1887

Abstract

The replication of coronaviruses involves proteolytic processing of the gene 1 translation products, pp1a and pp1ab. One of the key enzymes in this process is predicted to be a virus-encoded 3C-like proteinase. In this report, we describe a bacterial system that has allowed us to express and characterize a recombinant murine coronavirus (MHV- JHM) 3C-like proteinase. The partially purified protein has been shown to exhibit proteolytic activity in and mutation analysis has been used to demonstrate the indispensability of Cys- 3495 for enzymatic activity. Finally, the effect of class-specific proteinase inhibitors on the cleavage activity of the MHV 3C-like proteinase has been used to demonstrate the functional and structural homology of this enzyme to the picorna- virus 3C proteinases.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-78-1-71
1997-01-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/78/1/9010287.html?itemId=/content/journal/jgv/10.1099/0022-1317-78-1-71&mimeType=html&fmt=ahah

References

  1. Baum E. Z., Bebernitz G. A., Palant O., Mueller T., Plotch S. J. 1991; Purification, properties, and mutagenesis of poliovirus 3C protease. Virology 185:140–150
    [Google Scholar]
  2. Boursnell M. E. G., Brown T. D. K., Foulds I. J., Green P. F., Tomley F. M., Binns M. M. 1987; Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. Journal of General Virology 68:57–77
    [Google Scholar]
  3. Bredenbeek P. J., Pachuk C. J., Noten A. F. H., Charité J., Luytjes W., Weiss S. R., Spaan W. J. M. 1990; The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. Nucleic Acids Research 18:1825–1832
    [Google Scholar]
  4. Brierley I., Boursnell M. E. G., Binns M. M., Billimoria B., Blok V. C., Brown T. D. K., Inglis S. C. 1987; An efficient ribosomal frame- shifting signal in the polymerase-encoding region of the coronavirus IBV. EMBO Journal 6:3779–3785
    [Google Scholar]
  5. Brierley I., Digard P., Inglis S. C. 1989; Characterization of an efficient coronavirus ribosomal frameshift signal: requirement for an RNA pseudoknot. Cell 57:537–547
    [Google Scholar]
  6. Eleouet J.-F, Lambert P., Levy L., Vende P., Laude H. 1995; Complete sequence (20 kilobases) of the polyproteinencoding gene 1 of transmissible gastroenteritis virus. Virology 206:817–822
    [Google Scholar]
  7. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. 1989; Coronavirus genome : prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Research 17:4847–4861
    [Google Scholar]
  8. Grötzinger C., Heusipp G., Ziebuhr J., Harms U., Süss J., Siddell S. G. 1996; Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus 229E. Virology 222:227–235
    [Google Scholar]
  9. Herold J., Siddell S. G. 1993; An elaborated pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA. Nucleic Acids Research 21:5838–5842
    [Google Scholar]
  10. Herold J., Raabe T., Schelle-Prinz B., Siddell S. G. 1993; Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology 195:680–691
    [Google Scholar]
  11. Herold J., Siddell S. G., Ziebuhr J. 1996; Characterization of coronavirus RNA polymerase gene products. Methods in Enzymology 275: in press
    [Google Scholar]
  12. Lee H.-J., Shieh C.-K., Gorbalenya A. E., Koonin E. V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M. M. C. 1991; The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180:567–582
    [Google Scholar]
  13. Liu D. X., Brown T. D. K. 1995; Characterization and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein. Virology 209:420–427
    [Google Scholar]
  14. Liu D. X., Brierley I., Tibbles K. W., Brown T. D. K. 1994; A 100- kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. Journal of Virology 68:5772–5780
    [Google Scholar]
  15. Lu Y., Lu X., Denison M. R. 1995; Identification and characterization of a serine-like proteinase of the murine coronavirus MHV- A59. Journal of Virology 69:3554–3559
    [Google Scholar]
  16. Malcolm B. A., Chin S. M., Jewell D. A., Stratton-Thomas J. R., Thudium K. B., Ralston R., Rosenberg S. 1992; Expression and characterization of recombinant hepatitis A virus 3C proteinase. Biochemistry 31:3358–3363
    [Google Scholar]
  17. Tibbles K. W., Brierley I., Cavanagh D., Brown T. D. K. 1995; A region of the infectious bronchitis virus 1a polyprotein encoding the 3C- like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate. Journal of General Virology 76:3059–3070
    [Google Scholar]
  18. Tibbles K. W., Brierley I., Cavanagh D., Brown T. D. K. 1996; Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus. Journal of Virology 70:1923–1930
    [Google Scholar]
  19. Yao Z., Jones D. H., Grose C. 1992; Site-directed mutagenesis of herpesvirus glycoprotein phosphorylation sites by recombination polymerase chain reaction. PCR Methods and Applications 1:205–207
    [Google Scholar]
  20. Ziebuhr J., Herold J., Siddell S. G. 1995; Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. Journal of Virology 69:4331–4338
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-78-1-71
Loading
/content/journal/jgv/10.1099/0022-1317-78-1-71
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