Summary
The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9P35AcV5-1 and Sf9P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.
Similar content being viewed by others
References
Beidler, D. R.; Tewari, M.: Friesen, P. D.; Poirier, G.; Dixit, V. M. The Baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis. J. Biol. Chem. 270:16,526–16,528; 1995.
Berry, S. J. Insect nucleic acids. Comprehensive Insect Physiol. Biochem. Pharmacol. 10:219–253; 1985.
Bertin, J.; Mendrysa, S. M.; Lacount, D. J.; Gaur, S.; Krebs, J. F.; Armstrong, R. C.; Tomaselli, K. J.; Friesen, P. D. Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3-ICE-like protease. J. Virol. 70:6251–6259; 1996.
Blissard, G. W.; Rohrmann, G. F. Baculovirusgp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1. J. Virol. 65:5820–5827: 1991.
Bump, N. J.; Hackett, M.; Hugunin, M., et al. Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269:1885–1888; 1995.
Cartier, J. L.; Hersberger, P. A.; Friesen, P. D. Suppression of apoptosis in insect cells stably transfected with baculovirusp35: dominant interference by N-terminal sequencesp35 1–76. J. Virol. 68:7728–7737; 1994.
Chang, M.-J.; Kuzio, J.; Blissard, G. W. Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon. Virology 259:369–383 (1999).
Clem, R. J.; Fechheimer, M.; Miller, L. K. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254:1388–1390; 1991.
Cryns, V.; Yuan, J., Proteases to die for [Erratum appears in Genes Dev. 13(3):371;1999]. Genes Dev. 12:1551–1570; 1998.
Davis, T. R.; Trotter, K. M.; Granados, R. R.; Wood, H. A. Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion. Biotechnology (NY) 10:1148–1150; 1992.
Davis, T. R.; Wickham, T. J.; McKenna, K. A.; Granados, R. R.; Shuler, M. L.; Wood, H. A. Comparative recombinant protein production in eight insect cell lines. In Vitro Cell Dev. Biol. 29A:388–390; 1993.
Dyer, K. A.; Thornhill, W. B.; Riddiford, L. M.. DNA synthesis during the change to pupal commitment ofManduca epidermis. Dev. Biol. 84:425–431: 1981.
Efstratiadis, A.; Crain, W. R.; Britten, R. J.; Davidson, E. H.; Kafatos, F. C. DNA sequence organization in the lepidopteraAntheraea pernyi. Proc. natl. Acad. Sci. USA 73:2289–2293; 1976.
Fisher, A. J.; Cruz, W.; Zoog, S. J.; Schneider, C. L.; Friesen, P. D., Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J. 18:2031–2039; 1999.
Gage, L. P. TheBombyx mori genome: analysis by DNA reassociation kinetics. Chromosoma 45:27–42; 1974.
Gerbal, M.; Fournier, P.; Barry, P.; Mariller, M.; Odier, F., Devauchelle, G.; Duonor-Cerutti, M. Adaptation of an insect cell line ofSpodoptera frugiperda to grow at 37 degrees C: characterization of an endodiploid clone. In Vitro Cell. Dev. Biol. 36A:117–124; 2000.
Hay, B. A.; Wolff, T.; Rubin, G. M. Expression of baculovirus P35 prevents cell death inDrosophila. Development 120:2121–2129; 1994.
Hershberger, P. A.; Lacount, D. J.; Friesen, P. D. The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells. J. Virol. 68:3467–3477: 1994.
Hink, W. F. Established cell line from the cabbage looper,Trichoplusia ni. Nature 226:466–467; 1970.
Hohmann, A. W.; Faulkner, P. Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis. Virology 125:432–444, 1983.
Martin, S. J.; Green, D. R. Protease activation during apoptosis: death by a thousand cuts. Cell 82:349–352; 1995.
Martinou, I.; Fernandez, P. A.; Missotten, M.; White, E.; Allet, B.; Sadoul, R.; Martinou, J. C. Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation. J. Cell Biol. 128:201–208; 1995.
Monsma, S. A.; Blissard, G. W. Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. J. Virol. 69:2583–2595; 1995.
Monsma, S. A.; Oomens, A. G. P.; Blissard, G. W. The GP64 envelope fusion protein is an essential baculovirus protein required for cell to cell transmission of infection. J. Virol. 70:4607–4616; 1996.
O'Reilly, D. R.; Miller, L. K.; Luckow, V. A. Baculovirus expression vectors, a laboratory manual. New York: W. H. Freeman; 1992.
Rabizadeh, S.; Lacount, D. J.; Friesen, P. D.; Bredesen, D. E. Expression of the baculovirus p35 gene inhibits mammalian neural cell death. J. Neurochem. 61:2318–2321; 1993.
Rasch, E. M. The DNA content of sperm and hemocyte nuclei of the silkworm,Bombyx mori L. Chromosoma 45:1–26; 1974.
Reed, J. C. Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 124:1–6; 1994.
Robertson, N. M.; Zangrilli, J.; Fernandes Alnemri, T.; Friesen, P. D.; Litwack, G.; Alnemri, E. S. Baculovirus P35 inhibits the glucocorticoid-mediated pathway to cell death. Cancer Res., 57:43–47; 1997.
Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning. Cold Spring, Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Singh, R. P.; Emery, A. N., Al-Rubeai, M. Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor genebcl-2. Biotechnol. Bioeng. 52:166–175; 1996.
Sugimoto, A.; Friesen, P. D.; Rothman, J. H. Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematodeCaenorhabditis elegans. EMBO J. 13:2023–2028; 1994.
Summers, M. D.; Smith, G. E. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin No. 1555; 1987.
Wang, P.; Granados, R. R.; Shuler, M. L. Studies on serum-free culture of insect cells for virus propagation and recombinant protein production. J. Invertebr. Pathol. 59:46–53; 1992.
Wickham, T. J. Baculovirus-insect cell interactions in producing heterologous proteins: attachment, infection, and expression in different cell lines. Ithaca, NY: Department of Chemical Engineering, Cornell University; 1991:208.
Wickham, T. J.; Davis, T.; Granados, R. R. Shuler, M. L.; Wood, H. A. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol. Prog. 8:391–396; 1992.
Xue, D.; Horvitz, H. R. Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature (Lond.) 377:248–251; 1995.
Yu, Z.; Podgwaite, J. D.; Wood, H. A. Genetic engineering of a Lymantria dispar nuclear polyhedrosis virus for expression of foreign genes. J. Gen. Virol. 73:1509–1514; 1992.
Zhou, Q.; Krebs, J. F.; Snipas, S. J.; Price, A.; Alnemri, E. S.; Tomaselli, K. J.; Salvesen, G. S. Interaction of the baculovirus anti-apoptotic protein p35 with caspaes. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 37:10,757–10,765; 1998.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lin, G., Li, G., Granados, R.R. et al. Stable cell lines expressing baculovirus P35: Resistance to apoptosis and nutrient stress, and increased glycoprotein secretion. In Vitro Cell.Dev.Biol.-Animal 37, 293–302 (2001). https://doi.org/10.1007/BF02577545
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02577545