrate, pH 7.2) at 37 °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 phenol extraction and ethanol precipitation in the presence of 5 μg of yeast tRNA. The RNase A-treated samples were analyzed on formaldehyde agarose gel, followed by autoradiography. In vitro transcribed RNAs were dissolved in 200 μl of 50 mm NaOAc, pH 5.0. 50 μl of 0.1 mNaIO4 (20 mm) was added and then incubated for 1 h at room temperature. Lysine (60 mm) was added to saturate the excess periodate and further incubated for 3 h at room temperature. The reactions were phenol extracted and precipitated with ethanol. The pellets were washed with 70% EtOH and dissolved in water and subsequently desalted with Bio-Gel P gel column (Bio-Rad). The periodate-treated RNAs were quantified spectrophotometrically and visualized on PAGE, 7 m urea gel by acridine orange. Previous studies showed that flavivirus RdRP activity was tightly associated with intracellular membranes in the cytoplasmic fractions of flavivirus-infected mammalian (monkey kidney cell line (Vero), or baby hamster kidney cell line (BHK-21)) cells (37Chu P.W. Westaway E.G. Virology. 1985; 140: 68-79Crossref PubMed Scopus (149) Google Scholar, 38Grun J.B. Brinton M.A. J. Virol. 1986; 60: 1113-1124Crossref PubMed Google Scholar, 39Chu P.W. Westaway E.G. Virology. 1987; 157: 330-337Crossref PubMed Scopus (76) Google Scholar, 40Bartholomeusz A.I. Wright P.J. Arch. Virol. 1993; 128: 111-121Crossref PubMed Scopus (85) Google Scholar, 62Grun J.B. Brinton M.A. J. Virol. 1987; 61: 3641-3664Crossref PubMed Google Scholar,63Grun J.B. Brinton M.A. J. Gen. Virol. 1988; 69: 3121-3127Crossref PubMed Scopus (33) Google Scholar). The earlier studies examined the incorporation of labeled nucleotides into the three RNA species that were synthesized from endogenous viral RNA templates. We sought to develop an in vitro RdRP assay that could utilize exogenous RNA templates that contain either 3′- or 5′-untranslated regions or both to determine the sequence requirements for RNA synthesis and to characterize the proteins that interact with these elements. We chose to use an established Aedes albopictus (C6/36) cell line for preparation of cytoplasmic extracts from DEN2-infected cells (see "Experimental Procedures") because the viral titers are significantly higher in C6/36 cells than in vertebrate cells (64Mussgay M. Enzmann P.J. Horzinek M.C. Weiland E. Prog. Med. Virol. 1975; 19: 257-323PubMed Google Scholar). We constructed cDNA cl
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