Journal of General Virology (2008), 89, 2923–2932 DOI 10.1099/vir.0.2008/006254-0 Correspondence Birgit Bradel-Tretheway Birgit_BradelTretheway @urmc.rochester.edu The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit Birgit G. Bradel-Tretheway, Z. Kelley, Shikha Chakraborty-Sett, Toru Takimoto, Baek Kim and Stephen Dewhurst Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 6C and in the intestinal tract of birds at close to 41 6C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/ H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/ chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30– 42 6C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 6C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex. Received 4 August 2008 Accepted 28 August 2008 INTRODUCTION Among the viral factors that contribute to virulence and host range of influenza A viruses (IAVs) are the viral polymerase proteins (Almond, 1977; Gabriel et al., 2005, 2007; Hatta et al., 2001; Murakami et al., 1988). The IAV polymerase is a heterotrimeric protein (3P) complex comprised of PB1, PB2 and PA. PB1 contains conserved sequence elements found in other RNA-dependent RNA polymerases (Deng et al., 2005; Fodor & Brownlee, 2002; Lamb & Krug, 2001), and binds directly to the viral (vRNA) and complementary RNA (cRNA) promoter sequences. PA is needed for transcription and replication (Fodor et al., 2002; Hara et al., 2006; Perales et al., 2000; Regan et al., 2006), and for efficient nuclear accumulation of PB1 (Fodor & Smith, 2004; Sanz-Ezquerro et al., 1995). The third subunit, PB2, localizes to the nucleus Published online ahead of print on 11 September 2008 as DOI 10.1099/vir.0.2008/006254-0. A supplementary figure is available with the online version of this paper. 2008/006254 G 2008 SGM Printed in Great Britain and to mitochondria (Carr et al., 2006). It binds to cap structures of host mRNAs which are then cleaved by the endonucleolytic activity of PB1 (Guilligay et al., 2008; Li et al., 2001; Plotch et al., 1981). PB2 also plays a major role in host adaptation (Finkelstein et al., 2007; Gabriel et al., 2008; Hatta et al., 2001; Labadie et al., 2007; Li et al., 2005; Naffakh et al., 2000; Salomon et al., 2006). One factor that contributes to host adaptation is the temperature optimum for virus replication. IAV replicates in the upper respiratory tract of humans at 33 uC and in the intestinal tract of birds close to 41 uC. In cell culture, human IAV can replicate at temperatures ranging from 33 uC to 37 uC, but not at elevated temperatures such as 42 uC. In contrast, most avian IAVs exhibit cold sensitivity with reduced replication at 33 uC, but efficient replication at 37 uC or 42 uC (Massin et al., 2001; Murakami et al., 1988; Murphy et al., 1982a, b). It has been shown that amino acid residue 627 in PB2 is one of the main determinants for cold sensitivity of the avian IAV and for viral pathogenicity in mammalian hosts 2923