Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

Abstract

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

Fig. 1

Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the cluster to lysine mutations CL1–CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by arrows with the name of that particular mutation shown in brackets. (C) Amino-acid sequences of the carboxyl termini of the truncation and acidic cluster associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions. The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.

Fig. 2

Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A, B) Immunoblots of wild-type [3xFLAG So (WT)], cluster to lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol.

Fig. 3

Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S [3xFLAG So (WT)] (F1, F2), CL1 (A1, A2), CL2 (B1, B2), CL3 (C1, C2), CL4 (D1, D2), CL5 (E1, E2), CL6 (L1, L2), CL7 (M1, M2), T1214 (K1, K2), T1229 (J1, J2), T1238 (I1, I2), T1247(H1, H2) and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (G1, G2), which served as a negative control. At 48 h post-transfection, cells were immunohistochemically processed either under live conditions to show surface expression (A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2) or fixed and permeabilized conditions to show total expression (A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1).

Fig. 4

Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell-surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see Materials and methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild-type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio.

Fig. 5

Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and methods). Error bars shown represent the standard deviations calculated through comparison of the data from each of three experiments.

Fig. 6

Confocal microscopic visualization of endocytosed and intracellular distribution of SARS-CoV S glycoprotein mutants. Vero cells expressing wild-type SARS-CoV S glycoprotein [3xFLAG So (WT)] (A and B), CL6 (C and D), CL7 (E and F), T1229 (G and H), T1238 (I and J), and T1247 (K and L) were processed for confocal microscopy using two different methods in order to assess different properties of the mutants. Endocytosis patterns (A, C, E, G, I, and K) were visualized by adding anti-FLAG (green) antibody into the media 12 h prior to processing, enabling detection of the mutant protein after endocytosis from cellular surfaces. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). For total glycoprotein detection (B, D, F, H, J, and L), cells were fixed and permeabilized prior to labeling with anti-FLAG (green).

Fig. 7

Analysis of the endocytotic kinetic profile of the truncation mutants and the acidic cluster mutants using confocal microscopy. After transfection, SARS-CoV S glycoprotein wild-type [3xFLAG So (WT)] (A1–A4), T1229 (B1–B4), T1238 (C1–C4), T1247 (D1–D4), CL6 (E1–E4), and CL7 (F1–F4) expressing cells were incubated with an anti-FLAG monoclonal antibody (green) for 1 h and then returned to 37 °C for different times. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). Cell nuclei were labeled with To-Pro-3 Iodide (blue). Panels A1–A4, B1–B4, C1–C4, D1–D4, E1–E4, and F1–F4 correspond to 0-, 5-, 15-, and 60-min incubation times at 37 °C, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Cluster alignment of the carboxyl terminus of the SARS-CoV S and the MHV S glycoproteins. The shaded residues indicate the position of the cysteine-rich motif in their respective protein. The cysteine residues are bolded. The charged clusters are indicated by a bracket over the corresponding regions.