title: Acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells.
abstract: Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell-cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell-cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell-cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell-cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell-cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.
authors: Institute of Molecular and Cell Biology, Proteos, Singapore.
authors: Yoshiyuki
authors: Y
authors: Yamada
authors: Xiao Bo
authors: XB
authors: Liu
authors: Shou Guo
authors: SG
authors: Fang
authors: Felicia P L
authors: FP
authors: Tay
authors: Ding Xiang
authors: DX
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002459:Cell Fusion; D002478:Cells, Cultured; D017934:Coronavirus / Q000472:pathogenicity*; D018274:Electroporation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus