Mutagenesis of the active site coding region of the Autographa californicanucleopolyhedrovirus chiA gene

The chitinase of Autographa californica nucleopolyhedrovirus (AcMNPV) is re quired for the characteristic liquefaction of baculovirus-infected insect l arvae. Alignments of the putative active sites of a range of chitinases rev ealed two highly conserved residues, glutamate and aspartate, which have be en proposed to constitute the catalytic residues of the active site. These residues were mutated in the AcMNPV chitinase. Three recombinant viruses we re generated, AcchiA(D311G), AcchiA(E315G) and AcchiA(D31G E315G), which co ntained mutations at either the glutamate, the aspartate or both. It was de monstrated that chitinase protein production was unaffected by the mutation of these residues. However, mutation of both residues resulted in the atte nuation of chitinolytic activity and the cessation of liquefaction of Trich oplusia ni larvae infected with AcchiA(D311G) (E315G) Mutagenesis of the gl utamate residue led to a reduction in exochitinase activity and a delay in the appearance of endochitinase activity. In addition, larvae infected with this virus, AcchiA(E315G) 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 endochi tinolytic activity. Liquefaction of AcchiA(D311G)-infected larvae was obser ved at the same time as that of AcMNPV-infected larvae.