Identification and preliminary characterization of a chitinase gene in the Epinotia aporema granulovirus genome

SALVADOR RICARDO; FERRELLI MARÍA LETICIA; BERRETTA MARCELO FACUNDO; ROMANOWSKI VÍCTOR; SCIOCCO-CAP ALICIA

Trabzon

Congreso; 43th Annual Meeting of the Society for Invertebrate Pathology. 10th International Colloquium on Invertebrate Pathology and Microbial Control. The Final Meeting of COST862: Bacterial Toxins for Insect Control; 2010

Society for Invertebrate Pathology

The main function of baculoviral chitinase (V-CHIA) protein is to promote the final liquefaction of infected host larvae, facilitating the dispersion of the occlusion bodies in the environment. Among the twelve Granulovirus complete genome sequences available in the GenBank, only six appear to contain v-chiA. In this work, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and preliminarily characterized. The 1713 base-pair-long open reading frame (ORF) encodes a protein with a predicted molecular weight of 63 kDa. EpapGV CHIA sequence alignment resulted 61% identical to CpGV and AgSeGV V-CHIA, and Blastp search revealed high conservation among all baculovirus chitinases. Amino acid sequence analysis indicates that the C-terminal KDEL endoplasmic reticulum retention motif, which is conserved in most NPV chitinases, is absent in EpapGV V-CHIA, as also described for CpGV V-CHIA. The EpapGV v-chiA was cloned into a transfer vector which was co-transfected with a defective AcMNPV bacmid (bApGOZA) in order to generate a recombinant Ac-chiAEpapGV. Over-expression of chitinase was analyzed by SDS-PAGE and Western blot. Consistent with the absence of KDEL motif, it was detected in the supernatant at 48 h post-infection. Ac-chiAEpapGV polyhedra were purified, and the presence of chitinase was detected by Western blot. Enzyme activity assays in the infected cell supernatants were maximal between 27°C and 37°C. The enzyme remained active throughout the pH range 5-10. Finally, the effect of the over-expression of EpapGV chiA on peritrophic membranes and larval mortality response was evaluated.