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Persistent and Stable Gene Expression by a Cytoplasmic RNA Replicon Based on a Noncytopathic Variant Sendai Virus

2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês

10.1074/jbc.m702028200

1083-351X

Ken Nishimura, Hiroaki Segawa, Takahiro Goto, Mariko Morishita, Akinori Masago, Hitoshi Takahashi, Yoshihiro Ohmiya, Takemasa Sakaguchi, Masahiro Asada, Toru Imamura, K Shimotono, Kozo Takayama, Tetsuya Yoshida, Mahito Nakanishi,

Mosquito-borne diseases and control

Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 × 104 copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications. Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 × 104 copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications. Delivery and ectopic expression of foreign genes in mammalian cells is now standard in modern biology. Although transient expression of delivered genes is usually sufficient for basic research purposes, their sustained expression is desirable in various medical and industrial applications. For example, persistent expression of therapeutic genes in tissue cells in situ is crucial for gene therapy of congenital metabolic diseases (1Verma I.M. Weitzman M.D. Annu. Rev. Biochem. 2005; 74: 711-738Crossref PubMed Scopus (489) Google Scholar). Stable and strong gene expression is also required for large scale production of recombinant proteins in cultured mammalian cells (2Wurm F.M. Nat. Biotechnol. 2004; 22: 1393-1398Crossref PubMed Scopus (1510) Google Scholar). Persistent expression of exogenous genes is achieved routinely by integrating them as a part of the host chromosome, either actively by using integrating viral vectors (1Verma I.M. Weitzman M.D. Annu. Rev. Biochem. 2005; 74: 711-738Crossref PubMed Scopus (489) Google Scholar) or transposable elements (3Izsvak Z. Ivics Z. Mol. Ther. 2004; 9: 147-156Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) or passively without any special molecular device. Whereas the former occurs much more efficiently, exogenous DNA elements are inserted into the random location on the host chromosome in either case. Chromosomal insertion has several drawbacks in practical applications of transgene expression. Because the number of expression cassettes integrated into a cell by a single transfection event is usually limited (2Wurm F.M. Nat. Biotechnol. 2004; 22: 1393-1398Crossref PubMed Scopus (1510) Google Scholar), multiplication of the target genes by laborious drug selection is sometimes required to maximize the gene expression in industrial purposes. In addition, random chromosomal insertion of exogenous genetic elements occasionally induces uncontrollable activation (or suppression) of endogenous genes, and this may lead to fatal side effects in medical applications. For example, in a recent gene therapy trial, the retroviral transfer of therapeutic genes into bone marrow stem cells induced malignant T-cell lymphoma through accidental activation of the LMO2 gene (4Nienhuis A.W. Dunbar C.E. Sorrentino B.P. Mol. Ther. 2006; 13: 1031-1049Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Installing exogenous genes on stable autonomous DNA replicons, such as circular episomal DNA and linear human artificial chromosomes, may avoid these problems (5Jackson D.A. Juranek S. Lipps H.J. Mol. Ther. 2006; 14: 613-626Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), although the significance of these replicons in medical and industrial applications is not yet established. Recombinant adeno-associated virus vectors also frequently maintain their genome as an episome in the nucleus, but the mechanism underlying this persistence and the long term stability in the cells have not been established (1Verma I.M. Weitzman M.D. Annu. Rev. Biochem. 2005; 74: 711-738Crossref PubMed Scopus (489) Google Scholar). Sendai virus (SeV) 2The abbreviations used are:SeVSendai virusDIdefective-interferingIFNβinterferon βIRF-3interferon regulatory factor 3CHOChinese hamster ovaryEGFPenhanced green fluorescent proteinCLucC. noctiluca luciferasePG-FGF-1fibroblast growth factor-proteoglycan fusion proteinMOImultiplicity of infectionPIPES1,4-piperazinediethanesulfonic acidntnucleotide(s)RIG-1retinoic acid-inducible geneDAPI4′,6′-diamino-2-phenylindole 2The abbreviations used are:SeVSendai virusDIdefective-interferingIFNβinterferon βIRF-3interferon regulatory factor 3CHOChinese hamster ovaryEGFPenhanced green fluorescent proteinCLucC. noctiluca luciferasePG-FGF-1fibroblast growth factor-proteoglycan fusion proteinMOImultiplicity of infectionPIPES1,4-piperazinediethanesulfonic acidntnucleotide(s)RIG-1retinoic acid-inducible geneDAPI4′,6′-diamino-2-phenylindole is a nonsegmented negative strand RNA virus belonging to the Paramyxoviridae (6Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Fundamental Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia, PA2001: 689-724Google Scholar). It has a single-strand RNA genome (15,384 nucleotides) encoding eight proteins (NP, P, C, V, M, F, HN, and L). Replication and transcription of SeV occurs in the cytoplasm with extremely low cell specificity and species specificity, although SeV is neither pathogenic nor carcinogenic in human beings. These characteristics make it a candidate for producing harmless viral vectors suitable for medical and industrial applications. Most recombinant SeV vectors developed by reverse genetics have genetic back-grounds of wild-type cytopathic SeV strains (Z and Fushimi) (7Bitzer M. Armeanu S. Lauer U.M. Neubert W.J. J. Gene Med. 2003; 5: 543-553Crossref PubMed Scopus (61) Google Scholar, 8Griesenbach U. Inoue M. Hasegawa M. Alton E.W. Curr. Opin. Mol. Ther. 2005; 7: 346-352PubMed Google Scholar). The replication-competent first generation vectors were prepared from the full-length viral genome cDNA with inserted exogenous genes. The replication-defective second generation vectors were prepared from the cDNA by deletion of essential viral genes. These vectors have characteristics reflecting those of the parental SeV strains (efficient gene transduction and strong gene expression, with low cell specificity and species specificity), and a gene therapy trial using a second generation SeV vector has been approved recently in Japan. However, none of these vectors could sustain gene expression for more than 10 days because of the cytopathic nature of the parental SeV strain. Extensive deletion and alteration of the viral genes should reduce the cytotoxicity to some extent (9Yoshizaki M. Hironaka T. Iwasaki H. Ban H. Tokusumi Y. Iida A. Nagai Y. Hasegawa M. Inoue M. J. Gene Med. 2006; 8: 1151-1159Crossref PubMed Scopus (36) Google Scholar), but no SeV vector has been reported to express foreign genes stably in a wide variety of cells. Sendai virus defective-interfering interferon β interferon regulatory factor 3 Chinese hamster ovary enhanced green fluorescent protein C. noctiluca luciferase fibroblast growth factor-proteoglycan fusion protein multiplicity of infection 1,4-piperazinediethanesulfonic acid nucleotide(s) retinoic acid-inducible gene 4′,6′-diamino-2-phenylindole Sendai virus defective-interfering interferon β interferon regulatory factor 3 Chinese hamster ovary enhanced green fluorescent protein C. noctiluca luciferase fibroblast growth factor-proteoglycan fusion protein multiplicity of infection 1,4-piperazinediethanesulfonic acid nucleotide(s) retinoic acid-inducible gene 4′,6′-diamino-2-phenylindole In this article, we describe a novel RNA replicon based on a noncytopathic persistent variant SeV strain. We found that this unique virus strain established stable symbiosis with host cells by escaping from the antiviral reaction mediated by interferon regulatory factor 3 (IRF-3). This remarkable characteristic was maintained even after exogenous marker genes had been inserted into the virus genome, thus enabling us to express the marker genes stably for more than 6 months. We discuss the potential of this novel cytoplasmic RNA replicon in medical and industrial applications. Viruses and Cells—Sendai virus was grown in 10-day-old fertilized chicken eggs at 35.5 °C for 3 days (Z and Nagoya 1-60 strain, GenBank™ accession number AB275417) or at 32 °C for 5 days (Cl.151 strain, GenBank™ accession number AB275416) and was purified by sucrose step gradient centrifugation as described (10Kato K. Nakanishi M. Kaneda Y. Uchida T. Okada Y. J. Biol. Chem. 1991; 266: 3361-3364Abstract Full Text PDF PubMed Google Scholar). Vaccinia virus MVAGKT7 (provided by Dr. G. R. Kovacs) was propagated using chicken embryo fibroblasts as described (11Kovacs G.R. Parks C.L. Vasilakis N. Udem S.A. J. Virol. Methods. 2003; 111: 29-36Crossref PubMed Scopus (25) Google Scholar). LLCMK2 and CV-1 cells were cultured in Eagle's minimum essential medium, COS-1 and NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium, Chinese hamster ovary (CHO)-K1 cells were cultured in Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1), and U937 cells were cultured in RPMI 1640 medium at 37 °C in an atmosphere containing 5% CO2. Each medium was supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). cDNA Cloning—All of the recombinant DNA experiments were performed according to the guidelines of the Institutional Recombinant DNA Experiment Committee of the National Institute of Advanced Industrial Science and Technology. Genomic RNA of SeV was isolated from the purified virion as described (12Kondo T. Yoshida T. Miura N. Nakanishi M. J. Biol. Chem. 1993; 268: 21924-21930Abstract Full Text PDF PubMed Google Scholar). First strand cDNA corresponding to the SeV genome RNA was synthesized using the Superscript III first strand synthesis system (Invitrogen) according to the manufacturer's protocol. Double-stranded genome cDNA was cloned into pBluescript II SK (+) (Stratagene, La Jolla, CA) as three overlapping clones (nucleotide numbers 1-2875, 2870-10484, and 10479-15384) after PCR amplification using Pfu Ultra high fidelity DNA polymerase (Stratagene). The nucleotide sequences of four independent SeV cDNA clones were determined by Takara Bio Inc. (Otsu, Japan). For constructing full-length genome cDNA of the Z strain, pSeV (+) (provided by Dr. A. Kato) was used as a template for PCR. A full-length SeV genome cDNA was finally assembled on λ DASH II (Stratagene). The primers used in this article are summarized in supplemental Table S2, and the detailed procedure of the cloning and assembly of the full-length SeV genome cDNA is described in supplemental Fig. S1. To construct the SeV vector, marker genes were inserted into a unique NotI site created in SeV genomic cDNA as described (13Hasan M.K. Kato A. Shioda T. Sakai Y. Yu D. Nagai Y. J. Gen. Virol. 1997; 78: 2813-2820Crossref PubMed Scopus (87) Google Scholar). cDNAs encoding enhanced green fluorescent protein (EGFP), Cypridina noctiluca luciferase (CLuc), and fibroblast growth factor 1-proteoglycan fusion protein (PG-FGF-1) were amplified by PCR to add the transcription initiation signal (S) and termination signal (T), using pEGFP-1 (Clontech, Mountain View, CA), pCLm (14Nakajima Y. Kobayashi K. Yamagishi K. Enomoto T. Ohmiya Y. Biosci. Biotechnol. Biochem. 2004; 68: 565-570Crossref PubMed Scopus (91) Google Scholar), and pMex-RyuII (15Yoneda A. Asada M. Oda Y. Suzuki M. Imamura T. Nat. Biotechnol. 2000; 18: 641-644Crossref PubMed Scopus (33) Google Scholar) as templates, respectively. The detailed procedure for the construction of the vector cDNA with marker genes is described in supplemental Fig. S2. Rescue of Recombinant SeV by Reverse Genetics—Recombinant SeV and the derivatives were rescued essentially as described (11Kovacs G.R. Parks C.L. Vasilakis N. Udem S.A. J. Virol. Methods. 2003; 111: 29-36Crossref PubMed Scopus (25) Google Scholar). LLCMK2 cells (1 × 106 cells/well) in 6-well plates were infected with MVAGKT7 at a multiplicity of infection (MOI) of 10 plaque-forming units/cell and incubated at 37 °C for 1 h. The culture medium was removed, and the cells were supplemented with 2 ml of fresh medium and transfected with SeV genome cDNA. A complex of Lipofectamine 2000 (Invitrogen) (10 μl), λ phage DNA encoding SeV genomic cDNA (5 μg), pGEM-NP (2 μg), pGEM-P (1 μg), and pGEM-L (2 μg) (pGEM-NP, -P, and -L were provided by Dr. D. Kolakofsky) was prepared according to the protocol provided by the supplier, added to each well of cell culture, and incubated for 4 h at 37 °C. The complex was removed, and the cells were incubated in fresh medium for 20 h at 32 or 37 °C. A cell lysate was prepared and inoculated into 10-day-old fertilized chicken eggs as described (13Hasan M.K. Kato A. Shioda T. Sakai Y. Yu D. Nagai Y. J. Gen. Virol. 1997; 78: 2813-2820Crossref PubMed Scopus (87) Google Scholar). The eggs were incubated at 35.5 °C for 3 days (Z and Nagoya strains and the derivatives) or at 32 °C for 5 days (Cl.151 strain and the derivatives), after which the viruses were recovered from the allantoic fluid. The titer of SeV in allantoic fluid was determined by measuring hemagglutinating activity (10Kato K. Nakanishi M. Kaneda Y. Uchida T. Okada Y. J. Biol. Chem. 1991; 266: 3361-3364Abstract Full Text PDF PubMed Google Scholar). The recombinant virus seeds were propagated in fertilized chicken eggs once and then purified using sucrose step density centrifugation as described (10Kato K. Nakanishi M. Kaneda Y. Uchida T. Okada Y. J. Biol. Chem. 1991; 266: 3361-3364Abstract Full Text PDF PubMed Google Scholar). The presence of defective-interfering (DI) genomic RNA in the virus preparation was determined as described (16Strahle L. Garcin D. Kolakofsky D. Virology. 2006; 351: 101-111Crossref PubMed Scopus (159) Google Scholar). Cytotoxic Assay—Because the ratio of plaque-forming units and viral particles differed significantly between different SeV strains and because Cl.151 did not make plaques at 37 °C, we used the 50% tissue culture infectivity dose (TCID50) as an index of viral infectivity. This was determined from the ratio of the cells expressing NP protein in the culture examined with an indirect immunofluorescent assay using a mouse anti-NP monoclonal antibody as described (17Eguchi A. Kondoh T. Kosaka H. Suzuki T. Momota H. Masago A. Yoshida T. Taira H. Ishii-Watabe A. Okabe J. Hu J. Miura N. Ueda S. Suzuki Y. Taki T. Hayakawa T. Nakanishi M. J. Biol. Chem. 2000; 275: 17549-17555Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). One TCID50 unit of SeV suspension of the Cl.151 and Z strains corresponds to 1.25 and 0.125 pg of viral protein/cell, respectively. Cytotoxicity was determined using a cytotoxicity detection kit (Roche Applied Science) that measures lactose dehydrogenase activity as described (9Yoshizaki M. Hironaka T. Iwasaki H. Ban H. Tokusumi Y. Iida A. Nagai Y. Hasegawa M. Inoue M. J. Gene Med. 2006; 8: 1151-1159Crossref PubMed Scopus (36) Google Scholar). Detection of EGFP—To measure EGFP production in cultured cells, the cells were fixed with 2% paraformaldehyde at room temperature for 10 min and mounted in VECTASHIELD with 4′,6′-diamino-2-phenylindole HCl (Vector Laboratories, Burlingame, CA). The EGFP signal was then examined by fluorescence microscopy as described (18Fujita S. Eguchi A. Okabe J. Harada A. Sasaki K. Ogiwara N. Inoue Y. Ito T. Matsuda H. Kataoka K. Kato A. Hasegawa M. Nakanishi M. Biol. Pharm. Bull. 2006; 29: 1728-1734Crossref PubMed Scopus (10) Google Scholar). Animal Use—All of the animal experiments were performed according to the regulations of the Institutional Animal Care and Use Committee of the Advanced Industrial Science and Technology with permission of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Eight-week-old male Wistar rats were fasted for 24 h and then anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg; Dainippon Sumitomo Pharma, Osaka, Japan). The animals were restrained in a supine position on a thermocontrolled sheet to maintain body temperature during the experiment. Three hundred micrograms (protein) of SeV vector suspended in 0.3 ml of buffered salt solution (150 mm NaCl, 10 mm Tris-HCl, 1 mm CaCl2, pH 7.4) was administered directly through a 3-cm-long tube through the anus. The animals were euthanized at the times described in Fig. 2B, and tissue specimens were prepared and observed as described (19Goto T. Morishita M. Nishimura K. Nakanishi M. Kato A. Ehara J. Takayama K. Pharm. Res. 2006; 23: 384-391Crossref PubMed Scopus (44) Google Scholar), except that the thickness of the transverse section was 20 μm. Determination of Secreted Marker Proteins—CLuc activity was determined as described (14Nakajima Y. Kobayashi K. Yamagishi K. Enomoto T. Ohmiya Y. Biosci. Biotechnol. Biochem. 2004; 68: 565-570Crossref PubMed Scopus (91) Google Scholar) with modification: 50 μl of filtrated culture medium was mixed with 50 μl of C. noctiluca luciferin (ATTO, Tokyo, Japan; 216 nm in 10 mm Tris-HCl, pH 7.4) at room temperature, and the emission was determined with a luminometer (Aloka, Tokyo, Japan). The amount of C. noctiluca luciferase was quantified using purified enzyme (ATTO) as a standard. To express PG-FGF-1 from the conventional DNA-based vector, the full-length PG-FGF-1 cDNA was cloned into the EcoRI/NotI site of pMKIT-neo (provided by Dr. K. Maruyama) to create pMKIT-neo-PG-FGF-1, in which the cDNA was driven by the strong SRα promoter (20Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). CHO cells (6.5 × 105 cells/well with 0.8 ml medium) were cultured in 12-well plates for 8 h. The cells were transfected with the complex of 2 μg of pMKIT-neo-PG-FGF-1 and 4 μl of Lipofectamine 2000 prepared in 200 μl of Opti-MEM according to the protocol provided by the supplier. The cells were incubated for 14 h, the medium was replaced with 1 ml of Opti-MEM, and the cells were incubated for a further for 48 h for transient expression. Under this condition, about 80% of the cells expressed the marker gene. To ensure stable expression, transfected cells were selected further with G418 (800 μg/ml), and a clonal cell line expressing the highest level of PG-FGF-1 was cultured for 48 h as described above. PG-FGF-1 was determined using an enzyme-linked immunosorbent assay as described (15Yoneda A. Asada M. Oda Y. Suzuki M. Imamura T. Nat. Biotechnol. 2000; 18: 641-644Crossref PubMed Scopus (33) Google Scholar). Interferon β Promoter Activation Assay—A 172-bp KpnI/HindIII fragment containing the 140-bp human interferon β (IFNβ) promoter (from -119 bp to +21 bp, +1 = transcription start site) was excised from the pGL3-IFNβ-promoter-luc (provided by Dr. A. Matsuda) and inserted into a KpnI/HindIII site of pGL4.12 (Promega, Madison, WI) to create pIV3. Nineteen micrograms of pIV3 and 1 μg of pRSVHyg were cotransfected into LLCMK2 cells with DOTAP transfection reagent (Roche Applied Science) according to the manufacturer's protocol. One of the hygromycin-resistant clones (LLCMK2/pIV3/clone 16) containing the complete IFNβ promoter-luciferase transcription unit was used in this study. Luciferase activity was determined using a luciferase assay system (Promega), and protein concentration was measured using a BCA assay kit (Pierce). The IFNβ promoter was activated either by virus infection or by transfecting with a 147-nt 5′-triphosphate single-stranded RNA synthesized in vitro using a RiboMAX Large Scale RNA production system-T7 (Promega) (21Hornung V. Ellegast J. Kim S. Brzozka K. Jung A. Kato H. Poeck H. Akira S. Conzelmann K.K. Schlee M. Endres S. Hartmann G. Science. 2006; 314: 994-997Crossref PubMed Scopus (1863) Google Scholar, 22Pichlmair A. Schulz O. Tan C.P. Naslund T.I. Liljestrom P. Weber F. Reis e Sousa C. Science. 2006; 314: 997-1001Crossref PubMed Scopus (1745) Google Scholar) with the aid of Lipofectamine 2000 (Invitrogen) or by adding poly(rI): poly(rC) (Sigma-Aldrich) to the culture medium (23Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3082) Google Scholar) as described in the figure legend. LLCMK2/pIV3/16/RIG-IC/clone 20 was the cell line derived from LLCMK2/pIV3/clone 16 and expressed RIG-IC (dominant-negative form of RIG-I) (23Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3082) Google Scholar) stably. This cell line was established by cotransfecting a RIG-IC expression cassette pNH63-8 (Flag-RIG-IC cDNA driven by CAG promoter) pRSV-Neo and selected by G-418 (1600 μg/ml) as described above. Determination of Viral RNA—Total cellular RNA was purified with ISOGEN (Nippon Gene, Tokyo, Japan) according to the supplier's protocol. Copy numbers of Cluc mRNA and SeV genome RNA in the cells were determined by a quantitative S1 nuclease assay using a double-strand DNA probe, which was 32P-labeled at the 5′ end with [γ-32P]ATP (185 TBq/mmol), as described (24Murray M.G. Anal. Biochem. 1986; 158: 165-170Crossref PubMed Scopus (111) Google Scholar). Details of the S1 assay, including the structure of the probes against Cluc mRNA, SeV genomic RNA, and monkey β-actin mRNA, are described in supplemental Fig. S3. Five or 10 μg of total RNA was hybridized with 2 fmol each of the Cluc probe and monkey β-actin probe or with 2 fmol each of SeV genome probe and monkey β-actin probe in 10 μl of hybridization buffer (3 m sodium trichloroacetate, 50 mm PIPES-NaOH, 5 mm EDTA, pH 7.0) at 45 °C for 16 h. The mixture was digested with S1 nuclease (400 units, in 200 μl of 250 mm NaCl, 40 mm sodium acetate, 1 mm ZnCl2, pH 5.5) at 37 °C for 60 min. Digested materials were recovered with ethanol precipitation and analyzed on a prewarmed, denatured 5% polyacrylamide gel containing 8 m urea. Signals corresponding to the protected probes were quantified using the STORM 830 image analyzer (Molecular Dynamics, Sunnyvale, CA). The results were normalized against monkey β-actin mRNA, and the copy numbers were estimated from the signal of predetermined single-strand RNA synthesized in vitro using a Ribo-MAX large scale RNA production system-T7 (Promega) according to the supplier's protocol. Characterization of the Genome Structure of SeV Clone 151 Strain—A noncytopathic persistent variant SeV strain Cl.151 (25Yoshida T. Nagai Y. Maeno K. Iinuma M. Hamaguchi M. Matsumoto T. Nagayoshi S. Hoshino M. Virology. 1979; 92: 139-154Crossref PubMed Scopus (73) Google Scholar) was isolated originally from the parental cytopathic Nagoya strain (26Matsumoto T. Nagata I. Kariya Y. Ohashi K. Nagoya J. Med. Sci. 1954; 17: 93-97Google Scholar, 27Matsumoto T. Yamamoto N. Maeno K. J. Immunol. 1961; 87: 590-598PubMed Google Scholar) as a variant that caused stable persistent infection in baby hamster kidney cells at a nonpermissive temperature (38 °C). Cl.151 can establish persistent infection even in sensitive CV-1 cells without cytopathic effects (Fig. 1C). We showed previously that amino acid substitutions in the M protein of Cl.151 lead to temperature-sensitive virion production (12Kondo T. Yoshida T. Miura N. Nakanishi M. J. Biol. Chem. 1993; 268: 21924-21930Abstract Full Text PDF PubMed Google Scholar). Defective virion production might be responsible for the reduced cytotoxicity because accumulation of unassembled NP protein affects the relative levels of transcription and replication (6Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Fundamental Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia, PA2001: 689-724Google Scholar, 28Gubbay O. Curran J. Kolakofsky D. J. Gen. Virol. 2001; 82: 2895-2903Crossref PubMed Scopus (56) Google Scholar). However, the recombinant M-deleted SeV is still highly cytopathic, although it is defective in virion production, as is Cl.151 (29Inoue M. Tokusumi Y. Ban H. Shirakura M. Kanaya T. Yoshizaki M. Hironaka T. Nagai Y. Iida A. Hasegawa M. J. Gene Med. 2004; 6: 1069-1081Crossref PubMed Scopus (31) Google Scholar). Therefore, SeV injures the infected cells by a mechanism that is independent of virus morphogenesis. To uncover the mechanism underlying the noncytopathic persistent nature of Cl.151, we cloned a cDNA corresponding to the full-length genome RNA of Cl.151 and characterized it in comparison with that of the parental Nagoya strain. Although no deletion or insertion was identified, we found 50 nucleotide substitutions throughout the viral genome, 35 of which led to the amino acid substitutions (Fig. 1A and supplemental Table S1). Because the characteristics of different SeV strains are affected largely by external factors such as the presence of DI genome RNA, we then characterized the recombinant virus recovered from the cloned cDNA by reverse genetics to examine whether these substitutions alone were responsible and sufficient for exhibiting the phenotype of Cl.151. Recombinant SeV was recovered from the full-length genome cDNA by converting it to the antigenome RNA in the cytoplasm with the aid of T7 RNA polymerase. Simultaneous expression of NP, P, and L proteins in the cell encapsulates the de novo synthesized antigenome RNA and initiates the virus replication in the cytoplasm (30Kato A. Sakai Y. Shioda T. Kondo T. Nakanishi M. Nagai Y. Genes Cells. 1996; 1: 569-579Crossref PubMed Scopus (212) Google Scholar). One of the drawbacks of the original procedure is that full-length SeV genome cDNA (15384 bp) cloned into plasmid vectors was unstable in bacterial cells and frequently caused partial deletion during amplification (data not shown). To overcome this problem, we reconstructed the full-length SeV genome cDNAs on a nonlysogenic λ phage vector, which maintained the long SeV cDNA more stably in Escherichia coli than did plasmid vectors (Fig. 1B and supplemental Fig. S1). Antigenome RNA is transcribed from this cDNA under the control of the T7 promoter and is trimmed precisely by hairpin ribozyme derived from the tobacco ringspot virus (31Hampel A. Tritz R. Biochemistry. 1989; 28: 4929-4933Crossref PubMed Scopus (334) Google Scholar). We recovered virus at 32 °C, but not at 37 °C (the standard temperature for recovering wild-type SeV) from Cl.151 genomic cDNA; this is consistent with the temperature-sensitive replication of Cl.151. In addition, this recombinant SeV had characteristics indistinguishable from the original Cl.151: it infected various cultured cells persistently at nonpermissive temperature (≥37 °C) without cytopathic effects (Fig. 1C), and virion production occurred only at the permissive temperature (32 °C) (data not shown). We conclude that this cloned cDNA contains all the genetic information necessary and sufficient for reproducing the characteristics of Cl.151. Mechanism Underlying the Noncytopathic Phenotype of SeV Cl.151 Strain—Wild-type SeV induces apoptotic death in infected cells. This phenomenon is triggered by caspases 3, 8, and 9 (32Bitzer M. Armeanu S. Prinz F. Ungerechts G. Wybranietz W. Spiegel M. Bernlohr C. Cecconi F. Gregor M. Neubert W.J. Schulze-Osthoff K. Lauer U.M. J. Biol. Chem. 2002; 277: 29817-29824Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 33Bitzer M. Prinz F. Bauer M. Spiegel M. Neubert W.J. Gregor M. Schulze-Osthoff K. Lauer U. J. Virol. 1999; 73: 702-708Crossref PubMed Google Scholar), which are induced as one of the IRF-3-mediated antiviral responses, which include IFNβ induction (34Heylbroeck C. Balachandran S. Servant M.J. DeLuca C. Barber G.N.