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: Hyun Chul Song, Mi-Young Seo, Konrad Stadler, Byoung J Yoo, Qui-Lim Choo, Stephen R Coates, Yasushi Uematsu, Takashi Harada, Catherine E Greer, John M Polo, Piero Pileri, Markus Eickmann, Rino Rappuoli, Sergio Abrignani, Michael Houghton, Jang H Han
• Journal: Journal of virology (J Virol) Vol. 78 Issue 19 Pg. 10328-35 (Oct 2004) ISSN: 0022-538X [Print] United States
• PMID: 15367599 (Publication Type: Journal Article)

Chemical References

• Antigens, Viral
• Culture Media
• DNA, Complementary
• DNA, Viral
• Membrane Glycoproteins
• Protein Subunits
• Recombinant Proteins
• Spike Glycoprotein, Coronavirus
• Viral Envelope Proteins
• spike glycoprotein, SARS-CoV
• spike protein, mouse hepatitis virus
• Glycoside Hydrolases
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

Topics

• Animals
• Antigens, Viral (chemistry, genetics, immunology, metabolism)
• COS Cells
• Cell Line
• Chlorocebus aethiops
• Cricetinae
• Culture Media (chemistry)
• DNA, Complementary
• DNA, Viral (genetics, metabolism)
• Endoplasmic Reticulum (chemistry)
• Glycoside Hydrolases (metabolism)
• Golgi Apparatus (chemistry)
• Membrane Glycoproteins (chemistry, genetics, immunology, metabolism)
• Molecular Weight
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase (metabolism)
• Protein Folding
• Protein Processing, Post-Translational
• Protein Structure, Tertiary
• Protein Subunits (analysis)
• Protein Transport
• Recombinant Proteins (chemistry, genetics, immunology, metabolism)
• Severe acute respiratory syndrome-related coronavirus (genetics)
• Spike Glycoprotein, Coronavirus
• Viral Envelope Proteins (chemistry, genetics, immunology, metabolism)