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. 1998 Oct 1;17(19):5844–5852. doi: 10.1093/emboj/17.19.5844

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.

M L Li 1, B C Ramirez 1, R M Krug 1
PMCID: PMC1170912  PMID: 9755184

Abstract

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.

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Selected References

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

  1. Autexier C., Greider C. W. Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro. Nucleic Acids Res. 1998 Feb 1;26(3):787–795. doi: 10.1093/nar/26.3.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhattacharyya A., Blackburn E. H. A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2823–2827. doi: 10.1073/pnas.94.7.2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  4. Cianci C., Tiley L., Krystal M. Differential activation of the influenza virus polymerase via template RNA binding. J Virol. 1995 Jul;69(7):3995–3999. doi: 10.1128/jvi.69.7.3995-3999.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Detjen B. M., St Angelo C., Katze M. G., Krug R. M. The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex. J Virol. 1987 Jan;61(1):16–22. doi: 10.1128/jvi.61.1.16-22.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Favre A., Saintomé C., Fourrey J. L., Clivio P., Laugâa P. Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions. J Photochem Photobiol B. 1998 Feb;42(2):109–124. doi: 10.1016/s1011-1344(97)00116-4. [DOI] [PubMed] [Google Scholar]
  7. Fiering S., Northrop J. P., Nolan G. P., Mattila P. S., Crabtree G. R., Herzenberg L. A. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 1990 Oct;4(10):1823–1834. doi: 10.1101/gad.4.10.1823. [DOI] [PubMed] [Google Scholar]
  8. Fodor E., Seong B. L., Brownlee G. G. Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter. J Gen Virol. 1993 Jul;74(Pt 7):1327–1333. doi: 10.1099/0022-1317-74-7-1327. [DOI] [PubMed] [Google Scholar]
  9. Gilley D., Blackburn E. H. Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase. Mol Cell Biol. 1996 Jan;16(1):66–75. doi: 10.1128/mcb.16.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hagen M., Chung T. D., Butcher J. A., Krystal M. Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity. J Virol. 1994 Mar;68(3):1509–1515. doi: 10.1128/jvi.68.3.1509-1515.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hale S. P., Auld D. S., Schmidt E., Schimmel P. Discrete determinants in transfer RNA for editing and aminoacylation. Science. 1997 May 23;276(5316):1250–1252. doi: 10.1126/science.276.5316.1250. [DOI] [PubMed] [Google Scholar]
  12. Klumpp K., Ruigrok R. W., Baudin F. Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J. 1997 Mar 17;16(6):1248–1257. doi: 10.1093/emboj/16.6.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lin L., Hale S. P., Schimmel P. Aminoacylation error correction. Nature. 1996 Nov 7;384(6604):33–34. doi: 10.1038/384033b0. [DOI] [PubMed] [Google Scholar]
  14. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  15. Plotch S. J., Bouloy M., Ulmanen I., Krug R. M. A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell. 1981 Mar;23(3):847–858. doi: 10.1016/0092-8674(81)90449-9. [DOI] [PubMed] [Google Scholar]
  16. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prescott J., Blackburn E. H. Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro. Genes Dev. 1997 Feb 15;11(4):528–540. doi: 10.1101/gad.11.4.528. [DOI] [PubMed] [Google Scholar]
  18. Shah K., Wu H., Rana T. M. Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences. Bioconjug Chem. 1994 Nov-Dec;5(6):508–512. doi: 10.1021/bc00030a005. [DOI] [PubMed] [Google Scholar]
  19. Shih S. R., Krug R. M. Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against the cap-snatching reaction catalyzed by the same polymerase proteins. Virology. 1996 Dec 15;226(2):430–435. doi: 10.1006/viro.1996.0673. [DOI] [PubMed] [Google Scholar]
  20. Shih S. R., Nemeroff M. E., Krug R. M. The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6324–6328. doi: 10.1073/pnas.92.14.6324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith G. L., Levin J. Z., Palese P., Moss B. Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses. Virology. 1987 Oct;160(2):336–345. doi: 10.1016/0042-6822(87)90004-3. [DOI] [PubMed] [Google Scholar]
  22. Tavis J. E., Ganem D. Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template. J Virol. 1996 Sep;70(9):5741–5750. doi: 10.1128/jvi.70.9.5741-5750.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tavis J. E., Massey B., Gong Y. The duck hepatitis B virus polymerase is activated by its RNA packaging signal, epsilon. J Virol. 1998 Jul;72(7):5789–5796. doi: 10.1128/jvi.72.7.5789-5796.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tiley L. S., Hagen M., Matthews J. T., Krystal M. Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs. J Virol. 1994 Aug;68(8):5108–5116. doi: 10.1128/jvi.68.8.5108-5116.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ulmanen I., Broni B. A., Krug R. M. 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 U S A. 1981 Dec;78(12):7355–7359. doi: 10.1073/pnas.78.12.7355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wang Z., Rana T. M. RNA-protein interactions in the Tat-trans-activation response element complex determined by site-specific photo-cross-linking. Biochemistry. 1998 Mar 24;37(12):4235–4243. doi: 10.1021/bi972889a. [DOI] [PubMed] [Google Scholar]

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