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Generation of Influenza a Viruses Entirely from Cloned Cdnas Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9345–9350, August 1999 Microbiology Generation of influenza A viruses entirely from cloned cDNAs GABRIELE NEUMANN*, TOKIKO WATANABE*†,HIROSHI ITO*, SHINJI WATANABE*†,HIDEO GOTO*, PENG GAO*, MARK HUGHES*, DANIEL R. PEREZ‡,RUBEN DONIS‡,ERICH HOFFMANN§,GERD HOBOM§, AND YOSHIHIRO KAWAOKA*¶ *Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, 2015 Linden Drive West, Madison, WI 53706; †Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan; ‡Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, P.O. Box 830905, Lincoln, NE 68583-0905; and §Institut fu¨r Mikro- und Molekularbiologie, Frankfurter Strasse 107, 35392 Giessen, Germany Communicated by Paul Ahlquist, University of Wisconsin, Madison, WI, May 27, 1999 (received for review March 23, 1999) ABSTRACT We describe a new reverse-genetics system ruses, which contain six (Thogotovirus), seven (influenza C that allows one to efficiently generate influenza A viruses virus), or eight (influenza A and B viruses) segments of entirely from cloned cDNAs. Human embryonic kidney cells negative-sense RNA, have been produced entirely from cloned (293T) were transfected with eight plasmids, each encoding a cDNAs. This lag in progress has been felt most acutely in viral RNA of the A͞WSN͞33 (H1N1) or A͞PR͞8͞34 (H1N1) efforts to control influenza virus infections. virus, flanked by the human RNA polymerase I promoter and Palese and colleagues (13) pioneered the reverse-genetics, the mouse RNA polymerase I terminator—together with plas- helper virus-dependent system for influenza A virus (Fig. 1A). mids encoding viral nucleoprotein and the PB2, PB1, and PA In their approach, a ribonucleoprotein (RNP) complex is viral polymerases. This strategy yielded >1 ؋ 103 plaque- generated by in vitro vRNA synthesis in the presence of forming units (pfu) of virus per ml of supernatant at 48 hr purified polymerase and NP proteins and then used to trans- posttransfection. The addition of plasmids expressing all of fect eukaryotic cells. Subsequent infection with influenza A the remaining viral structural proteins led to a substantial helper virus results in the generation of viruses possessing a increase in virus production, 3 ؋ 104–5 ؋ 107 pfu͞ml. We also gene derived from cloned cDNA. A second method, developed used reverse genetics to generate a reassortant virus contain- by Neumann et al. (14), is based on the in vivo synthesis of ing the PB1 gene of the A͞PR͞8͞34 virus, with all other genes vRNA by RNA polymerase I (Fig. 1B), a cellular enzyme that representing A͞WSN͞33. Additional viruses produced by this transcribes ribosomal RNA that lacks both a 5Ј cap and a 3Ј method had mutations in the PA gene or possessed a foreign poly(A) tail. Transfection of cells with a plasmid containing epitope in the head of the neuraminidase protein. This effi- cloned influenza virus cDNAs, flanked by RNA polymerase I cient system, which does not require helper virus infection, promoter and terminator sequences, followed by influenza should be useful in viral mutagenesis studies and in the virus infection, led to the production of transfectant viruses. production of vaccines and gene therapy vectors. With both methods, however, transfectants must be selected from a vast background of helper viruses, which requires a The ability to generate infectious RNA viruses from cloned strong selection system and complicates the generation of cDNAs has contributed greatly to our biological understand- growth-defective viruses. ing of these pathogens and, hence, to improved methods of We report here the generation of influenza A viruses disease control (1). However, this progress had been relatively entirely from cloned cDNAs. The reverse-genetics approach limited for negative-sense as compared with positive-sense we describe is highly efficient and can be used to introduce RNA viruses, because neither the genomic viral RNA (vRNA) mutations into any gene segment and to develop influenza nor the antigenomic complementary RNA (cRNA) of nega- virus-based gene delivery systems. tive-sense RNA viruses can serve as a direct template for protein synthesis. Rather, the vRNA, after its encapsidation by MATERIALS AND METHODS viral nucleoprotein (NP), must be transcribed into positive- sense mRNA by the viral RNA polymerase complex. Thus, the Cells and Viruses. 293T human embryonic kidney cells and minimal replication unit is formed by the genomic vRNA Madin–Darby canine kidney cells (MDCK) were maintained complexed with NP and the polymerase proteins. Despite in DMEM supplemented with 10% FCS and in MEM con- these obstacles, reverse-genetics methods have been estab- taining 5% newborn calf serum, respectively. The 293T cell lished to produce nonsegmented, negative-sense RNA viruses, line is a derivative of 293, into which the gene for the including rabies virus (2), vesicular stomatitis virus (3, 4), temperature-sensitive simian virus 40 T antigen has been measles virus (5), respiratory syncytial virus (6), Sendai virus inserted. This line produces replication-competent T antigen (7, 8), rinderpest virus (9), human parainfluenza virus type 3 in large amounts at 37°C (15). All cells were maintained at ͞ ͞ (10), and simian virus 5 (11). 37°C in 5% CO2. Influenza viruses A WSN 33 (H1N1) and Generating segmented, negative-sense RNA viruses from A͞PR͞8͞34 (H1N1) were propagated in 10-day-old eggs. cloned cDNAs poses a more formidable challenge, as one must Construction of Plasmids. To generate RNA polymerase I produce a separate vRNA for each gene segment. In one study, constructs, we cloned cDNAs derived from A͞WSN͞33 or Bridgen and Elliott (12) produced a Bunyamwera virus (family A͞PR͞8͞34 viral RNA between the promoter and terminator Bunyaviridae) from cloned cDNAs encoding three segments of sequences of RNA polymerase I. Briefly, the cloned cDNAs negative-sense vRNA; however, the efficiency of virus recov- were amplified by PCR with primers containing BsmBI sites, ery was low, and there have been no reports of an engineered Bunyamwera mutant. By contrast, none of the orthomyxovi- Abbreviations: cRNA, complementary RNA; MDCK, Madin–Darby canine kidney; HA, hemagglutinin; NA, neuraminidase; NP, nucleo- The publication costs of this article were defrayed in part by page charge protein; pfu, plaque-forming units; RNP, ribonucleoprotein complex; VLP, virus-like particle; vRNA, viral RNA. payment. This article must therefore be hereby marked ‘‘advertisement’’ in A Commentary on this article begins on page 8804. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. E-mail: kawaokay@ PNAS is available online at www.pnas.org. svm.vetmed.wisc.edu. 9345 Downloaded by guest on September 25, 2021 9346 Microbiology: Neumann et al. Proc. Natl. Acad. Sci. USA 96 (1999) promoter and the mouse RNA polymerase I terminator, separated by BsmBI sites (Fig. 2). The PB2, PB1, PA, hem- agglutinin (HA), NP, neuraminidase (NA), M, and NS genes of the A͞WSN͞33 strain were PCR-amplified by use of the following plasmids: pSCWPB2, pGW-PB1, and pSCWPA (all obtained from Debi Nayak at the University of California, Los Angeles) and pWH17, pWNP152, pT3WNA15 (16), pGT3WM, and pWNS1, respectively. The PB1 gene of influenza A͞PR͞8͞34 virus was amplified by using pcDNA774(PB1) (17) as a template. The sequences of the primers will be provided on request. To ensure that the genes were free of unwanted mutations, we sequenced PCR-derived fragments with an autosequencer (Applied Biosystems) according to the protocol recommended by the manufacturer. The cDNAs encoding the HA, NP, NA, and M1 genes of A͞WSN͞33 virus were cloned as described (18) and subcloned into the eukary- otic expression vector pCAGGS͞MCS (controlled by the chicken ␤-actin promoter) (19), resulting in pEWSN-HA, pCAGGS-WSN-NP0͞14, pCAGGS-WNA15, and pCAGGS- WSN-M1–2͞1, respectively. The M2 and NS2 genes from the A͞PR͞8͞34 virus were amplified by PCR and cloned into pCAGGS͞MCS, yielding pEP24c and pCA-NS2. Finally, pcDNA774(PB1), pcDNA762(PB2), and pcDNA787(PA) were used to express the PB2, PB1, and PA proteins under control of the cytomegalovirus promoter, as reported else- where (17). Generation of Infectious Influenza Particles. 293T cells (1 ϫ 6 FIG. 1. Schematic diagram of established reverse-genetics systems. In 10 ) were transfected with a maximum of 17 plasmids in the RNP transfection method (A), purified NP and polymerase proteins different amounts (as described in Results), with the use of are assembled into RNPs with the use of in vitro-synthesized vRNA. Cells Trans IT LT-1 (Panvera, Madison, WI) according to the are transfected with RNPs, followed by helper virus infection. In the RNA manufacturer’s instructions. Briefly, DNA and transfection polymerase I method (B), a plasmid containing the RNA polymerase I reagent were mixed (2 ␮l of Trans IT LT-1 per ␮g of DNA), promoter, a cDNA encoding the vRNA to be rescued, and the RNA incubated at room temperature for 45 min, and added to the polymerase I terminator are transfected into cells. Intracellular transcrip- cells. Six hours later, the DNA–transfection reagent mixture tion by RNA polymerase I yields synthetic vRNA, which is packaged into ͞ progeny virus particles upon infection with helper virus. With both was replaced by Opti-MEM (GIBCO BRL) containing 0.3% methods, transfectant viruses (i.e.,
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