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Journal of Virology, December 2008, p. 12406-12415, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01605-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Yutaka Takemoto,{dagger} Wataru Mitsuhashi,* Ritsuko Murakami, Hirosato Konishi, and Kazuhisa Miyamoto

National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan

Received 28 July 2008/ Accepted 24 September 2008

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 Asn191 is replaced with Gln, and {Delta}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-Glu161 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.

* Corresponding author. Mailing address: National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan. Phone: 81 29 838 6081. Fax: 81 29 838 6028. E-mail: mitsuhas{at}affrc.go.jp

{triangledown} Published ahead of print on 1 October 2008.

{dagger} Present address: National Institute of Advanced Industrial Science and Technology, Japan.

Journal of Virology, December 2008, p. 12406-12415, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01605-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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