title: Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry.
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.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Canada.
authors.forename: Han-Xin
authors.initials: HX
authors.lastname: Lin
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.forename: Xinming
authors.initials: X
authors.lastname: Tu
authors.forename: Xuesen
authors.initials: X
authors.lastname: Zhao
authors.forename: Chih-Heng
authors.initials: CH
authors.lastname: Hsieh
authors.forename: Lauren
authors.initials: L
authors.lastname: Griffin
authors.forename: Murray
authors.initials: M
authors.lastname: Junop
authors.forename: Chengsheng
authors.initials: C
authors.lastname: Zhang
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D058957:Coronavirus NL63, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism*; D017384:Sequence Deletion; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus