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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 U5-7 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 U5-7 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 gone 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. (C) 2001 Elsevier Science B.V. All rights reserved.

Type

Conference paper

Publication Date

2001

Volume

1219

Pages

427 - 434

Keywords

VACCINE, MESSENGER-RNA, IN-VITRO, POLYADENYLATION, RNA-POLYMERASE-II, POLY(A) TAIL, SEQUENCES, A VIRUS, VIRION RNA, poly(U)-tail, 3' end processing, attenuated virus