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Overlapping signals for translational regulation and packaging of influenza A virus segment 2

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  • H.M. Wise
  • C. Barbezange
  • B.W. Jagger
  • R.M. Dalton
  • J.R. Gog
  • M.D. Curran
  • J.K. Taubenberger
  • E.C. Anderson
  • P. Digard

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http://nar.oxfordjournals.org/content/39/17/7775
Original languageUndefined/Unknown
Pages (from-to)7775-7790
Number of pages16
JournalNucleic Acids Research
Volume39
Issue number17
DOIs
StatePublished - 2011

Abstract

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.

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