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Journal of Virology, July 2009, p. 6673-6680, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00212-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

Thierry Rolling, Iris Koerner, Petra Zimmermann, Kristian Holz, Otto Haller, Peter Staeheli, and Georg Kochs^*

Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, 79104 Freiburg, Germany

Received 29 January 2009/ Accepted 8 April 2009

High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.

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* Corresponding author. Mailing address: Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, University of Freiburg, Hermann-Herder-Strasse 11, 79104 Freiburg, Germany. Phone: 49-761-2036533. Fax: 49-761-2036562. E-mail: georg.kochs{at}uniklinik-freiburg.de

Published ahead of print on 29 April 2009.

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Journal of Virology, July 2009, p. 6673-6680, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00212-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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