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Journal of Virology, December 2008, p. 11869-11879, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01634-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus{triangledown}

Edward C. Hutchinson,1 Martin D. Curran,2 Eliot K. Read,1 Julia R. Gog,3 and Paul Digard1*

Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP,1 Health Protection Agency, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW,2 DAMTP, Centre for Mathematical Sciences, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, United Kingdom3

Received 31 July 2008/ Accepted 12 September 2008

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

* Corresponding author. Mailing address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. Phone: 44 1223 336920. Fax: 44 1223 336926. E-mail: pd1{at}mole.bio.cam.ac.uk

{triangledown} Published ahead of print on 24 September 2008.

Journal of Virology, December 2008, p. 11869-11879, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01634-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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