The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process.
ABSTRACT The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.
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ABSTRACT: The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac78 gene is one of the baculovirus core genes. Recent studies showed that ac78 is essential for budded virion (BV) production and the embedding of occlusion-derived virion (ODV) into occlusion body during the AcMNPV life cycle. Here, we report that an ac78-knockout AcMNPV (vAc78KO) constructed in this study had different phenotypes than those described in the previous studies. A few infectious BVs were detected using titer assays, immunoblot analyses and plaque assays, indicating that ac78 is not essential for BV formation. Electron microscopy confirmed that the ac78 deletion did not affect nucleocapsid assembly and ODV formation. However, the numbers of multiple nucleocapsid-enveloped ODVs and ODV-embedded occlusion bodies were significantly decreased. Subsequently, the highly conserved amino acid residues 2-25 and 64-88 of Ac78, which are homologous to an oxidoreductase and cytochrome c oxidase, respectively, were demonstrated to play a crucial role in the morphogenesis of multiple nucleocapsid-enveloped ODV. Immunoblot analysis found that Ac78 was an ODV envelope-associated protein. Consistently, amino acid residues 56-93 of Ac78 were identified as an inner nuclear membrane sorting motif, which may direct the localization of Ac78 to the ODV envelope. In vivo infectivity assays showed that the occlusion bodies of vAc78KO were unable to establish primary infection in the midgut of Trichoplusia ni larvae. Taken together, our results suggest that ac78 plays an important role in BV production and proper multiple nucleocapsid-enveloped ODV formation, as well as AcMNPV primary infection in vivo.Virus Research 07/2014; · 2.83 Impact Factor
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ABSTRACT: Autographa californica nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene, and its function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that the deletion of the chitin-binding domain of Ac83 resulted in the failure of AcMNPV oral infection of Trichoplusia ni larvae but AcMNPV remained infectious following intrahemocoelic injection, implying that the domain is involved in the binding of occlusion-derived virion to the peritrophic membrane and/or other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelope of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.Journal of Virology 07/2013; · 4.65 Impact Factor
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ABSTRACT: The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections.Virus Genes 12/2013; · 1.84 Impact Factor