The N-Terminal Region of an Entomopoxvirus Fusolin Is Essential for the Enhancement of Peroral Infection, whereas the C-Terminal Region Is Eliminated in Digestive Juice

Abstract

The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn 191 is replaced with Gln, and ⌬SIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu 161 to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.

Recent Advances in Studies for the Application of a Protein Produced by Entomopoxviruses (Poxviridae) for Insect-Pest Control

“…My group has identified the region of AcEPV fusolin, which is involved in the viral enhancing activity by assaying the activity of various AcEPV fusolin mutants using a bioassay system including BmNPV and B. mori larvae 22 . Namely, the N-terminal region (ca.…”

“…Furthermore, Hukuhara et al (2001) 9 expressed a PsEPV fusolin gene that had been introduced into E. coli, and it was found that the protein produced by this gene enhanced PuNPV infection in P. separata larvae by nearly 270-fold. My group has expressed the AcEPV fusolin gene in a baculovirus foreign gene expression system, and found that the resulting protein enhanced BmNPV infection of B. mori third-instar larvae by about 320-fold, but the degree of the enhancement appeared higher in the case of second instar larvae 22 . Furthermore, the enhancement of the infectivity of AcEPV by AcEPV fusolin produced by a baculovirus expression system was observed in A. cuprea larvae 22 .…”

“…250 amino acid residues from the N-terminus of a translated form) containing a signal peptide is essential for the activity and N-glycosylation is important; the rest of the fusolin is non-essential. It was found that the N-terminal region binds to chitin in vitro and that glycosylation is important for chitin binding 22 . This essential region contains some conserved regions among fusolins and GP37s of baculoviruses, and the regions constitute a chitin-binding domain.…”

“…Therefore this domain should bind to chitin, and this binding should be associated with PM disruption, since one main component of the PM is chitin. In addition, the non-essential region is easily eliminated by serine protease(s) in insect digestive juice, whereas the essential region is more stable, and the glycosylation is required for the stability of the N-terminal region 22 . A diagram of the structure of AcEPV fusolin showing the functional region is illustrated in Fig.…”

“…1C), whereas the spindles do not. In fields, spheroids may play a role in the protection of virions against sunlight (ultraviolet radiation) and high temperature 1 , and the function of spindles may be to enhance EPV infectivity 11,14,21,22 .…”