Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein.
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.
Song
Hyun Chul
HC
Vaccines Research, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608, USA.
Seo
Mi-Young
MY
Stadler
Konrad
K
Yoo
Byoung J
BJ
Choo
Qui-Lim
QL
Coates
Stephen R
SR
Uematsu
Yasushi
Y
Harada
Takashi
T
Greer
Catherine E
CE
Polo
John M
JM
Pileri
Piero
P
Eickmann
Markus
M
Rappuoli
Rino
R
Abrignani
Sergio
S
Houghton
Michael
M
Han
Jang H
JH
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*
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