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

• Published in: Nucleic acids research Vol. 39; no. 17; pp. 7775 - 7790
• Main Authors: Wise, Helen M., Barbezange, Cyril, Jagger, Brett W., Dalton, Rosa M., Gog, Julia R., Curran, Martin D., Taubenberger, Jeffery K., Anderson, Emma C., Digard, Paul
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.09.2011
• Subjects: Amino Acid Sequence; Base Sequence; Codon, Initiator; Codon, Terminator; Gene Expression Regulation, Viral; HEK293 Cells; Humans; Influenza A virus; Influenza A virus - genetics; Influenza A virus - metabolism; Influenza A virus - physiology; Molecular Sequence Data; Mutation; Open Reading Frames; Peptide Biosynthesis; Peptide Chain Initiation, Translational; Peptides - genetics; Regulatory Sequences, Ribonucleic Acid; RNA; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Viral - chemistry; RNA, Viral - metabolism; Viral Proteins - biosynthesis; Viral Proteins - genetics; Virion - physiology; Virus Assembly
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkr487

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.