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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

By Birgit G. Bradel-Tretheway, Z. Kelley, Shikha Chakraborty-Sett, Toru Takimoto, Baek Kim and Stephen Dewhurst

Abstract

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °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 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °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

Topics: Jgv Direct
Publisher: Society for General Microbiology
OAI identifier: oai:pubmedcentral.nih.gov:3067610
Provided by: PubMed Central

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Citations

  1. (2002). A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs.
  2. (1977). A single gene determines the host range of influenza virus.
  3. (1981). A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.
  4. (2006). Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.
  5. Bradel-Tretheway and others 2932
  6. (2006). Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses.
  7. (2005). Characterization of the 1918 influenza virus polymerase genes.
  8. (2006). Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA.
  9. (1988). Difference in growth behavior of human, swine, equine, and avian influenza viruses at a high temperature.
  10. (2007). Differential polymerase activity in avian and mammalian cells determines host range of influenza virus.
  11. (2000). Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses.
  12. Genomic signatures of human versus avian influenza A viruses.
  13. (2007). Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice.
  14. (2007). Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells.
  15. (2008). Human infection with highly pathogenic H5N1 influenza virus.
  16. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.
  17. (2005). In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex.
  18. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.
  19. (2002). Influenza virus replication.
  20. (2008). Interaction of polymerase subunit PB2 and NP with importin a1 is a determinant of host range of influenza A virus.
  21. (2005). Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes.
  22. (2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.
  23. (2005). Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.
  24. (1992). Nuclear transport of influenza virus polymerase PA protein.
  25. (2001). Orthomyxoviridae: the viruses and their replication.
  26. (2006). other authors
  27. other authors (2004). Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia.
  28. other authors (2006). The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/ 1203/04.
  29. other authors (2007). Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.
  30. other authors (2008). The structural basis for cap binding by influenza virus polymerase subunit PB2.
  31. (2004). PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice.
  32. (2007). Persistent host markers in pandemic and H5N1 influenza viruses.
  33. (1997). Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-b but interacts differently with RanBP1.
  34. (1982). Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.
  35. (2001). Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses.
  36. (2003). Role of PB2 in IAV temperature sensitivity http://vir.sgmjournals.org 2931Heim,
  37. (2006). Role of Ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex.
  38. (2000). Sialic acid species as a determinant of the host range of influenza A viruses.
  39. (2001). The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits.
  40. (1997). The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA.
  41. (2003). The influenza virus gene pool in a poultry market in South Central China.
  42. (2004). The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex.
  43. (2000). The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis.
  44. (2002). The RNA polymerase of influenza A virus is stabilized by interaction with its viral RNA promoter.
  45. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification.
  46. (2005). The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.
  47. (2007). Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer.
  48. (1982). Virulence of avian influenza A viruses for squirrel monkeys.