Spike protein, S, of human coronavirus HKU1: role in viral life cycle and application in antibody detection.
We recently described the discovery, genome, clinical features, genotypes and evolution of a novel and global human respiratory virus named human coronavirus HKU1 (HCoV-HKU1) which is not yet culturable. We expressed a C-terminal FLAG-tagged CoV-HKU1 spike (S) protein by the Semliki Forest Virus (SFV) system and investigated its maturation profile. Pulse chase labeling revealed that S-FLAG was expressed as high-mannose N-glycans of monomers and trimers. It was predominantly cleaved into subdomains S1 and S2 during maturation. S1 was secreted into the medium. Immunofluorescence analysis visualized S along the secretory pathway from endoplasmic reticulum to plasma membrane. Cleavage of S and release of HCoV-HKU1 S pseudotyped virus were inhibited by furin or furin-like enzyme inhibitors. The cell-based expressed full-length S-FLAG could be recognized by the convalescent serum obtained from a patient with HCoV-HKU1 pneumonia. The data suggest that the native form of HCoV-HKU1 spike expressed in our system can be used in developing serological diagnostic assay and in understanding the role of S in the viral life cycle.
Chan
Che-Man
CM
Department of Microbiology, The University of Hong Kong, Hong Kong Special Administration Region, Hong Kong.
Woo
Patrick C Y
PC
Lau
Susanna K P
SK
Tse
Herman
H
Chen
Hong-Lin
HL
Li
Feng
F
Zheng
Bo-Jian
BJ
Chen
Ling
L
Huang
Jian-Dong
JD
Yuen
Kwok-Yung
KY
D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D017934:Coronavirus / Q000145:classification* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006224:Cricetinae; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007114:Immunization; D007668:Kidney / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010829:Physiological Phenomena / Q000235:genetics*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D064370:Spike Glycoprotein, Coronavirus; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000914:Antibodies, Viral; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D016650:Fluorescein-5-isothiocyanate