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.

Fig. 1

Schematic diagram of genome organization of NL63 and domain architecture of spike (S) protein. Signal: signal peptide; RBD: receptor-binding domain; FP: fusion peptide; HR: heptad repeat; TM: transmembrane domain; Cyto: cytoplasmic tail. Twenty-one residues that have been previously shown to be important for RBD–hACE2 interaction are indicated within three receptor binding motifs (RBMs) (Li et al., 2007, Lin et al., 2008).

Fig. 2

Development of NL63 pseudotype virus system. (A) Pseudotype virus entry of 23 randomly selected NL63 S gene variants. The Luc activity was measured and normalized to that of variant No. 5 that consistently showed the highest virus entry level. (B) Yield and infectivity of pseudotype virus made by transfection of different amounts of pCAGGS vector and wt S plasmid DNA (0.04–4.0 μg in each well of 6-well plate). (C) Amount of S protein incorporated into virions. The density of protein bands was quantified by Quantity One, and the relative values were shown at the bottom of the gel. HIV-1 p24 protein was detected as a loading control.

Fig. 3

Analysis of the cytoplasmic tail of S protein in protein accumulation, receptor binding and pseudotype virus entry. (A) Schematic diagram of the truncated S mutants. (B) Pseudotype virus entry. (C) Amount of S protein incorporated into virions. (D) S protein accumulation in cell lysate and on cell surface. (E) Receptor binding ability. The density of protein bands was quantified by Quantity One, and the relative values were shown at the bottom of each gel.

Fig. 4

Viral entry mediated by NL63 S, SARS S and VSVG. Huh-7 (A) and 293T/hACE2 cells (B) were infected with same amount of pseudotyped NL63 and SARS viruses (20,000 cpm/well in a 24-well plate) and less amount of VSVG viruses (2000 cpm/well), respectively, and luciferase activity was measured at 48 h post-infection.

Fig. 5

The roles of 19 residues in protein accumulation, receptor binding and pseudotype virus entry. (A) Results for 13 residues that were previously identified in a minimum RBD context (aa 476–616) (Lin et al., 2008). (B) Results for 6 residues that were previously tested in double-substitution mutants in a larger RBD context (aa 301–749) (Li et al., 2007).

Fig. 6

Structure models of CBM–hACE2 interaction. (A) Conserved binding motif (CBM) of NL63 and SARS-CoV S protein (upper panel) and the structure model of SARS-CoV CBM–hACE2 interaction (lower panel). The underlined residues in SARS-CoV CBM represent those residues in contact with hACE2 revealed by the RBD–hACE2 complex crystal structure of RBD–hACE2 complex (Li et al., 2005a). The underlined residues in the NL63 CMB are those that were experimentally shown to be important for hACE2 binding (Fig. 4). (B) Structural basis for the enhanced receptor binding of N578Y. The left panel illustrates that residue Y475 of SARS-CoV forms a hydrogen bond with Y83 of hACE2 and has two weak Van der Waals interactions with T27 and K31 of hACE2. The right panel shows the loss of hydrogen bond when Y475 is changed to N475, mimicking the corresponding site in NL63 (N578), while the weak interactions with T27 and K31 are still retained. (C) Structural basis for the drastically reduced receptor binding of E582A. The left panel shows the hydrogen bond formed between N479 of SARS-CoV and H34 of hACE2. The middle panel illustrates that when N479 is changed to E479, mimicking the corresponding site in NL63 (E582), the contact between virus and receptor is strengthened by formation of a salt bridge that is stronger than a single hydrogen bond. The right panel depicts the loss of contact when E479 is changed to A479.