IDENTIFICATION AND CHARACTERIZATION OF THE CYDIA-POMONELLA GRANULOVIRUS CATHEPSIN AND CHITINASE GENES

A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpG V) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs), ORF7L is pred icted to encode a 66.7 kDa protein (594 amino acid residues) that is 5 7% identical (amino acid sequence) to the chiA gene (ORF126) of Autogr apha 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 r elated to AcMNPV ORF145 of unknown function. Phylogenetic trees were c onstructed for both chitinase and cathepsin sequences from baculovirus es and other species. In both cases, the baculovirus sequences were mo nophyletic but with a deep division between the GVs and NPVs, suggesti ng both genes were present in an ancestral virus prior to the separati on of the two genera, However, these studies did not provide definitiv e evidence for the origin of either protein in baculoviruses. To inves tigate CpGV cathepsin function, a rescue experiment was performed usin g 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 Bm NPV infected insects, confirming that CpGV cath encodes a functional c athepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infect ed cells showed that CpGV cath transcription was initiated from a cons ensus late transcription motif (ATAAG) within the CpGV sequences, indi cating that a CpGV late promoter motif was recognized in this NPV syst em.