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

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

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

    Glenn A. Marsh,<sup>1</sup><sup>,</sup><sup>,#</sup> Ra&#250;l Rabad&#225;n,<sup>3</sup><sup>,</sup> Arnold J. Levine,<sup>3</sup> and Peter Palese<sup>1</sup><sup>,2</sup><sup>*</sup> Departments of Microbiology,<sup>1</sup> Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, New York 10029,<sup>2</sup> Institute for Advanced Study, Einstein Dr., Princeton, New Jersey 08540<sup>3</sup>
    Received 18 October 2007/ Accepted 7 December 2007
    <!-- ABS --> The genome of the influenza A virus is composed of eight different<sup> </sup>segments of negative-sense RNA. These eight segments are incorporated<sup> </sup>into budding virions in an equimolar ratio through a mechanism<sup> </sup>that is not fully understood. Two different models have been<sup> </sup>proposed for packaging the viral ribonucleoproteins into newly<sup> </sup>assembling virus particles: the random-incorporation model and<sup> </sup>the selective-incorporation model. In the last few years, increasing<sup> </sup>evidence from many different laboratories that supports the<sup> </sup>selective-incorporation model has been accumulated. In particular,<sup> </sup>different groups have shown that some large viral RNA regions<sup> </sup>within the coding sequences at both the 5' and 3' ends of almost<sup> </sup>every segment are sufficient for packaging foreign RNA sequences.<sup> </sup>If the packaging regions are crucial for the viability of the<sup> </sup>virus, we would expect them to be conserved. Using large-scale<sup> </sup>analysis of influenza A virus sequences, we developed a method<sup> </sup>of identifying conserved RNA regions whose conservation cannot<sup> </sup>be explained by population structure or amino acid conservation.<sup> </sup>Interestingly, the conserved sequences are located within the<sup> </sup>regions identified as important for efficient packaging. By<sup> </sup>utilizing influenza virus reverse genetics, we have rescued<sup> </sup>mutant viruses containing synonymous mutations within these<sup> </sup>highly conserved regions. Packaging of viral RNAs in these viruses<sup> </sup>was analyzed by reverse transcription using a universal primer<sup> </sup>and quantitative PCR for individual segments. Employing this<sup> </sup>approach, we have identified regions in the polymerase gene<sup> </sup>segments that, if mutated, result in reductions of more than<sup> </sup>90&#37; in the packaging of that particular polymerase viral RNA.<sup> </sup>Reductions in the level of packaging of a polymerase viral RNA<sup> </sup>frequently resulted in reductions of other viral RNAs as well,<sup> </sup>and the results form a pattern of hierarchy of segment interactions.<sup> </sup>This work provides further evidence for a selective packaging<sup> </sup>mechanism for influenza A viruses, demonstrating that these<sup> </sup>highly conserved regions are important for efficient packaging.

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