Spike protein, S, of human coronavirus HKU1: role in viral life cycle and application in antibody detection.

Chan, Che-Man; Woo, Patrick C Y; Lau, Susanna K P; Tse, Herman; Chen, Hong-Lin; Li, Feng; Zheng, Bo-Jian; Chen, Ling; Huang, Jian-Dong; Yuen, Kwok-Yung

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.