title: Overlapping signals for translational regulation and packaging of influenza A virus segment 2.
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.
authors.affiliation: Department of Pathology, University of Cambridge, CB2 1QP, UK.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Wise
authors.forename: Cyril
authors.initials: C
authors.lastname: Barbezange
authors.forename: Brett W
authors.initials: BW
authors.lastname: Jagger
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Dalton
authors.forename: Julia R
authors.initials: JR
authors.lastname: Gog
authors.forename: Martin D
authors.initials: MD
authors.lastname: Curran
authors.forename: Jeffery K
authors.initials: JK
authors.lastname: Taubenberger
authors.forename: Emma C
authors.initials: EC
authors.lastname: Anderson
authors.forename: Paul
authors.initials: P
authors.lastname: Digard
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018387:Codon, Initiator; D018388:Codon, Terminator; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010452:Peptide Biosynthesis; D010442:Peptide Chain Initiation, Translational; D010455:Peptides / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
chemical_list: D018387:Codon, Initiator; D018388:Codon, Terminator; D010455:Peptides; D012333:RNA, Messenger; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D014764:Viral Proteins