title: Interaction of Autographa californica multiple nucleopolyhedrovirus cathepsin protease progenitor (proV-CATH) with insect baculovirus chitinase as a mechanism for proV-CATH cellular retention.
abstract: The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA-proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA-green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA-proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA-proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, 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: D000818:Animals; D002460:Cell Line; D002688:Chitinases / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007313:Insecta; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D002688:Chitinases; D003546:Cysteine Endopeptidases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C417028:V-CATH protein, Autographa californica nucleopolyhedrovirus