Expression of the Severe Acute Respiratory Syndrome Coronavirus 3a Protein and the Assembly of Coronavirus-Like Particles in the Baculovirus Expression System

  • Sehaam Khan
  • Mah-Lee Ng
  • Yee-Joo Tan
Part of the Methods in Molecular Biology book series (MIMB, volume 379)


The Bac-to-Bac Baculovirus expression system was used to generate a recombinant baculovirus capable of expressing the severe acute respiratory syndrome (SARS)-coronavirus (CoV) 3a protein. Using the same expression system, two structural proteins, membrane (M) and envelope (E), were co-expressed to form SARS-CoV virus-like particles (VLPs) within an insect cell. Expression of viral proteins was confirmed by Western blot analysis and the formation of VLPs was studied by transmission electron microscopy.

Key Words

Bac-to-Bac Baculovirus expression system severe acute respiratory syndrome coronavirus SARS-CoV 3a protein membrane protein (M) envelope protein (E) virus-like particles (VLPs) transmission electron microscopy Western Blot 


  1. 1.
    Kost, T. A., Condreay, J. P., and Jarvis, D. L. (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol. 23, 567–575.CrossRefPubMedGoogle Scholar
  2. 2.
    Anderson, D., Harris, R., Polayes, D., et al. (1996) Rapid generation of recombinant baculovirus and expression of foreign genes using the Bac-to-Bac baculovirus expression system. Focus 17, 53–58.Google Scholar
  3. 3.
    Ciccarone, V. C., Polayes, D., and Luckow, V. A. (1997) Generation of recombinant baculovirus DNA, in E. coli Using Baculovirus Shuttle Vector (U. Reischt, ed.). Humana, Totowa,NJ.Google Scholar
  4. 4.
    Luckow, V. L. and Summers, M. D. (1988) Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47–55.CrossRefGoogle Scholar
  5. 5.
    O’Reilly, D. R., Miller, L. K., and Luckow, V. A. (eds.) (1994) Baculovirus Expression Vectors, A Laboratory Manual. Oxford University Press, Oxford,UKGoogle Scholar
  6. 6.
    Jarvis, D. L. (1997) Baculovirus expression vectors in The Baculoviruses (Miller, L. K. ed.). Plenum, New York, NY.Google Scholar
  7. 7.
    Altmann, F., Schwihla H., Staudacher, E., Glossl, J., and Marz, L. (1995) Insect cells contain an unusual, membrane bound β-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J. Biol. Chem. 270, 17,377–17,349.Google Scholar
  8. 8.
    Harper, D. M., Franco, E. L., Wheeler, C., et al. The GlaxoSmithKline HPV Vaccine Study Group. (2004) Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet. 364, 1757–1765.CrossRefPubMedGoogle Scholar
  9. 9.
    Jeong, S. H., Qiao, M., Nascimbeni, M., et al. (2004) Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J. Virol. 78, 6995–7003.CrossRefPubMedGoogle Scholar
  10. 10.
    Abe, T., Takahashi, H., Hamazaki, H., Miyano-Kurosaki, N., Matsuura, Y., and Takaku, H. (2003) Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice. J. Immunol. 171, 1133–1139.PubMedGoogle Scholar
  11. 11.
    Brett, I. C. and Johansson, B. E. (2005) Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system. Virology 339, 273–280.CrossRefPubMedGoogle Scholar
  12. 12.
    Fouchier, R. A., Kuiken, T., Schutten, M., et al. (2003) Aetiology: Koch’s postulates fulfilled for SARS virus. Nature 423, 240.CrossRefPubMedGoogle Scholar
  13. 13.
    Rota, P. A., Oberste, M. S., Monroe, S. S., et al. (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399.CrossRefPubMedGoogle Scholar
  14. 14.
    Marra, M. A., Jones, S. J., Astell, C. R., et al. (2003) The Genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404.CrossRefPubMedGoogle Scholar
  15. 15.
    Guo, J. P., Petric, M., Campbell, W., and McGeer, P. L. (2004) SARS corona virus peptides recognized by antibodies in the sera of convalescent cases. Virology 324, 251–256.CrossRefPubMedGoogle Scholar
  16. 16.
    Tan, Y. J., Goh, P. Y., Fielding, B. C. F., et al. (2004) Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers. Clin. Diagn. Lab. Immunol. 11, 362–371.PubMedGoogle Scholar
  17. 17.
    Yu, C.-J., Chen, Y.-C., Hsiao, C.-H., et al. (2004) Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus. FEBS Lett. 565, 111–116.CrossRefPubMedGoogle Scholar
  18. 18.
    Zeng, R., Yang, R. F., Shi, M. D., et al. (2004) Characterization of the 3a protein of SARS-associated coronavirus in infected Vero E6 cells and SARS patients. J. Mol. Biol. 341, 271–279.CrossRefPubMedGoogle Scholar
  19. 19.
    Tan, Y.-J., Teng, E., Shen, S., et al. (2004) A novel SARS coronavirus protein, U274, is transported to the cell surface and undergoes endocytosis. J. Virol. 78, 6723–6734.CrossRefPubMedGoogle Scholar
  20. 20.
    Ito, N., Mossel, E. C., Narayanan, K., et al. (2005) Severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein. J. Virol. 79, 3182–3186.CrossRefPubMedGoogle Scholar
  21. 21.
    Shen, S., Lin, P-S., Chao, Y-C., et al. (2005) The severe acute respiratory syndrome coronavirus 3a is a novel structural protein. Biochem. Biophys. Res. Commun. 330, 286–292.CrossRefPubMedGoogle Scholar
  22. 22.
    Huang. C., Narayanan, K., Ito, N., Peters, C. J., and Makino, S. (2006) Severe acute respiratory syndrome coronavirus 3a protein is released in membranous structures from 3a protein-expressing cells and infected cells. J. Virol. 80, 210–217.CrossRefPubMedGoogle Scholar
  23. 23.
    Oostra, M., de Haan, C. A. M., de Groot, R. J., and Rottier, P. J. M. (2005) Xth International Nidovirus Symposium: Toward Control of SARS and other Nidovirus Disease. Abstract, p. 3–3.Google Scholar
  24. 24.
    Ho, Y., Lin, P.-H., Liu, C. Y.,Y., Lee, S.-P., and Chao, Y.-C. (2004) Assembly of human severe acute respiratory syndrome coronavirus-like particles. Biochem. Biophys. Res. Commun. 318, 833–838.CrossRefPubMedGoogle Scholar
  25. 25.
    Mortola, E. and Roy, P. (2004) Efficient assembly and release of SARS coronavirus-like particles by heterologous expression system. FEBS Lett. 576, 174–178.CrossRefPubMedGoogle Scholar
  26. 26.
    Rottier, P. J. M. (1995) The coronavirus membrane protein, in The Coronaviridae (Siddell, S. G., ed.). Plenum, New York, NY.Google Scholar
  27. 27.
    Siddell, S. G. (1995) The small membrane protein in The Coronaviridae (Siddell, S. G., ed.). Plenum, New York, NY.Google Scholar
  28. 28.
    Raamsman, M. J. B., Locker, J. K., DeHooge, A., et al. (2000) Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J. Virol. 74, 2333–2342.CrossRefPubMedGoogle Scholar
  29. 29.
    Nal, B., Chan, C., Kien, F., et al. (2005) Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E. J. Gen. Virol. 86, 1423–1434.CrossRefPubMedGoogle Scholar
  30. 30.
    Carstens, E. B., Krebs, A., and Gallerneault, C. E. (1986) Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra. J. Virol. 58, 684–688.PubMedGoogle Scholar
  31. 31.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Habor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  32. 32.
    Luckow, V. A., Lee, C. S., Barry, G. F., and Olins, P. O. (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579.PubMedGoogle Scholar
  33. 33.
    Granados, R. R. and Federici, B. A. (eds.) (1986) The Biology of Baculoviruses. CRC, Boca Raton, FL.Google Scholar
  34. 34.
    Lee, Y. N., Chen, L. K., Ma, H. C., Yang, H. H., Li, H. P., and Lo, S. Y. (2005) Thermal aggregation of SARS-CoV membrane protein. J. Virol. Meth. 129, 152–161.CrossRefGoogle Scholar
  35. 35.
    Guan, M., Chen, H. Y., Tan, P. H., et al. (2004) Use of viral lysate antigen combined with recombinant protein in Western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome. Clin. Diag. Lab. Immun. 11, 1148–1153.Google Scholar
  36. 36.
    Ng, M.-L., Tan, S.-H., See, E.-E., Ooi, E.-E., and Ling, A.-E. (2003) Proliferative growth of SARS coronavirus in Vero E6 cells. J. Gen. Virol. 84, 3291–3303.CrossRefPubMedGoogle Scholar
  37. 37.
    Tan, B.-H., Nason, E., Staeuber, N., Jiang, W., Monastryrskaya, K., and Roy, P. (2001) RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells. J. Virol. 75, 3937–3947.CrossRefPubMedGoogle Scholar
  38. 38.
    Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Sehaam Khan
    • 1
  • Mah-Lee Ng
    • 2
  • Yee-Joo Tan
    • 1
  1. 1.Collaborative Antiviral Research Group, Institute of Molecular and Cell BiologyProteosSingapore
  2. 2.Department of Microbiology and Electron Microscopy Unit, Faculty of MedicineNational University of SingaporeSingapore

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