adaptation and temperature sensitivity of overall virus replication, through effects on RNA transcription (Chen et al., 2006; Finkelstein et al., 2007). Further studies to address this hypothesis are ongoing. At the mechanistic level, our results show that the thermotolerance-enhancing activity of the avian PB2 can be attributed to an increase in functional stability of the polymerase complex. Brownlee & Sharps (2002) have speculated that, in the absence of the vRNA promoter, the WSN polymerase complex is open and labile to heat inactivation. We showed that the half-life of the WSN complex in the absence of vRNA was roughly 10 min at 30 uC, whereas the functional half-life of the W/W/N complex exceeded 2 h. The short functional half-life of the WSN complex was not due to disintegration or disassem- bly of the tripartite polymerase protein complex, since the complex remained intact for at least 1 h. Our findings support the earlier hypothesis (Brownlee & Sharps, 2002) that avian IAV RNA polymerases may have increased heat stability, due to the need of avian viruses to replicate at elevated temperatures present in birds. Brownlee also hypothesized that highly pathogenic human influenza viruses may possess polymerases with increased thermostability, relative to less pathogenic strains. Our results provide support for both of these predictions, and suggest the need to examine the thermostability of RNA polymerases from additional viruses. In conclusion, the results reported in this paper define a new and previously unappreciated role for the influenza PB2 protein in determining the thermostability of the virus RNA polymerase. Future studies will determine the specific amino acid residues that contribute to this property, and will assess whether increased thermostability is associated with greater pathogenicity and/or host adaptation by emerging influenza viruses. ACKNOWLEDGEMENTS We thank Drs Y. Kawaoka (University of Wisconsin-Madison, WI, USA) and R. Webster (St Jude Children’s Research Hospital, Memphis, TN, USA) for reagents and Heidi Regan for help with transcription assays. This work was supported by the New York Influenza Center of Excellence (NYICE), under NIH contract HHSN266200700008C. REFERENCES Almond, J. W. (1977). A single gene determines the host range of influenza virus. Nature 270, 617–618. Brownlee, G. G. & Sharps, J. L. (2002). The RNA polymerase of influenza A virus is stabilized by interaction with its viral RNA promoter. J Virol 76, 7103–7113. Carr, S. M., Carnero, E., Garcia-Sastre, A., Brownlee, G. G. & Fodor, E. (2006). Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses. Virology 344, 492–508. Chen, G. W., Chang, S. C., Mok, C. K., Lo, Y. L., Kung, Y. N., Huang, J. H., Shih, Y. H., Wang, J. 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