title: Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein.
abstract: 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 approximately 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.
authors.affiliation: Vaccines Research, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608, USA.
authors.forename: Hyun Chul
authors.initials: HC
authors.lastname: Song
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Seo
authors.forename: Konrad
authors.initials: K
authors.lastname: Stadler
authors.forename: Byoung J
authors.initials: BJ
authors.lastname: Yoo
authors.forename: Qui-Lim
authors.initials: QL
authors.lastname: Choo
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Coates
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Uematsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Harada
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Greer
authors.forename: John M
authors.initials: JM
authors.lastname: Polo
authors.forename: Piero
authors.initials: P
authors.lastname: Pileri
authors.forename: Markus
authors.initials: M
authors.lastname: Eickmann
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.forename: Sergio
authors.initials: S
authors.lastname: Abrignani
authors.forename: Michael
authors.initials: M
authors.lastname: Houghton
authors.forename: Jang H
authors.initials: JH
authors.lastname: Han
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003470:Culture Media / Q000737:chemistry; D018076:DNA, Complementary; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000032:analysis; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000956:Antigens, Viral; D003470:Culture Media; D018076:DNA, Complementary; D004279:DNA, Viral; D008562:Membrane Glycoproteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase