Canadian Forest Service Publications
Role of interactions between Autographa californica multiple nucleopolyhedrovirus procathepsin and chitinase chitin-binding or active-site domains in viral cathepsin processing. 2013. Hodgson, J.J.; Arif, B.M.; Krell, J. Journal of Virology (6) 87:3471-3483.
Available from: Great Lakes Forestry Centre
Catalog ID: 34615
CFS Availability: PDF (request by e-mail)
The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.
Plain Language Summary
We studied the molecular biology of a virus that affects the alfalfa looper. The enzymes procathepsin and chitinase work together to help the spread of virus from insect to insect. Chitinase will bind to procathepsin and together they release enzymes that will attack insect tissues until the infected host becomes liquefied. There are two domains where chitinase will bind to procathespin. This paper studies whether both domains are needed or if one can be deleted without affecting enzyme activity.
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