Transcription of influenza A virus genes

Abstract

Background: Polyadenylation of eukaryotic mRNAs occurs by cleavage of the pre-mRNA downstream of a conserved AAUAAA hexamer, followed by polyadenylation of the upstream cleavage product by a poly(A) polymerase in a template-independent manner. By contrast, polyadenylation of influenza virus mRNA molecules is performed by the viral RNA polymerase by reiterative copying of a U 5–7 sequence near the 5′ end of the vRNA template. Methods: We used both in vitro and in vivo transcription assays to demonstrate that replacement of the viral U 6 poly(A) site with a A 6 sequence in vRNA results in transcription products with poly(U) tails. A recombinant influenza A/WSN/33 virus has been generated, by using a helper virus-dependent rescue method, in which the U 6 poly(A) site of the neuraminidase (NA) gene has been modified so that the virus expressed a poly(U)-tailed NA mRNA. The growth properties of the virus were characterised in cell culture and in an animal model. A plasmid-based transcription/replication assay was used to study whether influenza RNA polymerase transcripts are substrates for cleavage and polyadenylation by the eukaryotic 3′ end processing machinery. Results and Conclusions: Our studies show, firstly, that the U 5–7 sequence near the 5′ end of vRNA acts directly as a template for poly(A) addition. Secondly, we show that a recombinant virus expressing a poly(U)-tailed mRNA for the NA gene is attenuated in cell culture and in mice, suggesting a novel strategy for designing live attenuated influenza virus vaccines. Thirdly, we show that a poly(U)-tailed NA influenza RNA polymerase transcripts containing a eukaryotic poly(A) site, could be processed by the cellular polyadenylation machinery even although such molecules are not synthesized by the host RNA polymerase II.