Reprogramming the chiA expression profile of Autographa californica multiple nucleopolyhedrovirus

Abstract

Expression of chiA and v-cath RNA and enzyme activity in wild-type Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was compared with that of recombinant AcMNPV viruses reprogrammed for expression of the endogenous chiA. To establish a baseline for our recombinant AcMNPV studies, we compared, for the first time, the temporal expression profiles of both AcMNPV chiA transcription and translation simultaneously. The rate of intracellular chitinase accumulation during AcMNPV infection followed the same pattern observed for chiA transcription but was delayed by about 6 h. Replacement of 21 nucleotides containing the native late chiA and v-cath promoters with a selectable polh–EGFP cassette was sufficient to eliminate expression of both chiA and v-cath. Viruses were generated that express chiA from either the late p6.9 or very late polh promoters of AcMNPV, replacing the native chiA promoter. There was a marked difference in the temporal chiA transcription profiles from the native, p6.9 and polh promoters, resulting in respective specific activities of chitinase at 48 h p.i. of 62, 160 and 219 mU (mg lysate total protein)−1. Based on temporal analysis of v-cath transcription by Northern blot, AcMNPV v-cath was transcribed from 9 h p.i. in Sf21 cells. However, expression of v-cath RNA or enzyme from a reconstructed v-cath promoter in the chiA-reprogrammed viruses was not detected at 48 h of virus replication. Reprogramming for increased chitinase (and putatively cathepsin) expression with native baculovirus promoters might provide a means for designing environmentally benign biological insecticides.