A bacterially produced virus enhancing factor from an entomopoxvirus enhances nucleopolyhedrovirus infection in armyworm larvae.

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.

Enhanced fusion of a nucleopolyhedrovirus with cultured cells by a virus enhancing factor from an entomopoxvirus.

Fusion of Pseudaletia unipuncta nucleopolyhedrovirus with an armyworm cell line (SIE-MSH-805-F) was studied by means of three fluorescence assays that are based on the relief of fluorescence self-quenching of octadecylrhodamine B chloride (R18). A gradual increase in fluorescence intensity indicative of virus-cell fusion was observed by spectrofluorometry when R18-labeled polyhedron-derived virus was incubated with cultured cells. The fusion was enhanced by the virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus. Lysosomotropic agents had little effect on the virus-cell fusion. The percentage of positively fluorescent cells, as determined by flow cytometry, gradually increased after the addition of labeled virus and was higher in the presence of the EF than in its absence. Confocal microscopy of cultured cells that had been combined with labeled virus showed that the fluorescence appeared first on their surface. The plasma membrane of cultured cells had specific affinity to the EF, as revealed by indirect immunofluorescence microscopy.

Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an entomopoxvirus gene.

We have introduced an entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.

Detection of a virus enhancing factor in the spheroid, spindle, and virion of an entomopoxvirus.

Spheroids, spindles, and virions of an entomopoxvirus (EPV) enhanced the infectivity of a nuclear polyhedrosis virus (NPV) when they were perorally administered to larvae of the armyworm, Pseudaletia separata. Spheroids and spindles at the same dose exhibited nearly the same enhancing activity. When the dose of spheroids or spindles was reduced 10 times, the median infectious dose of the NPV was increased approximately 100 times. An antiserum against an enhancing factor detected the homologous antigen in spheroids, spindles, and tissue-derived EPV virions but not in spheroid-derived virions.