Skip to main content
SpringerLink
Log in

Influenza virus PB1 protein is the minimal and essential subunit of RNA polymerase

  • Original Papers
  • Published:
Archives of Virology Aims and scope Submit manuscript

Summary

RNA polymerase of influenza virus with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the genome RNA. The RNA polymerase with transcription activity was reconstituted from three P proteins, which were separately isolated from insect cells infected with recombinant baculoviruses, each carrying cDNA for one P protein. Nuclear extracts of the insect cells infected with each of the recombinant baculoviruses or various combinations of these viruses were examined for transcription and replication activities. The nuclear extract of cells expressing all three P proteins catalyzed model template-directed RNA synthesis in the absence of primers (an indication of RNA replication), supporting the notion that the complete set of three P proteins is required for RNA replication. All the nuclear extracts containing the PB1 subunit, including the extract containing PB1 alone, were able to catalyze model template-directed dinucleotide-primed RNA synthesis (an indication of transcription). These observations not only confirm that the PB1 protein is a catalytic subunit of influenza virus RNA polymerase, but also indicate that PB1 alone is able to catalyze RNA synthesis in the absence of PB2 and PA subunits.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Akkina RK, Richardson JC, Aguilera MC, Yang C-M (1991) Heterogeneous forms of polymerase proteins exist in influenza A virus- infected cells. Virus Res 19: 17–30

    PubMed  Google Scholar 

  2. Asano Y, Mizumoto K, Maruyama T, Ishihama A (1995) Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP. J Biochem 117: 677–682

    PubMed  Google Scholar 

  3. Beaton AR, Krug RM (1984) Synthesis of the templates for influenza virion RNA replication in vitro. Proc Natl Acad Sci USA 81: 4682–4686

    PubMed  Google Scholar 

  4. Braam J, Ulmanen I, Krug RM (1983) Molecular model of a eukaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34: 609–618

    PubMed  Google Scholar 

  5. Del Rio L, Martinez C, Domingo E, Ortin J (1985) In vitro synthesis of full-length influenza virus complementary RNA. EMBO J 4: 243–247

    PubMed  Google Scholar 

  6. Digard P, Block VC, Inglis SC (1989) Complex formation between influenza virus polymerase proteins expressed inXenopus oocytes. Virology 171: 162–169

    PubMed  Google Scholar 

  7. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 5: 1475–1488

    Google Scholar 

  8. Hay AJ, Lomnicizi B, Bellamy AR, Skehel JJ (1977) Transcription of the influenza virus genome. Virology 83: 337–355

    PubMed  Google Scholar 

  9. Honda A, Ueda K, Nagata K, Ishihama A (1988) RNA polymerase of influenza virus: Role of NP in RNA chain elongation. J Biochem 104: 1021–1026

    PubMed  Google Scholar 

  10. Honda A, Mukaigawa J, Yoshikawa A, Kato A, Ueda S, Nagata K, Krystal M, Nayak DP, Ishihama A (1990) Purification and molecular structure of RNA polymerase from influenza virus A/PR8. J Biochem 107: 624–628

    PubMed  Google Scholar 

  11. Ishihama A, Barbier P (1994) Molecular anatomy of viral RNA-directed RNA polymerases. Arch Virol 134: 235–258

    PubMed  Google Scholar 

  12. Ishihama A, Mizumoto K, Kawakami K, Kato A, Honda A (1986) Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus. J Biol Chem 261: 10417–10421

    PubMed  Google Scholar 

  13. Ishihama A, Nagata K (1988) Viral RNA polymerases. CRC Crit Rev Biochem 23: 27–76

    PubMed  Google Scholar 

  14. Kimura N, Fukushima A, Oda K, Nakada S (1993) An in vitro study of the replication origin in the influenza virus complementary RNA. J Biochem 133: 88–92

    Google Scholar 

  15. Kobayashi M, Tuchiya K, Nagata K, Ishihama A (1992) Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system. Virus Res 22: 235–245

    PubMed  Google Scholar 

  16. Krug RM, Ueda M, Palese P (1975) Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis. J Virol 16: 790–796

    PubMed  Google Scholar 

  17. Krug RM, Alonso-Caplen FV, Julkenun I, Katze MG (1989) Expression and replication of the influenza virus genome. In: Krug RM (ed) The influenza viruses. Plenum Press, New York, pp 89–152

    Google Scholar 

  18. Lamb RA (1989) Genes and proteins of the influenza viruses. In: Krug RM (ed) The influenza viruses. Plenum Press, New York, pp 1–87

    Google Scholar 

  19. Luytjes W, Krystal M, Enami M, Parvin JD, Palese P (1989) Amplification, expression and packaging of a foreign gene by influenza virus. Cell 59: 1107–1113

    PubMed  Google Scholar 

  20. Mahy BWJ, Barrett T, Nichol ST, Penn CR, Wolstenholme AJ (1981) Analysis of the functions of influenza virus genome RNA segments by use of temperature-sensitive mutants of fowl plaque virus, In: Bishop DHL, Compans RW (eds) The replication of negative stranded viruses. Elsevier/North-Holland, New York pp 379–387

    Google Scholar 

  21. Martin J, Albo C, Ortin J, Melero JA, Portela A (1992) In vitro reconstitution of active influenza virus ribonucleoprotein complexes using viral proteins purified from infected cells. J Gen Virol 73: 1855–1859

    PubMed  Google Scholar 

  22. Matsudaira P (1988) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035–10038

    Google Scholar 

  23. Mowshowitz SL (1981) RNA synthesis of temperature-sensitive mutants of WSN influenza virus. In: Bishop DHL, Compans RW (eds) The replication of negative stranded viruses. Elsevier/North-Holland, New York, pp 317–323

    Google Scholar 

  24. Murhammer DW, Goochee CF (1988) Scale up of insect cell cultures: protective effects of Pluronic F-68. BioTechnology 6: 1411–1418

    Google Scholar 

  25. Nagata K, Takeuchi K, Ishihama A (1989) In vitro synthesis of influenza viral RNA: biochemical complementation assay of factors required for influenza virus replication. J Biochem 106: 205–208

    PubMed  Google Scholar 

  26. Nakagawa Y, Kimura N, Toyoda T, Mizumoto K, Ishihama A, Oda K, Nakada (1995) The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis. J Virol 69: 728–733

    PubMed  Google Scholar 

  27. Parvin JD, Palese P, Honda A, Ishihama A, Krystal M (1989) Promoter analysis of influenza virus RNA polymerase. J Virol 63: 5142–5152

    PubMed  Google Scholar 

  28. Piccone ME, Fernandes-Sesma A, Palese P (1993) Mutational analysis of the influenza virus vRNA promoter. Virus Res 28: 99–112

    PubMed  Google Scholar 

  29. Shapiro GI, Krug RM (1988) Influenza virus RNA replication in vitro: synthesis of viral template RNAs and viron RNAs in the absence of an added primer. J Virol 62: 2285–2290

    PubMed  Google Scholar 

  30. Seong BL, Brownlee GG (1992) Nucleotides 9 to 11 of the influenza A virus RNA promoter are crucial for activity in vitro. J Gen Virol 73: 3115–3124

    PubMed  Google Scholar 

  31. Seong BL, Brownlee GG (1992) A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo. Virology 186: 247–260

    PubMed  Google Scholar 

  32. Seong BL, Kobayashi M, Nagata K, Brownlee GG, Ishihama A (1992) Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus. J Biochem 111: 496–499

    PubMed  Google Scholar 

  33. Szewczyk B, Laver WG, Summers DF (1988) Purification, thioredoxin renaturation and reconstituted activity of the three subunits of the influenza A virus RNA polymerase. Proc Natl Acad Sci USA 85: 7907–7911

    PubMed  Google Scholar 

  34. Takeuchi K, Nagata K, Ishihama A (1987) In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA. J Biochem 101: 239–256

    Google Scholar 

  35. Toyoda T, Kobayashi M, Ishihama A (1994) Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes. Arch Virol 136: 269–286

    PubMed  Google Scholar 

  36. Ulmanen I, Broni BA, Krug RM (1981) The role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription. Proc Natl Acad Sci USA 78: 7355–7359

    PubMed  Google Scholar 

  37. Ulmanen I, Broni BA, Krug RM (1983) Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. J Virol 45: 27–35

    PubMed  Google Scholar 

  38. Yamanaka K, Ogasawara N, Yoshikawa H, Ishihama A, Nagata K (1991) In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA. Proc Natl Acad Sci USA 88: 5369–5373

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan

    M. Kobayashi, T. Toyoda & A. Ishihama

  2. Department of Biology, Jichi Medical School, Tochigi, Japan

    M. Kobayashi

  3. Department of Virology, Kurume University School of Medicine, Kurume, Fukuoka, Japan

    T. Toyoda

Authors
  1. M. Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. Toyoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Ishihama
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kobayashi, M., Toyoda, T. & Ishihama, A. Influenza virus PB1 protein is the minimal and essential subunit of RNA polymerase. Archives of Virology 141, 525–539 (1996). https://doi.org/10.1007/BF01718315

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01718315

Keywords

Search

Navigation