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Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.

A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66.7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopolyhedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system.