1887

Abstract

A binary expression system has been used to study the pathway for proteolytic processing of the plum pox potyvirus (PPV) polyprotein. Trans cleavage at the carboxyl end of the cylindrical inclusion protein occurred, although with lower efficiency than that at the large nuclear inclusion protein-capsid protein junction. No trans cleavage at the carboxyl end of the small nuclear inclusion protein (NIa) was detected. The proteolytic activities at different cleavage sites of several deletion and point mutations of NIa protein have been analysed. The large ΔSX deletion and two different point mutations at His 239 abolished proteolytic activity at all sites. The effect of other mutations, particularly a Glu substitution for Asp 274, depended on the particular cleavage site analysed. The results obtained with the PPV NIa protein mutants were similar to those reported for comparable mutations in the tobacco etch virus 49K protease, despite differences in the sequences recognized for processing. No evident competitive inhibition of the proteolytic activity of PPV NIa protease by the presence of an excess of the different protease mutants could be demonstrated.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-12-2773
1990-12-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/12/JV0710122773.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-12-2773&mimeType=html&fmt=ahah

References

  1. Carrington J. C., Dougherty W. G. 1987; Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. Journal of Virology 61:2540–2548
    [Google Scholar]
  2. Carrington J. C., Dougherty W. G. 1988; A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processingr. Proceedings of the National Academy of Sciences U.S.A: 853391–3395
    [Google Scholar]
  3. Carrington J. C., Cary S. M., Dougherty W. G. 1988; Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49 kilodalton proteinase. Journal of Virology 62:2313–2320
    [Google Scholar]
  4. Carrington J. C., Cary S. M., Dougherty W. G. 1989; A second proteinase encoded by a plant potyvirus genome. EMBO Journal 8:365–370
    [Google Scholar]
  5. Dougherty W. G., Carrington J. C. 1988; Expression and function of potyviral gene products. Annual Review of Phytopathology 26:123–143
    [Google Scholar]
  6. Dougherty W. G., Parks T. D. 1989; 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 172:145–155
    [Google Scholar]
  7. Dougherty W. G., Carrington J. C., Cary S. M., Parks T. D. 1988; Biochemical and mutational analysis of a plant virus polyprotein cleavage site. EMBO Journal 7:1281–1287
    [Google Scholar]
  8. Dougherty W. G., Cary S. M., Parks T. D. 1989a; Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 171:356–364
    [Google Scholar]
  9. Dougherty W. G., Parks T. D., Cary S. M., Bazan J. F., Fletterick R. J. 1989b; Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase. Virology 172:302–310
    [Google Scholar]
  10. García J. A., Riechmann J. L., Laín S. 1989a; Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli. Virology 170:362–369
    [Google Scholar]
  11. García J. A., Riechmann J. L., Laín S. 1989b; Artificial cleavage site recognized by plum pox potyvirus protease in Escherichia coli. Journal of Virology 63:2457–2460
    [Google Scholar]
  12. García J. A., Riechmann J. L., Martín M. T., Laín S. 1989c; Proteolytic activity of the plum pox potyvirus NIa-protein on excess of natural and artificial substrates in Escherichia coli. FEBS tetters 257:269–273
    [Google Scholar]
  13. Hellmann G. M., Shaw J. G., Rhoads R. E. 1988; In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus encoded protease. Virology 163:554–562
    [Google Scholar]
  14. Hiebert E., Purcifull D. E., Christie R. G. 1984; Purification and immunological analysis of plant viral inclusion bodies. In Methods in Virology VIII pp 225–280 Maramorosch K., Koprowski H. Orlando: Academic Press;
    [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
    [Google Scholar]
  16. Laín S., Riechmann J. L., García J. A. 1989; The complete nucleotide sequence of plum pox potyvirus RNA. Virus Research 13:157–172
    [Google Scholar]
  17. Martín M. T., LóPez-Otín C., Laín S., García J. A. 1990; Determination of polyprotein processing sites by amino-terminal sequencing of nonstructural proteins encoded by plum pox potyvirus. Virus Research 15:97–106
    [Google Scholar]
  18. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103–119
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-12-2773
Loading
/content/journal/jgv/10.1099/0022-1317-71-12-2773
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error