Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif.
During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.
Saville
Giles P
GP
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK1.
Thomas
Carole J
CJ
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK1.
Possee
Robert D
RD
NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK2.
King
Linda A
LA
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK1.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002688:Chitinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000821:virology; D007814:Larva / Q000254:growth & development / Q000821:virology; D007915:Lepidoptera / Q000254:growth & development / Q000821:virology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D009842:Oligopeptides / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D018411:Spodoptera; D015996:Survival Rate; D013997:Time Factors
D009842:Oligopeptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; D002688:Chitinases; D003546:Cysteine Endopeptidases