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. 1996 Mar;70(3):1905–1911. doi: 10.1128/jvi.70.3.1905-1911.1996

The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.

J J Sanz-Ezquerro 1, T Zürcher 1, S de la Luna 1, J Ortín 1, A Nieto 1
PMCID: PMC190019  PMID: 8627716

Abstract

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.

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Selected References

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  1. Akkina R. K., Chambers T. M., Londo D. R., Nayak D. P. Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA. J Virol. 1987 Jul;61(7):2217–2224. doi: 10.1128/jvi.61.7.2217-2224.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barany F. Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene. 1985;37(1-3):111–123. doi: 10.1016/0378-1119(85)90263-x. [DOI] [PubMed] [Google Scholar]
  3. Biswas S. K., Nayak D. P. Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1. J Virol. 1994 Mar;68(3):1819–1826. doi: 10.1128/jvi.68.3.1819-1826.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blaas D., Patzelt E., Kuechler E. Identification of the cap binding protein of influenza virus. Nucleic Acids Res. 1982 Aug 11;10(15):4803–4812. doi: 10.1093/nar/10.15.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braam J., Ulmanen I., Krug R. M. Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell. 1983 Sep;34(2):609–618. doi: 10.1016/0092-8674(83)90393-8. [DOI] [PubMed] [Google Scholar]
  6. Bárcena J., Ochoa M., de la Luna S., Melero J. A., Nieto A., Ortín J., Portela A. Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro. J Virol. 1994 Nov;68(11):6900–6909. doi: 10.1128/jvi.68.11.6900-6909.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuerst T. R., Earl P. L., Moss B. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol. 1987 Jul;7(7):2538–2544. doi: 10.1128/mcb.7.7.2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hay A. J., Skehel J. J., McCauley J. Characterization of influenza virus RNA complete transcripts. Virology. 1982 Jan 30;116(2):517–522. doi: 10.1016/0042-6822(82)90144-1. [DOI] [PubMed] [Google Scholar]
  11. Herz C., Stavnezer E., Krug R., Gurney T., Jr Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell. 1981 Nov;26(3 Pt 1):391–400. doi: 10.1016/0092-8674(81)90208-7. [DOI] [PubMed] [Google Scholar]
  12. Huang T. S., Palese P., Krystal M. Determination of influenza virus proteins required for genome replication. J Virol. 1990 Nov;64(11):5669–5673. doi: 10.1128/jvi.64.11.5669-5673.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jackson D. A., Caton A. J., McCready S. J., Cook P. R. Influenza virus RNA is synthesized at fixed sites in the nucleus. Nature. 1982 Mar 25;296(5855):366–368. doi: 10.1038/296366a0. [DOI] [PubMed] [Google Scholar]
  14. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  15. Kroll D. J., Abdel-Malek Abdel-Hafiz H., Marcell T., Simpson S., Chen C. Y., Gutierrez-Hartmann A., Lustbader J. W., Hoeffler J. P. A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection. DNA Cell Biol. 1993 Jun;12(5):441–453. doi: 10.1089/dna.1993.12.441. [DOI] [PubMed] [Google Scholar]
  16. Luo G. X., Luytjes W., Enami M., Palese P. The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. J Virol. 1991 Jun;65(6):2861–2867. doi: 10.1128/jvi.65.6.2861-2867.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. López-Turiso J. A., Martínez C., Tanaka T., Ortín J. The synthesis of influenza virus negative-strand RNA takes place in insoluble complexes present in the nuclear matrix fraction. Virus Res. 1990 Jul;16(3):325–337. doi: 10.1016/0168-1702(90)90056-h. [DOI] [PubMed] [Google Scholar]
  18. Mena I., de la Luna S., Albo C., Martín J., Nieto A., Ortín J., Portela A. Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. J Gen Virol. 1994 Aug;75(Pt 8):2109–2114. doi: 10.1099/0022-1317-75-8-2109. [DOI] [PubMed] [Google Scholar]
  19. Nieto A., de la Luna S., Bárcena J., Portela A., Ortín J. Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit. J Gen Virol. 1994 Jan;75(Pt 1):29–36. doi: 10.1099/0022-1317-75-1-29. [DOI] [PubMed] [Google Scholar]
  20. Nieto A., de la Luna S., Bárcena J., Portela A., Valcárcel J., Melero J. A., Ortín J. Nuclear transport of influenza virus polymerase PA protein. Virus Res. 1992 Jun;24(1):65–75. doi: 10.1016/0168-1702(92)90031-4. [DOI] [PubMed] [Google Scholar]
  21. Osterlund M., Luthman H., Nilsson S. V., Magnusson G. Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA. Gene. 1982 Nov;20(1):121–125. doi: 10.1016/0378-1119(82)90093-2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Robertson J. S., Schubert M., Lazzarini R. A. Polyadenylation sites for influenza virus mRNA. J Virol. 1981 Apr;38(1):157–163. doi: 10.1128/jvi.38.1.157-163.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanz-Ezquerro J. J., de la Luna S., Ortín J., Nieto A. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. J Virol. 1995 Apr;69(4):2420–2426. doi: 10.1128/jvi.69.4.2420-2426.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shapiro G. I., Krug R. M. Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J Virol. 1988 Jul;62(7):2285–2290. doi: 10.1128/jvi.62.7.2285-2290.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shi L., Summers D. F., Peng Q., Galarz J. M. Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. Virology. 1995 Apr 1;208(1):38–47. doi: 10.1006/viro.1995.1127. [DOI] [PubMed] [Google Scholar]
  28. Stranden A. M., Staeheli P., Pavlovic J. Function of the mouse Mx1 protein is inhibited by overexpression of the PB2 protein of influenza virus. Virology. 1993 Dec;197(2):642–651. doi: 10.1006/viro.1993.1639. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. de la Luna S., Martín J., Portela A., Ortín J. Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses. J Gen Virol. 1993 Mar;74(Pt 3):535–539. doi: 10.1099/0022-1317-74-3-535. [DOI] [PubMed] [Google Scholar]

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