Introduction

Scientists faced several challenges in generating influenza viruses from plasmids. The genome of influenza viruses comprises 8 (influenza A and B viruses) or 7 (influenza C viruses) negative-strand viral RNAs (vRNAs), all of which are required to generate functional viruses. The negative-strand vRNA is not a template for translation and is therefore noninfectious; hence, the components of the viral replication machinery (i.e., the three polymerase subunits PB2, PB1, and PA, and the nucleoprotein NP) are required for viral replication and transcription. Thus, to artificially generate influenza A viruses, 12 components are needed—eight to synthesize the eight vRNAs, and four to synthesize the polymerase and NP proteins. These components must be delivered into the nucleus, the site of influenza virus replication and transcription. In addition, the artificially generated vRNAs need to mimic authentic influenza vRNAs, which do not possess 5'-cap and 3'-polyA structures. This is achieved by inclusion of the RNA polymerase I

Yoshihiro Kawaoka and Gabriele Neumann (eds.), Influenza Virus: Methods and Protocols,

Methods in Molecular Biology, vol. 865, DOI 10.1007/978-1-61779-621-0_12, © Springer Science+Business Media, LLC 2012

Fig. 1. Artificial generation of influenza viruses (reverse genetics). Cells are transfected with eight plasmids for the transcription of the eight viral RNAs (vRNAs), and with four plasmids for the expression of the polymerase and NP proteins. The polymerase and NP proteins amplify the transcribed vRNAs, resulting in the expression of all of the viral proteins and the amplification of the vRNAs, leading to influenza viruses. Figure modified after Neumann and Kawaoka, 2001, Virology 287:243-250.

Fig. 1. Artificial generation of influenza viruses (reverse genetics). Cells are transfected with eight plasmids for the transcription of the eight viral RNAs (vRNAs), and with four plasmids for the expression of the polymerase and NP proteins. The polymerase and NP proteins amplify the transcribed vRNAs, resulting in the expression of all of the viral proteins and the amplification of the vRNAs, leading to influenza viruses. Figure modified after Neumann and Kawaoka, 2001, Virology 287:243-250.

transcription system, in which influenza viral cDNAs are inserted between the RNA polymerase I promoter and terminator sequences (1). RNA polymerase I is an abundant nuclear enzyme that transcribes non-capped and non-polyadenylated ribosomal RNAs. This transcription system thus allows the artificial generation of authentic influenza vRNAs in the nucleus of plasmid-transfected cells. The PB2, PB1, PA, and NP proteins are provided by standard RNA polymerase Il-driven protein expression plasmids (1) (Fig. 1).

This basic approach is described in detail in this chapter. Variations of this theme include (1) vectors that produce both the vRNA and mRNA from one template ( 2); (2) vectors that combine RNA polymerase I transcription units (3); (3) an adeno-virus system for cell transduction, rather than plasmid transfection (4); and (4) a T7 RNA polymerase transcription system that uses this enzyme and a ribozyme sequence for vRNA synthesis ( 5). These variations are briefly described in Subheading 4.

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