title: Role of interactions between Autographa californica multiple nucleopolyhedrovirus procathepsin and chitinase chitin-binding or active-site domains in viral cathepsin processing.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Hodgson
authors.forename: Basil M
authors.initials: BM
authors.lastname: Arif
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Krell
mesh_terms: D002403:Cathepsins / Q000378:metabolism*; D002498:Centrifugation / Q000379:methods; D002688:Chitinases / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D018832:Molecular Chaperones / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational
chemical_list: D018832:Molecular Chaperones; D049452:Green Fluorescent Proteins; D002688:Chitinases; D002403:Cathepsins