The chitinase of nucleopolyhedrovirus (AcMNPV) is required for the characteristic liquefaction of baculovirus-infected insect larvae. Alignments of the putative active sites of a range of chitinases revealed two highly conserved residues, glutamate and aspartate, which have been proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses were generated, Ac , Ac and Ac , which contained mutations at either the glutamate, the aspartate or both. It was demonstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the attenuation of chitinolytic activity and the cessation of liquefaction of larvae infected with Ac . Mutagenesis of the glutamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, Ac , liquefied more slowly than those larvae infected with wild-type AcMNPV. Mutagenesis of the aspartate residue resulted in a reduction of exochitinase activity but an unexpected enhancement of endochitinolytic activity. Liquefaction of Ac -infected larvae was observed at the same time as that of AcMNPV-infected larvae.


Article metrics loading...

Loading full text...

Full text loading...



  1. Ahrens, C. H., Russell, R. L. Q., Funk, C. J., Evans, J. T., Harwood, S. H. & Rohrmann, G. F. (1997). The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome.Virology 229, 381-399.[CrossRef] [Google Scholar]
  2. Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.Virology 202, 586-605.[CrossRef] [Google Scholar]
  3. Fuchs, R. L., McPherson, S. A. & Drahos, D. J. (1986). Cloning of a Serratia marcescens gene encoding chitinase.Applied and Environmental Microbiology 51, 504-509. [Google Scholar]
  4. Gomi, S., Majima, K. & Maeda, S. (1999). Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus.Journal of General Virology 80, 1323-1337. [Google Scholar]
  5. Gooday, G. W. (1994). Physiology of microbial degradation of chitin and chitosan. In Biochemistry of Microbial Degradation, pp. 279-312. Edited by C. Ratledge. Dordrecht: Kluwer.
  6. Hawtin, R. E., Arnold, K., Ayres, M. D., Zanotto, P. M. D. A., Howard, S. C., Gooday, G. W., Chappell, L. H., Kitts, P. A., King, L. A. & Possee, R. D. (1995). Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.Virology 212, 673-685.[CrossRef] [Google Scholar]
  7. Hawtin, R. E., Zarkowska, T., Arnold, K., Thomas, C. J., Gooday, G. W., King, L. A., Kuzio, J. A. & Possee, R. D. (1997). Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes.Virology 238, 243-253.[CrossRef] [Google Scholar]
  8. Henrissat, B. (1990). Weak sequence homologies among chitinases detected by clustering analysis.Protein Sequences & Data Analysis 3, 523-526. [Google Scholar]
  9. Hunter, F. R., Crook, N. E. & Entwistle, P. F. (1984). Viruses as pathogens for the control of insects. In Microbial Methods for Environmental Biotechnology, pp. 323-347. Edited by J. M. Grainger & J. M. Lynch. New York & London: Academic Press.
  10. Kang, W., Tristem, M., Maeda, S., Crook, N. E. & O’Reilly, D. R. (1998). Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.Journal of General Virology 79, 2283-2292. [Google Scholar]
  11. King, L. A. & Possee, R. D. (1992).The Baculovirus Expression System. A Laboratory Guide. London: Chapman & Hall.
  12. Kuranda, M. J. & Robbins, P. W. (1991). Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. Journal of Biological Chemistry 266, 19758-19767. [Google Scholar]
  13. Kuzio, J., Pearson, M. N., Harwood, S. H., Funk, C. J., Evans, J. T., Slavicek, J. M. & Rohrmann, G. F. (1999). Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar.Virology 253, 17-34.[CrossRef] [Google Scholar]
  14. McCreath, K. J. & Gooday, G. W. (1992). A rapid and sensitive microassay for determination of chitinolytic activity.Journal of Microbiological Methods 14, 229-237.[CrossRef] [Google Scholar]
  15. Malcolm, B. A., Rosenberg, S., Corey, M. J., Allen, J. S., de Baetselier, A. & Kirsch, J. F. (1989). Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.Proceedings of the National Academy of Sciences, USA 86, 133-137.[CrossRef] [Google Scholar]
  16. Molano, J., Duran, A. & Cabib, E. (1977). A rapid and sensitive assay for chitinase using tritiated chitin.Analytical Biochemistry 83, 648-656.[CrossRef] [Google Scholar]
  17. Ohkawa, T., Majima, K. & Maeda, S. (1994). A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.Journal of Virology 68, 6619-6625. [Google Scholar]
  18. Payne, G., Ahl, P., Moyer, M., Harper, A., Beck, J., Meins, F.Jr & Ryals, J. (1990). Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.Proceedings of the National Academy of Sciences, USA 87, 98-102.[CrossRef] [Google Scholar]
  19. Perrakis, A., Tews, I., Dauter, Z., Oppenheim, A. B., Chet, I., Wilson, K. S. & Vorgias, C. E. (1994). Crystal structure of a bacterial chitinase at 2·3 Å resolution.Structure 2, 1169-1180.[CrossRef] [Google Scholar]
  20. Possee, R. D. (1986). Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector.Virus Research 5, 43-59.[CrossRef] [Google Scholar]
  21. Possee, R. D. & Howard, S. C. (1987). Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus.Nucleic Acids Research 15, 10233-10248.[CrossRef] [Google Scholar]
  22. St Leger, R. J., Staples, R. C. & Roberts, D. W. (1993). Entomopathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Aspergillus flavus produce multiple extracellular chitinase isozymes.Journal of Invertebrate Pathology 61, 81-84.[CrossRef] [Google Scholar]
  23. Slack, J. M., Kuzio, J. & Faulkner, P. (1995). Characterisation of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.Journal of General Virology 76, 1091-1098.[CrossRef] [Google Scholar]
  24. Sticher, L., Hofsteenge, J., Milani, A., Neuhaus, J. M. & Meins, F.Jr (1992). Vacuolar chitinases of tobacco: a new class of hydroxyproline-containing proteins.Science 257, 655-657.[CrossRef] [Google Scholar]
  25. Vaughn, J. L., Goodwin, R. H., Tompkins, G. J. & McCawley, P. (1977). The establishment of two insect lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae).In Vitro 13, 213-217.[CrossRef] [Google Scholar]
  26. Watanabe, T., Oyanagi, W., Suzuki, K. & Tanaka, H. (1990). Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.Journal of Bacteriology 172, 4017-4022. [Google Scholar]
  27. Watanabe, T., Oyanagi, W., Suzuki, K., Ohnishi, K. & Tanaka, H. (1992). Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases.Journal of Bacteriology 174, 408-414. [Google Scholar]
  28. Watanabe, T., Kobori, K., Miyashita, K., Fujii, T., Sakai, H., Uchida, M. & Tanaka, H. (1993). Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity.Journal of Biological Chemistry 268, 18567-18572. [Google Scholar]

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