Generation of influenza A viruses entirely from cloned cDNAs

G Neumann

T Watanabe

H Ito

S Watanabe

R Donis+3 authors

^{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 für Mikro- und Molekularbiologie, Frankfurter Strasse 107, 35392 Giessen, Germany}

^{To whom reprint requests should be addressed. E-mail:}ude.csiw.demtev.mvs@yakoawak.

Communicated by Paul Ahlquist, University of Wisconsin, Madison, WI

Received 1999 Mar 23; Accepted 1999 May 27.

Abstract

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 10^{plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 10}^{–5 × 10}^{pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.}

Abstract

The ability to generate infectious RNA viruses from cloned cDNAs has contributed greatly to our biological understanding of these pathogens and, hence, to improved methods of disease control (1). However, this progress had been relatively limited for negative-sense as compared with positive-sense RNA viruses, because neither the genomic viral RNA (vRNA) nor the antigenomic complementary RNA (cRNA) of negative-sense RNA viruses can serve as a direct template for protein synthesis. Rather, the vRNA, after its encapsidation by viral nucleoprotein (NP), must be transcribed into positive-sense mRNA by the viral RNA polymerase complex. Thus, the minimal replication unit is formed by the genomic vRNA complexed with NP and the polymerase proteins. Despite these obstacles, reverse-genetics methods have been established to produce nonsegmented, negative-sense RNA viruses, including rabies virus (2), vesicular stomatitis virus (3, 4), measles virus (5), respiratory syncytial virus (6), Sendai virus (7, 8), rinderpest virus (9), human parainfluenza virus type 3 (10), and simian virus 5 (11).

Generating segmented, negative-sense RNA viruses from cloned cDNAs poses a more formidable challenge, as one must produce a separate vRNA for each gene segment. In one study, Bridgen and Elliott (12) produced a Bunyamwera virus (family Bunyaviridae) from cloned cDNAs encoding three segments of negative-sense vRNA; however, the efficiency of virus recovery was low, and there have been no reports of an engineered Bunyamwera mutant. By contrast, none of the orthomyxoviruses, which contain six (Thogotovirus), seven (influenza C virus), or eight (influenza A and B viruses) segments of negative-sense RNA, have been produced entirely from cloned cDNAs. This lag in progress has been felt most acutely in efforts to control influenza virus infections.

Palese and colleagues (13) pioneered the reverse-genetics, helper virus-dependent system for influenza A virus (Fig. (Fig.11A). In their approach, a ribonucleoprotein (RNP) complex is generated by in vitro vRNA synthesis in the presence of purified polymerase and NP proteins and then used to transfect eukaryotic cells. Subsequent infection with influenza A helper virus results in the generation of viruses possessing a gene derived from cloned cDNA. A second method, developed by Neumann et al. (14), is based on the in vivo synthesis of vRNA by RNA polymerase I (Fig. (Fig.11B), a cellular enzyme that transcribes ribosomal RNA that lacks both a 5′ cap and a 3′ poly(A) tail. Transfection of cells with a plasmid containing cloned influenza virus cDNAs, flanked by RNA polymerase I promoter and terminator sequences, followed by influenza virus infection, led to the production of transfectant viruses. With both methods, however, transfectants must be selected from a vast background of helper viruses, which requires a strong selection system and complicates the generation of growth-defective viruses.

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Figure 1

Schematic diagram of established reverse-genetics systems. In the RNP transfection method (A), purified NP and polymerase proteins are assembled into RNPs with the use of in vitro-synthesized vRNA. Cells are transfected with RNPs, followed by helper virus infection. In the RNA polymerase I method (B), a plasmid containing the RNA polymerase I promoter, a cDNA encoding the vRNA to be rescued, and the RNA polymerase I terminator are transfected into cells. Intracellular transcription by RNA polymerase I yields synthetic vRNA, which is packaged into progeny virus particles upon infection with helper virus. With both methods, transfectant viruses (i.e., those containing RNA derived from cloned cDNA) are selected from the helper virus population.

We report here the generation of influenza A viruses entirely from cloned cDNAs. The reverse-genetics approach we describe is highly efficient and can be used to introduce mutations into any gene segment and to develop influenza virus-based gene delivery systems.

293T cells were transfected with eight RNA polymerase I plasmids encoding A/WSN/33 virus genes (Experiments 1 and 2) or with these plasmids except the PB1 gene, which was derived from A/PR/8/34 virus (Experiments 3 and 4), and only PA, PB1, PB2, and NP (Experiment 1) or nine (Experiments 2–4) protein expression plasmids as described in the text. At different time points, we titrated virus in the culture supernatant in MDCK cells. ND, not determined.

Acknowledgments

We thank Krisna Wells and Martha McGregor for excellent technical assistance and John Gilbert for editing the manuscript. We are also grateful to Dr. Debi Nayak for WSN polymerase plasmids, Dr. A. James Cooley for immunostaining pictures, and Yuko Kawaoka for illustrations. Automated sequencing was performed at the University of Wisconsin–Biotechnology Center. Support for this work came from National Institute of Allergy and Infectious Diseases Public Health Service research grants.

Acknowledgments

ABBREVIATIONS

cRNA	complementary RNA
MDCK	Madin–Darby canine kidney
HA	hemagglutinin
NA	neuraminidase
NP	nucleoprotein
pfu	plaque-forming units
RNP	ribonucleoprotein complex
VLP	virus-like particle
vRNA	viral RNA

ABBREVIATIONS

Footnotes

A Commentary on this article begins on page 8804.

Footnotes

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