Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes

Abstract

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the fulllength negative-strand RNA of the CAT gene positioned between the 5'-and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexjamethasone.

Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system

“…2), reporter gene activity was only detected if the CAT RNA was supplied as an in vitro assembled RNP, as reported by Luytjes et al (1989). However, in the expression system reported here and in those reported by Kimura et al (1992) and de la Luna et al (1993), naked RNAs were efficiently transcribed in vivo by the influenza virus replicase. These results contrast with the expression system based on four vaccinia virus recombinants (Huang et al, 1990) in which naked RNA was not expressed.…”

“…It was therefore concluded that CAT activity detected in the cell extracts was mediated by influenza virus components expressed from the cloned viral genes. Therefore, the NP and the three subunits of the influenza virus polymerase are the minimum set of proteins required for expression of the influenza virus genome, as shown previously (Huang et al, 1990;Kimura et al, 1992;de la Luna et al, 1993).…”

“…In the last few years however, several such systems have been described for members of the Orthomyxoviridae (Huang et al, 1990;Kimura et al, 1992;de la Luna et al, 1993), Rhabdoviridae (Pattnaik & Wertz, 1990;Pattnaik et al, 1992), Paramyxoviridae (Curran et al, 1991 ;Calain et al, 1992) and Bunyaviridae (Jin & Elliot, 1991) families. Thus, it has been possible to define the cis-acting RNA regions and viral proteins required for virus-specific RNA synthesis.…”

“…In the system described by Huang et al (1990) the influenza virus proteins were supplied through infection with four vaccinia virus recombinants, each containing one of four influenza virus genes. Kimura et al (1992) reported functional expression of the influenza virus polymerase in a cell line that expresses the proteins in response to dexamethasone. In the system described by de la Luna et al (1993) the viral proteins were provided by co-infection of cell cultures with a set of four simian virus (SV) 40 recombinant viruses.…”

“…Three systems where expression of synthetic influenza virus-like RNAs was driven by influenza virus proteins expressed from cloned cDNAs have been described (Huang et al, 1990;Kimura et al, 1992;de la Luna et al, 1993). In all three cases, the influenza virus nucleoprotein (NP), PA, PB1 and PB2 polypeptides were 0001-2292 © 1994 SGM with specific monoelonal antibodies raised against the NP, PA and PB2 polypeptides, or with a control monoclonal antibody (C) as indicated, the minimum set of viral components required for efficient expression of synthetic RNAs.…”