Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jan;70(1):241–247. doi: 10.1128/jvi.70.1.241-247.1996

Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein.

K Watanabe 1, H Handa 1, K Mizumoto 1, K Nagata 1
PMCID: PMC189810  PMID: 8523532

Abstract

Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.

Full Text

The Full Text of this article is available as a PDF (408.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albo C., Valencia A., Portela A. Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein. J Virol. 1995 Jun;69(6):3799–3806. doi: 10.1128/jvi.69.6.3799-3806.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bucher D., Popple S., Baer M., Mikhail A., Gong Y. F., Whitaker C., Paoletti E., Judd A. M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies. J Virol. 1989 Sep;63(9):3622–3633. doi: 10.1128/jvi.63.9.3622-3633.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carroll A. R., Wagner R. R. Role of the membrane (M) protein in endogenous inhibition of in vitro transcription by vesicular stomatitis virus. J Virol. 1979 Jan;29(1):134–142. doi: 10.1128/jvi.29.1.134-142.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chong L. D., Rose J. K. Membrane association of functional vesicular stomatitis virus matrix protein in vivo. J Virol. 1993 Jan;67(1):407–414. doi: 10.1128/jvi.67.1.407-414.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Enami K., Sato T. A., Nakada S., Enami M. Influenza virus NS1 protein stimulates translation of the M1 protein. J Virol. 1994 Mar;68(3):1432–1437. doi: 10.1128/jvi.68.3.1432-1437.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gregoriades A., Frangione B. Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion. J Virol. 1981 Oct;40(1):323–328. doi: 10.1128/jvi.40.1.323-328.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gregoriades A. Interaction of influenza M protein with viral lipid and phosphatidylcholine vesicles. J Virol. 1980 Nov;36(2):470–479. doi: 10.1128/jvi.36.2.470-479.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gregoriades A. The membrane protein of influenza virus: extraction from virus and infected cell with acidic chloroform-methanol. Virology. 1973 Aug;54(2):369–383. doi: 10.1016/0042-6822(73)90150-5. [DOI] [PubMed] [Google Scholar]
  9. Hankins R. W., Nagata K., Bucher D. J., Popple S., Ishihama A. Monoclonal antibody analysis of influenza virus matrix protein epitopes involved in transcription inhibition. Virus Genes. 1989 Nov;3(2):111–126. doi: 10.1007/BF00125124. [DOI] [PubMed] [Google Scholar]
  10. Honda A., Mukaigawa J., Yokoiyama A., Kato A., Ueda S., Nagata K., Krystal M., Nayak D. P., Ishihama A. Purification and molecular structure of RNA polymerase from influenza virus A/PR8. J Biochem. 1990 Apr;107(4):624–628. doi: 10.1093/oxfordjournals.jbchem.a123097. [DOI] [PubMed] [Google Scholar]
  11. Honda A., Uéda K., Nagata K., Ishihama A. Identification of the RNA polymerase-binding site on genome RNA of influenza virus. J Biochem. 1987 Nov;102(5):1241–1249. doi: 10.1093/oxfordjournals.jbchem.a122163. [DOI] [PubMed] [Google Scholar]
  12. Honda A., Uéda K., Nagata K., Ishihama A. RNA polymerase of influenza virus: role of NP in RNA chain elongation. J Biochem. 1988 Dec;104(6):1021–1026. doi: 10.1093/oxfordjournals.jbchem.a122569. [DOI] [PubMed] [Google Scholar]
  13. Ishihama A., Nagata K. Viral RNA polymerases. CRC Crit Rev Biochem. 1988;23(1):27–76. doi: 10.3109/10409238809103119. [DOI] [PubMed] [Google Scholar]
  14. Kato A., Mizumoto K., Ishihama A. Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus. Virus Res. 1985 Sep;3(2):115–127. doi: 10.1016/0168-1702(85)90002-4. [DOI] [PubMed] [Google Scholar]
  15. Martin K., Helenius A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell. 1991 Oct 4;67(1):117–130. doi: 10.1016/0092-8674(91)90576-k. [DOI] [PubMed] [Google Scholar]
  16. Martin K., Helenius A. Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol. 1991 Jan;65(1):232–244. doi: 10.1128/jvi.65.1.232-244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mizumoto K., Kaziro Y., Lipmann F. Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1693–1697. doi: 10.1073/pnas.79.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ogden J. R., Pal R., Wagner R. R. Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies. J Virol. 1986 Jun;58(3):860–868. doi: 10.1128/jvi.58.3.860-868.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Seong B. L., Brownlee G. G. 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. 1992 Jan;186(1):247–260. doi: 10.1016/0042-6822(92)90079-5. [DOI] [PubMed] [Google Scholar]
  20. Seong B. L., Kobayashi M., Nagata K., Brownlee G. G., Ishihama A. Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus. J Biochem. 1992 Apr;111(4):496–499. doi: 10.1093/oxfordjournals.jbchem.a123786. [DOI] [PubMed] [Google Scholar]
  21. Shimizu K., Handa H., Nakada S., Nagata K. Regulation of influenza virus RNA polymerase activity by cellular and viral factors. Nucleic Acids Res. 1994 Nov 25;22(23):5047–5053. doi: 10.1093/nar/22.23.5047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  23. Tucker P. A., Tsernoglou D., Tucker A. D., Coenjaerts F. E., Leenders H., van der Vliet P. C. Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding. EMBO J. 1994 Jul 1;13(13):2994–3002. doi: 10.1002/j.1460-2075.1994.tb06598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wakefield L., Brownlee G. G. RNA-binding properties of influenza A virus matrix protein M1. Nucleic Acids Res. 1989 Nov 11;17(21):8569–8580. doi: 10.1093/nar/17.21.8569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Watanabe K., Momose F., Handa H., Nagata K. Interaction between influenza virus proteins and pine cone antitumor substance that inhibits the virus multiplication. Biochem Biophys Res Commun. 1995 Sep 14;214(2):318–323. doi: 10.1006/bbrc.1995.2290. [DOI] [PubMed] [Google Scholar]
  26. White J. M. Viral and cellular membrane fusion proteins. Annu Rev Physiol. 1990;52:675–697. doi: 10.1146/annurev.ph.52.030190.003331. [DOI] [PubMed] [Google Scholar]
  27. Whittaker G., Kemler I., Helenius A. Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus. J Virol. 1995 Jan;69(1):439–445. doi: 10.1128/jvi.69.1.439-445.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Winter G., Fields S. Cloning of influenza cDNA ino M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein. Nucleic Acids Res. 1980 May 10;8(9):1965–1974. doi: 10.1093/nar/8.9.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yamanaka K., Ishihama A., Nagata K. Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. J Biol Chem. 1990 Jul 5;265(19):11151–11155. [PubMed] [Google Scholar]
  30. Yamanaka K., Ishihama A., Nagata K. Translational regulation of influenza virus mRNAs. Virus Genes. 1988 Oct;2(1):19–30. doi: 10.1007/BF00569734. [DOI] [PubMed] [Google Scholar]
  31. Ye Z. P., Baylor N. W., Wagner R. R. Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides. J Virol. 1989 Sep;63(9):3586–3594. doi: 10.1128/jvi.63.9.3586-3594.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ye Z. P., Pal R., Fox J. W., Wagner R. R. Functional and antigenic domains of the matrix (M1) protein of influenza A virus. J Virol. 1987 Feb;61(2):239–246. doi: 10.1128/jvi.61.2.239-246.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ye Z., Robinson D., Wagner R. R. Nucleus-targeting domain of the matrix protein (M1) of influenza virus. J Virol. 1995 Mar;69(3):1964–1970. doi: 10.1128/jvi.69.3.1964-1970.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhirnov O. P. Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. Virology. 1992 Jan;186(1):324–330. doi: 10.1016/0042-6822(92)90090-c. [DOI] [PubMed] [Google Scholar]
  35. Zvonarjev A. Y., Ghendon Y. Z. Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine. J Virol. 1980 Feb;33(2):583–586. doi: 10.1128/jvi.33.2.583-586.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES