Highly Conserved Regions of Influenza A Virus Polymerase Gene Segments Are Critical for Efficient Viral RNA Packaging

[Sally Furniss]

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Copyright © 2008, American Society for Microbiology. All Rights Reserved.[/SIZE]
Highly Conserved Regions of Influenza A Virus Polymerase Gene Segments Are Critical for Efficient Viral RNA Packaging<sup>

</sup>

Glenn A. Marsh,¹<sup>,

</sup>^,# Raúl Rabadán,³<sup>,

</sup> Arnold J. Levine,³ and Peter Palese¹^,2^* Departments of Microbiology,¹ Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029,² Institute for Advanced Study, Einstein Dr., Princeton, New Jersey 08540³
Received 18 October 2007/ Accepted 7 December 2007
<!-- ABS --> The genome of the influenza A virus is composed of eight differentsegments of negative-sense RNA. These eight segments are incorporatedinto budding virions in an equimolar ratio through a mechanismthat is not fully understood. Two different models have beenproposed for packaging the viral ribonucleoproteins into newlyassembling virus particles: the random-incorporation model andthe selective-incorporation model. In the last few years, increasingevidence from many different laboratories that supports theselective-incorporation model has been accumulated. In particular,different groups have shown that some large viral RNA regionswithin the coding sequences at both the 5' and 3' ends of almostevery segment are sufficient for packaging foreign RNA sequences.If the packaging regions are crucial for the viability of thevirus, we would expect them to be conserved. Using large-scaleanalysis of influenza A virus sequences, we developed a methodof identifying conserved RNA regions whose conservation cannotbe explained by population structure or amino acid conservation.Interestingly, the conserved sequences are located within theregions identified as important for efficient packaging. Byutilizing influenza virus reverse genetics, we have rescuedmutant viruses containing synonymous mutations within thesehighly conserved regions. Packaging of viral RNAs in these viruseswas analyzed by reverse transcription using a universal primerand quantitative PCR for individual segments. Employing thisapproach, we have identified regions in the polymerase genesegments that, if mutated, result in reductions of more than90% in the packaging of that particular polymerase viral RNA.Reductions in the level of packaging of a polymerase viral RNAfrequently resulted in reductions of other viral RNAs as well,and the results form a pattern of hierarchy of segment interactions.This work provides further evidence for a selective packagingmechanism for influenza A viruses, demonstrating that thesehighly conserved regions are important for efficient packaging.

http://jvi.asm.org/cgi/content/abstract/82/5/2295