title: 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.
abstract: Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 degrees C and in the intestinal tract of birds at close to 41 degrees C. 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 degrees C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 degrees C 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.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Birgit G
authors.initials: BG
authors.lastname: Bradel-Tretheway
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Kelley
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Shikha
authors.initials: S
authors.lastname: Chakraborty-Sett
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Toru
authors.initials: T
authors.lastname: Takimoto
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Dewhurst
mesh_terms: D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells; D004795:Enzyme Stability; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology*; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology*; D007668:Kidney / Q000166:cytology; D008168:Lung / Q000166:cytology; D051379:Mice; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase