Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene

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Abstract
The chitinase ofAutographa californicanucleopolyhedrovirus (AcMNPV) is required for the characteristic liquefaction of baculovirus-infected insect larvae. Alignments of the putative active sites of a range of chitinases revealed two highly conserved residues, glutamate and aspartate, which have been proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses were generated, AcchiAD311G, AcchiAE315Gand AcchiAD311G E315G, which contained mutations at either the glutamate, the aspartate or both. It was demonstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the attenuation of chitinolytic activity and the cessation of liquefaction ofTrichoplusia nilarvae infected with AcchiAD311G E315G. Mutagenesis of the glutamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, AcchiAE315G, liquefied more slowly than those larvae infected with wild-type AcMNPV. Mutagenesis of the aspartate residue resulted in a reduction of exochitinase activity but an unexpected enhancement of endochitinolytic activity. Liquefaction of AcchiAD311G-infected larvae was observed at the same time as that of AcMNPV-infected larvae.