Synthesis in Vitro of Rabbit Hemorrhagic Disease Virus Subgenomic RNA by Internal Initiation on (–)Sense Genomic RNA
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313674200
ISSN1083-351X
AutoresMònica Morales, Juan Bárcena, Miguel Ángel Ramírez, José Antonio Boga, Francisco Parra, Juan María Torres,
Tópico(s)Hepatitis B Virus Studies
ResumoRabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3′ one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3′-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (–)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (–)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae. Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3′ one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3′-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (–)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (–)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae. Positive-strand RNA viruses are defined by the translatability of their genomic RNAs (gRNA). 1The abbreviations used are: gRNA, genomic RNA; 3Dpol, RNA-dependent RNA polymerase; EMCV, encephalomyocarditis virus; ORF, open reading frame; RHDV, rabbit hemorrhagic disease virus; RdRp, RNA-dependent RNA polymerase; sgRNA, subgenomic RNA; ORF, open reading frame; VPg, virus-encoded protein. 1The abbreviations used are: gRNA, genomic RNA; 3Dpol, RNA-dependent RNA polymerase; EMCV, encephalomyocarditis virus; ORF, open reading frame; RHDV, rabbit hemorrhagic disease virus; RdRp, RNA-dependent RNA polymerase; sgRNA, subgenomic RNA; ORF, open reading frame; VPg, virus-encoded protein. For some of them, such as picornaviruses and flaviviruses, the gRNA is the only viral mRNA in the infected cells. Nevertheless, there are many other viruses in which one or more subgenomic mRNAs (sgRNA) are also synthesized. SgRNA of positive-strand viruses are 3′-co-terminal and identical to the gRNA for most of their length but have deletions at the 5′ ends (with respect to gRNA) to bring their 5′ ends close to the start codon of downstream (on genomic RNA) ORFs. In these cases, some of the viral genes are translated from the sgRNA species, thus creating the potential for independently regulating the levels of viral proteins in infected cells. SgRNA synthesis has been more studied in plant viruses than in animal viruses, probably because a greater percentage of all of the plant viruses make sgRNA and also because their smaller genomes and highly efficient replication make them more amenable to studies on RNA replication mechanisms, especially using cell-free extracts. Several basic mechanisms for generating subgenomic RNAs have been defined (1Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (155) Google Scholar). The most widely recognized model is internal initiation on longer-than-subgenomic-length (–)strand templates in which the RNA-dependent RNA polymerase (RdRp) internally initiates (+)strand sgRNA synthesis. Initiation of RNA synthesis occurs at selected sites called promoters, which have been characterized in several plant viruses (2Miller W.A. Dreher T.W. Hall T.C. Nature. 1985; 313: 68-70Crossref PubMed Scopus (218) Google Scholar, 3van der Kuyl A.C. Langereis K. Houwing C.J. Jaspars E.M. Bol J.F. Virology. 1990; 176: 346-354Crossref PubMed Scopus (48) Google Scholar, 4Song C. Simon A.E. J. Mol. Biol. 1995; 254: 6-14Crossref PubMed Scopus (116) Google Scholar, 5Guan H. Song C. Simon A.E. RNA. 1997; 3: 1401-1412PubMed Google Scholar, 6Wang J. Simon A.E. Virology. 1997; 232: 174-186Crossref PubMed Scopus (63) Google Scholar, 7Singh R.N. Dreher T.W. Virology. 1997; 233: 430-439Crossref PubMed Scopus (66) Google Scholar, 8Adkins S. Kao C.C. Virology. 1998; 252: 1-8Crossref PubMed Scopus (39) Google Scholar, 9Olsthoorn R.C. Mertens S. Brederode F.T. Bol J.F. EMBO J. 1999; 18: 4856-4864Crossref PubMed Scopus (123) Google Scholar, 10Osman T.A. Hemenway C.L. Buck K.W. J. Virol. 2000; 74: 11671-11680Crossref PubMed Scopus (51) Google Scholar, 11Panavas T. Pogany J. Nagy P.D. Virology. 2002; 296: 263-274Crossref PubMed Scopus (71) Google Scholar) and the alphavirus (12Levis R. Schlesinger S. Huang H.V. J. Virol. 1990; 64: 1726-1733Crossref PubMed Google Scholar). Most of the characterized viral RNA promoters are located at the 3′ end of the RNA templates, and they often have stem-loop structures or consist of short single-stranded regions with unique primary sequences (13Buck K.W. Adv. Virus Res. 1996; 47: 159-251Crossref PubMed Google Scholar, 14Dreher T.W. Annu. Rev. Phytopathol. 1999; 37: 151-174Crossref PubMed Scopus (189) Google Scholar, 15Kao C.C. Singh P. Ecker D.J. Virology. 2001; 287: 251-260Crossref PubMed Scopus (183) Google Scholar). The best characterized cases are the subgenomic promoters for brome mosaic virus and Turnip crinkle virus. The second mechanism is premature termination during (–)strand synthesis from the genomic RNA template, giving a subgenomic-length (–)strand (16Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar). This would then serve as template for end-to-end (+)strand synthesis.Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae (17Ohlinger V.F. Haas B. Meyers G. Thiel H-J. J. Virol. 1990; 64: 3331-3336Crossref PubMed Google Scholar, 18Parra F. Prieto M. J. Virol. 1990; 64: 4013-4015Crossref PubMed Google Scholar, 19Cubbit D. Bradley D.W. Carter M.J. Chiba S. Estes M.K. Saif L.J. Schaffer F.L. Smith A.W. Studdert M.J. Thiel. H.J. Arch. Virol. 1995; 10: 359-363Google Scholar). The genome of RHDV consists of a single positive-stranded RNA of 7.4 kb (20Meyers G. Wirblich C. Thiel H.J. Virology. 1991; 184: 664-676Crossref PubMed Scopus (255) Google Scholar) that has a virus-encoded protein, VPg (21Wirblich C. Thiel H.J. Meyers G. J. Virol. 1996; 70: 7974-7983Crossref PubMed Google Scholar), attached covalently to the 5′end (22Meyers G. Wirblich C. Thiel H.J. Virology. 1991; 184: 677-686Crossref PubMed Scopus (159) Google Scholar) and is polyadenylated at the 3′ end. Viral particles also encapsidate a VPg-linked polyadenylated subgenomic RNA of approximately 2.2 kb (22Meyers G. Wirblich C. Thiel H.J. Virology. 1991; 184: 677-686Crossref PubMed Scopus (159) Google Scholar), which is co-terminal with the 3′ end of the viral genome. Progress on the replication of caliciviruses has been negligible (23Clarke I.N. Lambden P.R. J. Gen. Virol. 1997; 78: 291-301Crossref PubMed Scopus (107) Google Scholar) compared with the advances made with the picornaviruses. Because of the lack of a cell culture system for most caliciviruses such as RHDV, recombinant DNA technology has been crucial for the production and characterization of viral proteins. The data obtained from the in vitro translation (21Wirblich C. Thiel H.J. Meyers G. J. Virol. 1996; 70: 7974-7983Crossref PubMed Google Scholar) and Escherichia coli expression studies (24Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1996; 70: 1261-1265Crossref PubMed Google Scholar) revealed that the viral RNA is translated into a polyprotein that is subsequently cleaved to give rise to mature structural and nonstructural proteins. The extensive sequence similarities between the RdRp of picornaviruses (3Dpol) and the RHDV polyprotein cleavage product p58 (21Wirblich C. Thiel H.J. Meyers G. J. Virol. 1996; 70: 7974-7983Crossref PubMed Google Scholar, 25König M. Thiel H.J. Meyers G. J. Virol. 1998; 72: 4492-4497Crossref PubMed Google Scholar) suggested that this polypeptide might have a similar role in RHDV genome replication. Recently, the structure of the recombinant RdRp from RHDV produced in E. coli has been determined by x-ray crystallography showing a close structural similarity to the RdRps from poliovirus and hepatitis C virus (26Ng K.K. Cherney M.M. López Vázquez A. Machín A. Alonso J.M. Parra F. James M.N. J. Biol. Chem. 2002; 277: 1381-1387Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Previous studies of the purified recombinant RHDV enzyme also showed RdRp activity acting on synthetic RHDV positive subgenomic RNA in the presence or absence of an oligo(U) primer (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar, 28López Vázquez A. Martín Alonso J.M. Parra F. Arch. Virol. 2001; 146: 59-69Crossref PubMed Scopus (20) Google Scholar). Templatesized products were synthesized in the oligo(U)-primed reactions as described for EMCV 3Dpol (29Sankar S. Porter A.G. J. Virol. 1991; 65: 2993-3000Crossref PubMed Google Scholar), whereas in the absence of added primer, RNA products up to twice the length of the template were made, suggesting that the newly made negative-strand RNA was covalently linked to the plus-strand RNA template (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar). Despite the structural and functional similarities found between the Picornaviridae and Caliciviridae, the synthesis of a sgRNA in the Caliciviridae denotes a significant discrepancy concerning their gene expression strategies.In this work, we have intended the study of RHDV sgRNA initiation using synthetic (–)RNAs of genomic length or having varying deletions at their 3′ regions considering that, in addition to their role as templates for (+)strand gRNA production, they might also have internal sequences involved in initiation of sgRNA synthesis.EXPERIMENTAL PROCEDURESConstruction of Plasmid pBac-3D—The coding region for RHDV RNA-dependent RNA polymerase (3Dpol) was cloned as a EcoRI-XbaI cassette by PCR amplification of an AST/89 RHDV isolate cDNA. This was done by using oligonucleotide primers 3D5′ (5′-tcacaggatccgccaccatgacatcaaacttcttctgc-3′) and 3D3′ (5′-gtgcgtctagatcactccataacattcac-3′), which also added the indicated restriction enzyme recognition sequences (underlined residues) at both ends of the amplified region. The resulting amplicon was EcoRI-XbaI digested, and the 1.5-kb fragment containing the RHDV RdRp gene (3Dpol) was inserted into EcoRI-XbaI-digested expression vector pBacPAK8 (Invitrogen) to generate the recombinant plasmid pBac-3D.Production of Recombinant RHDV 3Dpol—A recombinant baculovirus containing the RHDV 3Dpol-coding sequence under control of the polyhedrin promoter was made by co-transfection of Sf9 cells with plasmid pBac-3D and the β-galactosidase-containing AcNPV (Autographa californica nuclear polyhedrosis virus or baculovirus) DNA. The recombinant baculovirus Bac-3D was plaque-purified (30Summers M.D. Smith G.E. Virology. 1978; 84: 390-402Crossref PubMed Scopus (101) Google Scholar) and propagated using monolayers of Sf9 cells infected at multiplicities of 0.1–1.0 plaque-forming unit/cell for virus amplification or 1–10 plaque-forming unit/cell for protein expression assays. For screening of the recombinant clones, 1 h after infection, cells were overlaid with 1.5% agarose in Grace's medium containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) (120 μg/ml). Blue plaques could be detected 5–10 days post-infection. Occlusion negative plaques were identified under a microscope and were picked using sterile glass-disposable pipettes to remove the agarose directly over the plaque. The plaques were suspended in fresh medium and used for subsequent plaque assays or for virus amplification.Recombinant baculovirus-infected H5 cells were harvested, concentrated by centrifugation at 1000 × g for 10 min, and suspended in 10 mm sodium phosphate (pH 7.0) to a final concentration of 1 × 104 H5 cells/μl prior to sonication on ice using a 20-kilocycle sonifier (1-min burst followed by four 30-s bursts with 2-min cooling steps between bursts). The resulting crude cell extract was centrifuged at 50,000 × g for 20 min, and the supernatant was stored at –70 °C after addition of 5% glycerol and RNase inhibitor (PerkinElmer Life Sciences).In Vitro Synthesis of RHDV RNAs—Positive and negative sense gRNA, sgRNA, and a series of 3′-truncated (–)gRNAs or 5′-truncated (+)gRNAs were obtained (Fig. 4) by transcription in vitro of purified PCR fragments to which a modified T7 promoter was added using the appropriate primers (Table I). The PCR fragments used to produce full-length positive (gRNA+) or negative sense genomic RNA (gRNA–) were made using primer pairs RHDV10/RHDV14 and RHDV1/RHDV9, respectively (Table I). The PCR used to produce positive (sgRNA+) or negative sense subgenomic RNA (sgRNA–) were made using RHDV7/RHDV9 and RHDV13/RHDV14 primer pairs, respectively (Table I). The PCR used to produce a series of 5′-truncated (+)gRNAs (Fig. 4, RNA h and RNA i) were made using primer pairs RHDV11/14 and RHDV12/14, and the series of 3′-truncated (–)gRNAs (Fig. 4, RNA a, RNA b, RNA c, RNA d, RNA e, and RNA g) were made using RHDV2/9, RHDV3/9, RHDV4/9, RHDV5/9, RHDV6/9, RHDV7/9, and RHDV8/9 (Table I). The RNA transcripts were made using the large scale RNA production system Ribomax (Promega). The resulting positive and negative transcripts were analyzed by electrophoresis on denaturing agarose gels, and their concentrations were measured spectrophotometrically.Table IOligonucleotide primers used for PCR amplification of RHDV cDNAs for in vitro transcription of positive and negative synthetic RNAsNamePosition in RHDVPrimer sequenceaThe sequences are written from 5′ to 3′. Nucleotide residues not related to RHDV AST/89 sequence (GenBank™ accession number Z49271) are underlined. The T7 promoter sequence included in oligonucleotides is indicated in boldface.PolaritybPolarity refers to homology (+) or complementarity (-) to RHDV genomic RNA.RHDV 11-32GTGAAAATTATGGCGGCTATGTCGCGCCTTAC+RHDV 23763-3780ACATCAAACTTCTTCTGC+RHDV 35140-5154GCTGCCGCACATGGCC+RHDV 45196-5216GCAGGCGTACACCCAGTTTAG+RHDV 55246-5266TACTTGCAGATCGTAAGAGAG+RHDV 65266-5282GTCGTCTCGGTAGTACC+RHDV 75296-5310GTGAATGTTATGGAGGGCAAAGCCCG+RHDV 85758-5779CAATTCCCCCATGTTGTCATCG+RHDV 97437-7414ACCGCGGCCGCTAATACGACTCACTATAGGGGCGG(T)23ATAGCTTACTTTAAAC-RHDV 101-21CCGCGGCCGCTAATACGACTCACTATAGTGAAAATTATGGCGGCTATG+RHDV 115039-5061GCGCCGCGGCCGCTAATACGACTCACTATAGGCAGTTAGAGTGGAGCAAAACAACATCC+RHDV 125196-5216GCGCCGCGGCCGCTAATACGACTCACTATAGCAGGCGTACACCCAGTTTAG+RHDV 135296-5317GCGCCGCGGCCGCTAATACGACTCACTATAGTGAATGTTATGGAGGGCAAAG+RHDV 147437-7414(T)23ATAGCTTACTTTAAACTATAAACa The sequences are written from 5′ to 3′. Nucleotide residues not related to RHDV AST/89 sequence (GenBank™ accession number Z49271) are underlined. The T7 promoter sequence included in oligonucleotides is indicated in boldface.b Polarity refers to homology (+) or complementarity (-) to RHDV genomic RNA. Open table in a new tab Gel Electrophoresis and Protein Concentration—RNA was analyzed on 1% formaldehyde-agarose gels (31Sambrook J. Firtsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). SDS-PAGE was performed as described elsewhere (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206016) Google Scholar). The protein concentration was measured using the Bio-Rad protein assay.Preparation of Oligo(U)—Oligo(U) was made by alkali hydrolysis of poly(U) as described previously (33Plotch S.J. Palant O. Gluzman Y. J. Virol. 1989; 63: 216-225Crossref PubMed Google Scholar). The size of the resulting oligo(U) was investigated by end-labeling with [γ-32P]ATP (ICN) and electrophoresis on denaturing 6% polyacrylamide gels.Enzymatic Assays—In vitro RHDV polymerase assays were performed as described previously (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar) with some modifications. The reactions were carried out in a final volume of 50 μl containing appropriate concentrations of 3Dpol (1–10 μl of baculovirus-infected H5 cell-free extract), 50 mm HEPES, pH 8.0, 10 μm ATP, 10 μm CTP, 10 μm GTP, 5 μm UTP, 4 mm dithiothreitol, 3 mm magnesium acetate, 6 μm zinc chloride, 50 units of ribonuclease inhibitor (Promega), 25 μmol of [α-32 P]UTP, and 14–28 nm of the appropriate synthetic RNA template or poly(A) template in the absence (–) or presence (+) of oligo(U) primer. After incubation at 30 °C for 60 min, the reaction mixtures were phenolchloroform-extracted and ethanol-precipitated in the presence of 0.3 m sodium acetate (pH 6.0) and 20 μg of carrier tRNA. The sediments were dissolved in electrophoresis sample buffer, loaded onto 1.2% formaldehyde-agarose gels, and analyzed at 60–70 V. The gels were then dried, and the 32P-labeled RNA was detected by autoradiography.RESULTSProduction of Recombinant RHDV 3Dpol Using Baculovirus-infected Insect Cells—Previous functional (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar, 28López Vázquez A. Martín Alonso J.M. Parra F. Arch. Virol. 2001; 146: 59-69Crossref PubMed Scopus (20) Google Scholar) and structural (26Ng K.K. Cherney M.M. López Vázquez A. Machín A. Alonso J.M. Parra F. James M.N. J. Biol. Chem. 2002; 277: 1381-1387Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) studies on RHDV 3Dpol used a recombinant protein produced in E. coli, which nonetheless showed enzymatic activity on homopolymeric and heteropolymeric RNA templates. In this work, we have selected insect cells as the host system to produce recombinant 3Dpol. For this purpose, a suitable vector was made (see "Experimental Procedures") after PCR amplification of the previously identified 3Dpol-coding region. The resulting gene construct included an added ATG initiator codon in an optimized Kozak (34Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (991) Google Scholar) sequence context and was 5′- and 3′-flanked by appropriate restriction enzyme targets to facilitate directional cloning. After digestion using the restriction enzymes EcoRI and XbaI, the amplicon was directly cloned into EcoRI-XbaI-digested pBacPAK8 vector (Invitrogen) yielding the baculovirus transfer vector pBac-3D, which was sequenced to investigate the correct reading frame.A recombinant baculovirus was generated following co-transfection of Sf9 cells with transfer vector pBac-3D and infectious BacPAK6 DNA. Viral DNA recombination resulted in the replacement of polyhedrin gene sequences by the RHDV 3Dpol construct from the pBac-3D transfer vector.Expression of the recombinant RHDV 3Dpol gene cloned in baculovirus Bac-3D was expected to produce a 58-kDa polypeptide in the infected cells. In good agreement to this prediction, the protein pattern of baculovirus-infected cell-free extracts after SDS-PAGE and Coomassie Blue staining showed a protein product of the expected size, which was increasingly predominant at longer infections times (Fig. 1). It should be mentioned that the recombinant 3Dpol produced in insect cells was expected to be almost identical in amino acid sequence (only an initiator Met was added) to the authentic RHDV polymerase obtained after proteolytic processing of the viral polyprotein (24Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1996; 70: 1261-1265Crossref PubMed Google Scholar, 35Wirblich C. Sibilia M. Boniotti B. Rossi C. Thiel H.J. Meyers G. J. Virol. 1995; 69: 7159-7168Crossref PubMed Google Scholar).Fig. 1SDS-PAGE analysis of recombinant RHDV 3Dpol produced in insect cells. The protein pattern was obtained after separation and Coomassie Blue staining of cell-free extract samples from mock infected (lane 1) or Bac-3D-infected H5 (lanes 2–6) at 1, 2, 3, 4, and 5 days post-infection respectively. The numbers on the left indicate the protein markers for molecular mass (in kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Recombinant RHDV 3Dpol Activity Assay—As a first approach to investigate the biological activity of recombinant RHDV 3Dpol produced in baculovirus-infected insect cells, we used a previously described poly(A)-dependent oligo(U)-primed poly(U) polymerase assay (29Sankar S. Porter A.G. J. Virol. 1991; 65: 2993-3000Crossref PubMed Google Scholar, 27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar). Extracts of H5 cells mockinfected or infected with recombinant baculovirus Bac-3D containing equivalent amounts of total soluble protein were assayed for poly(A)-dependent poly(U) polymerase activity in the presence and absence of an oligo(U) primer (Fig. 2). The results indicated that poly(U) polymerase activity was detectable in H5 cells 1 day post-infection and increased with time up to day 3 post-infection (Fig. 2). This observation was consistent with the relative amounts of 3Dpol in the cell extracts, as deduced from the intensity pattern of the protein band observed after SDS-PAGE analysis (Fig. 1, lanes 2–6). As described earlier for recombinant RdRp produced in E. coli, the poly(U) polymerase activity found in the infected-H5 extracts was dependent on the presence of an added oligo(U) primer, an omission of which resulted in an almost complete loss of activity (Fig. 2). It should be mentioned that activity levels were not proportional to the volume of cell extract in the assay reaction when samples over 10 μl of H5 extracts were used, indicating the presence of an inhibitory component in the cell extract (data not shown). For this reason, in subsequent analyses using hetero-polymeric RNA templates, 1–10 μl of H5 extracts will be used.Fig. 2Poly(U)-polymerase activity in mock or Bac-3D-infected H5 cell free extracts. Crude extracts were assayed for poly(U)-polymerase activity using a poly(A) template and an oligo(U) primer as described under "Experimental Procedures." Incubations contained mock-infected H5 (▵) and Bac-3D-infected H5 cells harvested at day 1 (▴), day 2 (□), day 3 (⋄), and day 4 (•) post-infection in the presence of oligo(U) primer or day 4 post-infection in the absence of oligo(U) primer (▪). The reactions were stopped at the indicated times of incubation and assayed for acid-precipitable radioactivity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RdRp Activity on Heteropolymeric RNA Templates—To investigate whether the recombinant enzyme produced in insect cells exhibited RdRp activity on heteropolymeric RNA templates, synthetic RHDV genomic and subgenomic RNAs of positive and negative polarity were made by in vitro transcription of purified PCR fragments to which a modified T7 minimal promoter sequence was added (see "Experimental Procedures"). The synthetic RHDV genomic and subgenomic RNAs of positive polarity transcribed in vitro where identical in nucleotide sequence with respect to the authentic viral gRNA and sgRNA and differed only in that the synthetic transcripts lacked a covalently linked VPg protein at their 5′ ends and in that the 3′ terminus had a shorter poly(A) tail of only 23 residues. The synthetic RHDV genomic and subgenomic RNAs of negative polarity were complementary to the positive gRNA and sgRNA transcripts for most of their lengths but differed in a few additional residues (<7) added to the 5′-poly(U) as a consequence of the sequence GGGGCGG included in the oligonucleotide used to make the amplicons (Table 1) in order to allow efficient T7 transcription initiation. The size and quantity of the RNA templates used and the labeled RNA products synthesized in the RdRp assays were analyzed by formaldehyde-agarose gel electrophoresis.The RHDV RdRp produced in baculovirus-infected H5 cells was able to use (+)- and (–)-single-stranded RNA templates in the absence of added primers. As has been described previously using the bacterially produced RHDV RdRp (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar), the major labeled products in the presence of either gRNA or sgRNA of (+) polarity were of twice the length (∼15 and 4.4 kb, respectively) of the input template (Fig. 3A, lanes 1–2). When (–)gRNA was used as template (Fig. 3B, lane 1), the major synthesized product was of the size of sgRNA (2.2 kb). In the presence of (–)sgRNA as the template, the products, if any, were poorly labeled indicating low polymerization efficiency, being the major observed band of twice the length (4.4 kb) of the input template (Fig. 3B, lane 2). These data suggested that the subgenomic RNA of RHDV was synthesized in vitro by internal initiation of replication on a (–)RNA template of genomic length.Fig. 3RHDV RdRp assays using heteropolymeric RNA templates. The reactions were made in the absence of added RNA template (lanes 3) or in the presence of synthetic gRNA (lanes 1) or sgRNA (lanes 2) of positive (panel A) or negative polarity (panel B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Deletion Mapping of a Putative Subgenomic Promoter—To further investigate initiation of sgRNA synthesis on (–)gRNA templates and aiming to map the minimum sequence required for full function of the putative internal promoter, a series of deletion mutants were constructed in which the 3′-terminal sequence of (–)gRNA was progressively deleted (Fig. 4, transcripts a–g), approaching (transcripts a–e), reaching (transcript f), or surpassing (transcripts g) the transcriptional start residue (+1) for sgRNA. A series of (+)transcripts identical in nucleotide sequence to the RHDV sgRNA (Fig. 4, transcript j) or including 100 (transcript i) or 257 (transcript h) 5′-additional genomic residues were also made. The truncated RNA transcripts of positive and negative polarity made by in vitro transcription of appropriate amplicons were used as templates in the absence of added primers in in vitro RHDV RdRp assays.The major labeled products obtained using (+)RNA templates h, i, and j (Fig. 4) were of twice the length of the input RNA (Fig. 5B, lanes h–j), suggesting that de novo initiation, premature termination, or template terminal-labeling have not occurred in these in vitro reactions. Moreover, the presence of double-sized products suggested a template-primed synthesis mechanism (15Kao C.C. Singh P. Ecker D.J. Virology. 2001; 287: 251-260Crossref PubMed Scopus (183) Google Scholar) as described earlier for the RHDV enzyme produced in bacteria (27López Vázquez A. Martín Alonso J.M. Casais R. Boga J.A. Parra F. J. Virol. 1998; 72: 2999-3004Crossref PubMed Google Scholar).Fig. 5Subgenomic promoter mapping using RHDV RdRp in vitro assays. The reactions were made in the absence (lanes N) or presence of negative (A) or positive polarity (B) RNA templates. Lowercase case letters on the lanes indicate the RNA transcripts (see Fig. 4) used as template.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast with the results found using (+)RNA templates, the use of several (–)RNA templates (Fig. 4, RNA a–d) in the RdRp reaction mixtures yielded a common major product of 2.2 kb, which was smaller than the input templates (Fig. 5A, lanes a–d). In these reactions, a second slow-moving band of up to twice the size of the RNA template was also present (Fig. 5A, lanes a–d). These results suggested that under the experimental conditions used RHDV RdRp could concomitantly perform a template-primed synthesis, giving rise to the minor double-sized products, and a de novo synthesis initiation at an internal promoter
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