Mutational analysis of active site residues of chitinase from Bombyx mori nucleopolyhedrovirus.
Infection of Bombyx mori larvae with B. mori nucleopolyhedrovirus (BmNPV) results in liquefaction of the host. This process is attributed to the synergistic action of two virus-encoded genes, chitinase (v-chiA) and cathepsin (v-cath). Previous studies have suggested that Autographa californica nucleopolyhedrovirus (AcMNPV) CATH cannot be processed within infected cells in the absence of AcMNPV CHIA. To investigate the interactions between V-CHIA and V-CATH, we generated a recombinant BmNPV (103ChiAmut) in which the residues of the active site of BmNPV chiA were mutated (D302NE306Q) and the gene was driven by its own promoter at the native locus. Mutation at the active site of BmNPV CHIA resulted in complete loss of chitinolytic activity. Bombyx mori larvae infected with 103ChiAmut survived longer than larvae infected with wild-type BmNPV and did not undergo terminal liquefaction after death. Cysteine protease activity and Western blot analysis showed that, in cells infected with v-chiA-deleted BmNPV (ChiAD), BmNPV CATH was not processed properly and was accumulated as a detergent-insoluble form, suggesting that BmNPV CHIA plays a crucial role in V-CATH processing. In cells infected with 103ChiAmut, BmNPV CATH formed insoluble aggregates, suggesting that active site-mutated BmNPV CHIA loses its additional role as a molecular chaperon during V-CATH processing.
Daimon
Takaaki
T
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
Katsuma
Susumu
S
Shimada
Toru
T
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D012831:Bombyx / Q000821:virology; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis; D007814:Larva / Q000821:virology; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D011499:Protein Processing, Post-Translational; D016019:Survival Analysis; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D014764:Viral Proteins; D002688:Chitinases; D003546:Cysteine Endopeptidases