3D structure of the influenza virus polymerase complex: Localization of subunit domains

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP–mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.

[1]  N. Habuka,et al.  Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. , 1999, Structure.

[2]  Maite Huarte,et al.  PA Subunit from Influenza Virus Polymerase Complex Interacts with a Cellular Protein with Homology to a Family of Transcriptional Activators , 2001, Journal of Virology.

[3]  K. Yano,et al.  Identification of Hsp90 as a Stimulatory Host Factor Involved in Influenza Virus RNA Synthesis* , 2002, The Journal of Biological Chemistry.

[4]  R. Krug,et al.  The active sites of the influenza cap‐dependent endonuclease are on different polymerase subunits , 2001, The EMBO journal.

[5]  R. Webster,et al.  Localization of RNA polymerases on influenza viral ribonucleoproteins by immunogold labeling. , 1988, Virology.

[6]  E. V. Makeyev,et al.  A mechanism for initiating RNA-dependent RNA polymerization , 2001, Nature.

[7]  J. Ortega,et al.  Three‐dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle , 2001, EMBO reports.

[8]  T. Deng,et al.  A Single Amino Acid Mutation in the PA Subunit of the Influenza Virus RNA Polymerase Inhibits Endonucleolytic Cleavage of Capped RNAs , 2002, Journal of Virology.

[9]  D. Nayak,et al.  Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1 , 1994, Journal of virology.

[10]  J. Ortín,et al.  Characterization of Influenza Virus PB1 Protein Binding to Viral RNA: Two Separate Regions of the Protein Contribute to the Interaction Domain , 1999, Journal of Virology.

[11]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[12]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[13]  I Sauvaget,et al.  Identification of four conserved motifs among the RNA‐dependent polymerase encoding elements. , 1989, The EMBO journal.

[14]  George G. Brownlee,et al.  Direct Evidence that the Poly(A) Tail of Influenza A Virus mRNA Is Synthesized by Reiterative Copying of a U Track in the Virion RNA Template , 1999, Journal of Virology.

[15]  M. Katze,et al.  The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex , 1987, Journal of virology.

[16]  F. Baudin,et al.  Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles. , 1995, The Journal of general virology.

[17]  Charles A. Lesburg,et al.  Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site , 1999, Nature Structural Biology.

[18]  J. Ortín,et al.  Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure. , 1996, Nucleic acids research.

[19]  C. Cianci,et al.  Differential activation of the influenza virus polymerase via template RNA binding , 1995, Journal of virology.

[20]  R. Krug,et al.  Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. de la Luna,et al.  Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant. , 1996, The Journal of general virology.

[22]  F. Rey,et al.  Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  L. Poon,et al.  Polyuridylated mRNA Synthesized by a Recombinant Influenza Virus Is Defective in Nuclear Export , 2000, Journal of Virology.

[24]  P. L. Boutz,et al.  Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins , 1998, Journal of Virology.

[25]  David A. Steinhauer,et al.  Genetics of influenza viruses. , 2002 .

[26]  J. Ortega,et al.  Ultrastructural and Functional Analyses of Recombinant Influenza Virus Ribonucleoproteins Suggest Dimerization of Nucleoprotein during Virus Amplification , 2000, Journal of Virology.

[27]  J. Wood,et al.  Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system. , 2005, Vaccine.

[28]  M. Lai.  Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. , 1998, Virology.

[29]  S. Cusack,et al.  Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. , 1994, The EMBO journal.

[30]  I. Schulze,et al.  Isolation and characterization of the ribonucleoprotein of influenza virus. , 1969, Virology.

[31]  J. Hansen,et al.  Structure of the RNA-dependent RNA polymerase of poliovirus. , 1997, Structure.

[32]  F. Baudin,et al.  Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure , 1997, The EMBO journal.

[33]  J M Carazo,et al.  Pattern recognition and classification of images of biological macromolecules using artificial neural networks. , 1994, Biophysical journal.

[34]  M. B. Leahy,et al.  A Hairpin Loop at the 5′ End of Influenza A Virus Virion RNA Is Required for Synthesis of Poly(A)+ mRNA In Vitro , 1999, Journal of Virology.

[35]  M. James,et al.  Crystal Structures of Active and Inactive Conformations of a Caliciviral RNA-dependent RNA Polymerase* , 2002, The Journal of Biological Chemistry.

[36]  J M Carazo,et al.  3D reconstruction in electron microscopy using ART with smooth spherically symmetric volume elements (blobs). , 1998, Ultramicroscopy.

[37]  R. Lazzarini,et al.  Polyadenylation sites for influenza virus mRNA , 1981, Journal of virology.

[38]  D. Pérez,et al.  A 48-amino-acid region of influenza A virus PB1 protein is sufficient for complex formation with PA , 1995, Journal of virology.

[39]  Marco,et al.  Xmipp: An Image Processing Package for Electron Microscopy , 1996, Journal of structural biology.

[40]  J. Ortín,et al.  The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA , 1997, Journal of virology.

[41]  A. Ishihama,et al.  Molecular assembly of the influenza virus RNA polymerase: determination of the subunit-subunit contact sites. , 1996, The Journal of general virology.

[42]  José María Carazo,et al.  A variant to the “random approximation” of the reference-free alignment algorithm , 1996 .

[43]  R. Krug,et al.  RNA‐dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA‐binding sites , 1998, The EMBO journal.

[44]  S. Inglis,et al.  Complex formation between influenza virus polymerase proteins expressed in Xenopus oocytes , 1989, Virology.

[45]  J. Ortega,et al.  The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis , 2000, Journal of Virology.

[46]  J. Ortín,et al.  Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates , 1999, The EMBO journal.

[47]  R. Krug,et al.  Are the 5′ ends of influenza viral mrnas synthesized in vivo donated by host mRNAs? , 1979, Cell.

[48]  S. de la Luna,et al.  Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro , 1994, Journal of virology.

[49]  J. Finch,et al.  Does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral RNA? , 1983, Cell.