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Journal of Virology, October 2004, p. 10328-10335, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10328-10335.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Synthesis and Characterization of a Native, Oligomeric Form of Recombinant Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein

Hyun Chul Song,1 Mi-Young Seo,1 Konrad Stadler,2 Byoung J. Yoo,3 Qui-Lim Choo,1 Stephen R. Coates,1 Yasushi Uematsu,2 Takashi Harada,2 Catherine E. Greer,1 John M. Polo,1 Piero Pileri,2 Markus Eickmann,4 Rino Rappuoli,1,2 Sergio Abrignani,2 Michael Houghton,1 and Jang H. Han1*

Vaccines Research, Chiron Corporation, Emeryville, California,1 IRIS, Chiron Vaccines, Siena, Italy,2 Division of Natural Sciences, Daegu University, Kyungbuk, Korea,3 Institute for Virology, University of Marburg, Marburg, Germany4

Received 30 March 2004/ Accepted 18 May 2004

We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ~500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.

* Corresponding author. Mailing address: Vaccines Research, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608. Phone: (510) 923-2937. Fax: (510) 923-2586. E-mail: jang_han{at}chiron.com.

Journal of Virology, October 2004, p. 10328-10335, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10328-10335.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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