We have identified a novel protein that promotes nucleopolyhedrovirus (BmNPV) replication . This protein was purified from heat-treated haemolymph of larvae by gel filtration and ion exchange chromatography, and designated as promoting protein (PP). The molecular mass of native PP estimated by column chromatography and that of denatured PP estimated by SDS–PAGE were 9600 Da and 15200 Da, respectively, suggesting that native PP is composed of a single polypeptide and may behave in the column as if it is a smaller protein because of its conformation and/or adsorptive nature. Addition of the PP to the culture medium of SES-BoMo-15A cells derived from embryos resulted in the strong promotion of BmNPV replication. The promoting activity positively correlated with the amount of PP in the culture medium up to 1 μg/ml, above which maximum virus replication occurred and resulted in the highest budded virus production and polyhedrin promoter-mediated luciferase gene expression of 10000-fold and 6000-fold higher than those without PP, respectively. A cDNA encoding the PP precursor (prePP) was successfully cloned and sequenced. Comparison between the amino acid sequence deduced from the nucleotide sequence of prePP cDNA and the N-terminal 18 amino acids determined for the purified PP indicated that the prePP (154 amino acids) consisted of a mature PP polypeptide (136 amino acids) with a signal sequence at the N terminus. Recombinant PP expressed from the cDNA using a baculovirus vector was similar in molecular mass, immunoreactivity and promoting activity to the native PP.


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  1. Derksen, A. C. G. & Granados, R. R. (1988). Alteration of a lepidopteran peritrophic membrane by baculovirus and enhancement of viral infection.Virology 167, 242-250.[CrossRef] [Google Scholar]
  2. de Wet, J. R., Wood, K. V., Deluca, M., Helinski, D. R. & Subramani, S. (1987). Firefly luciferase gene: structure and expression in mammalian cells.Molecular and Cellular Biology 7, 725-737. [Google Scholar]
  3. Gallo, L. G., Corsaro, B. G., Hughies, P. R. & Granados, R. R. (1991). In vitro enhancement of baculovirus infection by the viral enhancing factor of a granulosis of the cabbage looper, Trichoplusia ni. (Lepidoptera: Noctuidae).Journal of Invertebrate Pathology 58, 203-210.[CrossRef] [Google Scholar]
  4. Gardiner, G. R. & Stockdale, H. (1975). Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis virus.Journal of Invertebrate Pathology 25, 363-370.[CrossRef] [Google Scholar]
  5. Grace, T. D. C. (1958). Effects of various substances on growth of silkworm tissue in vitro.Australian Journal of Biological Sciences 11, 407-417. [Google Scholar]
  6. Grace, T. D. C. (1962). Establishment of four strains of cells from insect tissues grown in vitro.Nature 195, 788-789.[CrossRef] [Google Scholar]
  7. Grace, T. D. C. (1967). Establishment of a line of cells from the silkworm, Bombyx mori.Nature 216, 613. [Google Scholar]
  8. Granados, R. R. & McKenna, K. A. (1995). Insect cell culture methods and their use in virus research. In Baculovirus Expression System and Biopesticides, pp. 13-39. Edited by M. L. Shuler, H. A. Wood, R. R. Granados & D. A. Hammer. New York: Wiley-Liss.
  9. Hukuhara, T. & Zhu, Y. (1989). Enhancement of the in vitro infectivity of a nuclear polyhedrosis virus by a factor in the capsule of a granulosis virus.Journal of Invertebrate Pathology 54, 71-78.[CrossRef] [Google Scholar]
  10. Hukuhara, T., Tamura, K., Zhu, Y., Abe, H. & Tanada, Y. (1987). Synergistic factors show specificity in enhancing nuclear polyhedrosis virus infections.Applied Entomology and Zoology 22, 235-236. [Google Scholar]
  11. Inoue, H. & Mitsuhashi, J. (1988). Characterization of a new continuous cell line from silkworm (Bombyx mori) embryos.Applied Entomology and Zoology 23, 8-14. [Google Scholar]
  12. Inoue, H., Shimizu, K. & Kuwabara, N. (1989).In vitro effect of insect hemolymph on the production of polyhedra of a nuclear polyhedrosis virus.Applied Entomology and Zoology 24, 486-488. [Google Scholar]
  13. Inoue, H., Taniai, K. & Kobayashi, J. (1990). Establishment and characterization of substrate depending cell lines of Bombyx mori.Bulletin of the National Institute of Sericulture and Entomological Science 1, 13-25. [Google Scholar]
  14. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680-685.[CrossRef] [Google Scholar]
  15. Lepore, L. S., Roelvink, P. R. & Granados, R. R. (1996). Enhancin, the granulovirus protein that facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease.Journal of Invertebrate Pathology 68, 131-140.[CrossRef] [Google Scholar]
  16. Maeda, S. (1989). Expression of foreign genes in insect cells using baculovirus vectors. In Insect Cell Biochemistry, pp. 1-31. Edited by K. Maramorosch & A. H. McIntosh. Boca Raton, FL: CRC Press.
  17. Mitsuhashi, J. (1984). Isolation of continuous cell line from larval fat bodies of an arctii, Spilarctia seriatopunctata (Insecta, Lepidoptera, Arctiidae).Zoological Science 1, 415-419. [Google Scholar]
  18. Mitsuhashi, J. & Maramorosch, K. (1964). Leafhopper tissue culture: embryonic, nymphal, and imaginal tissues from aseptic insects.Contributions from Boyce Thompson Institute 22, 435-460. [Google Scholar]
  19. Ohba, M. & Tanada, Y. (1983). A synergistic factor enhances the in vitro infection of an insect baculovirus.Naturwissenschaften 70, 613-615.[CrossRef] [Google Scholar]
  20. Ohba, M. & Tanada, Y. (1984). A synergistic factor of an insect granulosis virus agglutinates insect cells.Experientia 40, 742-744.[CrossRef] [Google Scholar]
  21. O’Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992).Baculovirus Expression Vectors: A Laboratory Manual, p. 347. New York: Freeman.
  22. Reed, L. J. & Muench, H. (1938). A simple method for estimating fifty percent endpoints.American Journal of Hygiene 27, 493-497. [Google Scholar]
  23. Suzuki, T., Kanaya, T., Okazaki, H., Ogawa, K., Usami, A., Watanabe, H., Kadono-Okuda, K., Yamakawa, M., Sato, H., Mori, H., Takahashi, S. & Oda, K. (1997). Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.Journal of General Virology 78, 3073-3080. [Google Scholar]
  24. Tanada, Y. (1985). A synopsis of studies on the synergistic property of an insect baculovirus: a tribute to Edward A. Steinhaus.Journal of Invertebrate Pathology 45, 125-138. [Google Scholar]
  25. Tanada, Y., Himeno, M. & Omi, E. M. (1973). Isolation of a factor from the capsule of a granulosis virus, synergistic factor a nuclear polyhedrosis virus of the armyworm.Journal of Invertebrate Pathology 21, 31-40.[CrossRef] [Google Scholar]
  26. Uchima, K., Egerter, D. E. & Tanada, Y. (1989). Synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta: its uptake and enhancement of virus infection in vitro.Journal of Invertebrate Pathology 54, 156-164.[CrossRef] [Google Scholar]
  27. Watanabe, H. (1987). Effect of the concentration of fetal bovine serum in a culture medium on the susceptibility of Bombyx mori cells to a nuclear polyhedrosis virus.Applied Entomology and Zoology 22, 397-398. [Google Scholar]
  28. Watanabe, Y., Yoshihara, T. & Inoue, H. (1988). Infection of a Bombyx cell line adapted to different media with a nuclear polyhedrosis virus.Journal of Sericultural Science of Japan 57, 227-231. [Google Scholar]
  29. Wyatt, S. S. (1956). Culture in vitro of tissue from the silkworm Bombyx mori L.Journal of General Physiology 39, 841-852.[CrossRef] [Google Scholar]
  30. Xu, J. & Hukuhara, T. (1994). Biochemical properties of an enhancing factor of an entomopoxvirus.Journal of Invertebrate Pathology 63, 14-18.[CrossRef] [Google Scholar]

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