Deletion of the Autographa californica nucleopolyhedrovirus chitinase KDEL motif and in vitro and in vivo analysis of the modified virus.
Infection of insect larvae with Autographa californica nucleopolyhedrovirus (AcMNPV) results in the liquefaction of the host, a process involving the action of virus-encoded chitinase and cathepsin gene products. Chitinase is localized in the endoplasmic reticulum (ER) during infection because of the presence of a C-terminal ER retrieval motif (KDEL). In this study, the KDEL coding region was removed from the chitinase gene so that expression of the modified chitinase remained under the control of its own gene promoter, at its native locus. The deletion of KDEL resulted in the redistribution of chitinase within the cell during virus infection. Chitinase lacking the KDEL motif was detectable at the plasma membrane and was also evident in the culture medium of virus-infected cells from as early as 12 h post-infection (p.i.). Secretion of chitinase from the cell continued up to 72 h p.i., until cytolysis. The biological activity of the recombinant virus in Trichoplusia ni larvae was enhanced, with a significant reduction in the lethal dose and lethal time associated with infection. Furthermore, a reduction in feeding damage caused by infected larvae was observed compared to AcMNPV-infected individuals.
Saville
Giles P
GP
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK.
Patmanidi
Alexandra L
AL
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK.
Possee
Robert D
RD
NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK.
King
Linda A
LA
School of Biological and Molecular Sciences, Gipsy Lane Campus, Oxford Brookes University, Oxford OX3 0BP, UK.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D009842:Oligopeptides; D021382:Protein Sorting Signals; D017384:Sequence Deletion; D018411:Spodoptera; D014774:Virulence
D004279:DNA, Viral; D009842:Oligopeptides; D021382:Protein Sorting Signals; C073762:lysyl-aspartyl-glutamyl-leucine; D002688:Chitinases