PMID:21798295

Citation	Lin, HX, Feng, Y, Tu, X, Zhao, X, Hsieh, CH, Griffin, L, Junop, M and Zhang, C (2011) Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry. Virus Res. 160:283-93
Abstract	The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.
Links	PubMed Online version:10.1016/j.virusres.2011.06.029
Keywords	Amino Acid Substitution; Cell Line; Coronavirus NL63, Human/genetics; Coronavirus NL63, Human/physiology; Humans; Membrane Glycoproteins/genetics; Membrane Glycoproteins/metabolism; Mutant Proteins/genetics; Mutant Proteins/metabolism; Protein Binding; Receptors, Virus/metabolism; Sequence Deletion; Spike Glycoprotein, Coronavirus; Viral Envelope Proteins/genetics; Viral Envelope Proteins/metabolism; Virus Internalization
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CVHNL:SPIKE		GO:0019062: virion attachment to host cell	ECO:0000315:		P		Figures 3c and d show that high expression levels on cell surface are positively correlated with high S protein levels in the virion, therefore, showing this protein helps with attachment/infection.	complete CACAO 12619

References

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