Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 May;9(5):1347–1353. doi: 10.1002/j.1460-2075.1990.tb08249.x

Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing.

J C Carrington 1, D D Freed 1, C S Oh 1
PMCID: PMC551818  PMID: 2184028

Abstract

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus-encoded proteinases (NIa and HC-Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N-terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC-Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC-Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC-Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC-Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC-Pro had no effect on N-terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC-Pro N-terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.

Full text

PDF
1347

Images in this article

1348Fig. 1.
on p.1348
1349Fig. 2.
on p.1349
1349Fig.3
on p.1349
1350Fig.4
on p.1350
1350Fig.5
on p.1350
1351Fig.6
on p.1351
1351Fig.7
on p.1351

Selected References

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

  1. A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  2. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  3. Carrington J. C., Cary S. M., Dougherty W. G. Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase. J Virol. 1988 Jul;62(7):2313–2320. doi: 10.1128/jvi.62.7.2313-2320.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrington J. C., Cary S. M., Parks T. D., Dougherty W. G. A second proteinase encoded by a plant potyvirus genome. EMBO J. 1989 Feb;8(2):365–370. doi: 10.1002/j.1460-2075.1989.tb03386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrington J. C., Dougherty W. G. Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol. 1987 Aug;61(8):2540–2548. doi: 10.1128/jvi.61.8.2540-2548.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carrington J. C., Freed D. D. Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region. J Virol. 1990 Apr;64(4):1590–1597. doi: 10.1128/jvi.64.4.1590-1597.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carrington J. C., Freed D. D., Sanders T. C. Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro. J Virol. 1989 Oct;63(10):4459–4463. doi: 10.1128/jvi.63.10.4459-4463.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dougherty W. G., Carrington J. C., Cary S. M., Parks T. D. Biochemical and mutational analysis of a plant virus polyprotein cleavage site. EMBO J. 1988 May;7(5):1281–1287. doi: 10.1002/j.1460-2075.1988.tb02942.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dougherty W. G., Cary S. M., Parks T. D. Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology. 1989 Aug;171(2):356–364. doi: 10.1016/0042-6822(89)90603-x. [DOI] [PubMed] [Google Scholar]
  11. Dougherty W. G., Parks T. D. Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays. Virology. 1989 Sep;172(1):145–155. doi: 10.1016/0042-6822(89)90116-5. [DOI] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. García J. A., Riechmann J. L., Laín S. Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli. Virology. 1989 Jun;170(2):362–369. doi: 10.1016/0042-6822(89)90426-1. [DOI] [PubMed] [Google Scholar]
  14. Hellmann G. M., Shaw J. G., Rhoads R. E. In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease. Virology. 1988 Apr;163(2):554–562. doi: 10.1016/0042-6822(88)90296-6. [DOI] [PubMed] [Google Scholar]
  15. Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Neurath H. Evolution of proteolytic enzymes. Science. 1984 Apr 27;224(4647):350–357. doi: 10.1126/science.6369538. [DOI] [PubMed] [Google Scholar]
  18. Nicklin M. J., Kräusslich H. G., Toyoda H., Dunn J. J., Wimmer E. Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4002–4006. doi: 10.1073/pnas.84.12.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Strebel K., Beck E. A second protease of foot-and-mouth disease virus. J Virol. 1986 Jun;58(3):893–899. doi: 10.1128/jvi.58.3.893-899.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Töpfer R., Matzeit V., Gronenborn B., Schell J., Steinbiss H. H. A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res. 1987 Jul 24;15(14):5890–5890. doi: 10.1093/nar/15.14.5890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ypma-Wong M. F., Filman D. J., Hogle J. M., Semler B. L. Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs. J Biol Chem. 1988 Nov 25;263(33):17846–17856. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES