Spindles of an Entomopoxvirus Facilitate Its Infection of the Host Insect by Disrupting the Peritrophic Membrane

Abstract

The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.Entomopoxviruses (EPVs) are poxviruses that infect insects of orders such as Coleoptera, Lepidoptera, Orthoptera, and Diptera. Many EPVs form two types of proteinaceous crystalline bodies: spheroids and spindles. The spheroid contains virions, whereas the spindle does not; thus, spheroids are agents of infection. The major constituent proteins of the spheroid and spindle are spheroidin and fusolin, respectively, and these two are the most abundant proteins among those produced by EPVs. Peroral administration of the spindles of a coleopteran EPV, Anomala cuprea EPV (AcEPV), to Bombyx mori and Spilosoma imparilis, lepidopteran hosts of two species of nucleopolyhedroviruses (NPVs), enhances the infectivity of not only the occluded NPVs of these two species (16, 19) but also of a nonoccluded NPV of B. mori (4), although AcEPV itself cannot infect these two insects. However, these are artificial phenomena that occur by experimental combination of the spindles with NPVs; these insects on plants rarely ingest AcEPV spindles that are present in soils, together with the NPVs. Also, this mechanism of enhanced infectivity of NPVs is considered to be worthless for the survival of AcEPVs that cannot infect the insects. NPVs and EPVs are taxonomically distant DNA insect viruses. NPVs belong to the family Baculoviridae, whereas EPVs belong to the Poxviridae. However, AcEPV spindles also enhance the infectivity of AcEPV in its host insect, the larva of the cupreous chafer A. cuprea, strongly suggesting that the natural biological function of EPV spindles is to enhance the infectivity of EPVs (15,20). (17) showed that the peritrophic membrane (PM) of B. mori larvae was disinte...

Peritrophic membrane structure of Aedes aegypti (Diptera: Culicidae) mosquitoes after infection with dengue virus type 2 (D2-16681)

“…Litomosoides chagasfilhoi microfilariae can invade the midgut epithelium during PM development of Culex quinquefasciatus, but when the PM was fully formed, microfilariae were no longer able to cross it (Santos et al, 2006). On the other hand, it may belong to the third strategy as Mitsuhashi et al (2007) demonstrated that fusolin, the constitutive protein of the spindles of entomopoxvirus, may bind with chitin in the PM, thus enhancing disruption in the host insect. In larval Ae.…”

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

“…Infected cells were harvested 72 h postinoculation. Preliminary Western blot analysis results using an antibody to the AncuEPV spindles (26) indicated that the recombinant fusolin was contained only in the insoluble fraction of infected cells. Therefore, protein purification was performed under a denaturing condition as described below.…”

“…Precision Plus all blue standard (Bio-Rad, Hercules, CA) was used as molecular mass standard. For Western blot analysis, protein samples were run on a low-bis polyacrylamide gel and blotted onto a polyvinylidene difluoride (PVDF) membrane (FluoroTrans; (26) in Tris-buffered saline containing 0.05% Tween 20 at room temperature for 1 h. Binding of the antibody was probed with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (Promega, Madison, WI) and visualized using a 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (BCIP-NBT) solution kit for alkaline phosphatase staining (Nakalai Tesque, Tokyo, Japan).…”

“…The spindles of several EPVs have been shown to strongly enhance the infectivity of several insect viruses (2,9,23,25,27,29,30,44). We previously demonstrated that the peritrophic membranes (PMs) of Bombyx mori and Anomala cuprea larvae are disintegrated by peroral administration of Anomala cuprea entomopoxvirus (AncuEPV) spindles (26,27). Thus, disintegration of the PM is thought to be a mechanism by which spindles enhance virus infectivity.…”

The Baculoviral Genome

“…Fusolin was demonstrated to form spindle-like bodies that enhance the entomopoxvirus oral infection in host larvae. The mechanism of action for these spindles appears to be associated with the disruption of the peritrophic membrane (PM) allowing the virions to reach the midgut epithelial cells (Mitsuhashi et al, 2007). Except for the case of CfDEFMNPV (Li et al, 2000), in baculoviruses no spindle bodies have been observed, although GP37 was found to be associated with OBs in AcMNPV (Vialard et al, 1990).…”