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. 2007 Apr 10;360(2):264-74.
doi: 10.1016/j.virol.2006.10.034. Epub 2006 Nov 28.

Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

Chad M Petit  1 , Vladimir N Chouljenko, Arun Iyer, Robin Colgrove, Michael Farzan, David M Knipe, K G Kousoulas
Affiliations

Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

Chad M Petit et al. Virology. .

Abstract

The SARS-coronavirus (SARS-CoV) is the etiological agent of the 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. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of (3)H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

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Figures

Fig. 1
Fig. 1
Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses. A schematic diagram of the SARS–CoV S protein from amino acid 1 to amino acid 1255 is shown at the top of the figure. A vertical line demarcates the potential cleavage between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS–CoV S glycoprotein is shown enlarged below aligned with the same region of the S glycoprotein specified by other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV′, human coronavirus 229E [HCoV–229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV–OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks (Abraham et al., 1990, Binns et al., 1985, Delmas et al., 1992, Kunkel and Herrler, 1993, Luytjes et al., 1987, Marra et al., 2003, Mounir and Talbot, 1993, Parker et al., 1989, Raabe et al., 1990, Rasschaert and Laude, 1987).
Fig. 2
Fig. 2
Schematic diagram of the SARS–CoV S glycoprotein endodomain and the cysteine cluster to alanine mutations. Amino acid sequences of the carboxyl termini and the cysteine cluster-to-alanine mutations are shown for the wild-type as well as the mutant proteins. The cysteine clusters (CRM1 and CRM2) and the charged rich regions of the S proteins are encompassed in brackets and labeled. The transmembrane portion of the endodomain is italicized and underlined. Amino acids mutated to alanines for the mCL-I, mCL-II, mCL-III, and mCL-IV cluster mutations are in bold.
Fig. 3
Fig. 3
Western blot analysis of the expressed mutant SARS–CoV mutant glycoproteins. Immunoblots of wild-type [So–3xF(WT)] and cysteine to alanine mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control in which mock-transfected Vero cells were probed with the monoclonal antibody to the SARS–CoV S glycoprotein.
Fig. 4
Fig. 4
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 (SARS So 3xF) (E1, E2), mCL-I (A1, A2), mCL-II (B1, B2), mCL-III (C1, C2), mCL-IV (D1, D2), and a wild-type SARS–CoV optimized S labeled with a 3xFLAG carboxyl tag (F1, F2), which served as a negative control. At 48 h post-transfection, cells were immunohistochemically processed under live conditions to detect cell-surface expression (A1, B1, C1, D1, E1, and F1), or permeabilized conditions to detect total expression (A2, B2, C2, D2, E2, and F2) using the anti-FLAG antibody.
Fig. 5
Fig. 5
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 and total cell expression of the S glycoprotein was measured by immunohistochemistry. For cell-surface expression the transfected cell monolayers were reacted with anti-FLAG antibody at room temperature under live conditions. For total S glycoprotein detection, cells were fixed and permeabilized prior to reaction with the anti-FLAG antibody. A ratio between the amount of S detected on cell-surfaces to total cellular expression of S was calculated and normalized to the wild-type protein, and expressed as a percentage ratio of the wild-type S. 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 as a percentage of the wild-type.
Fig. 6
Fig. 6
Incorporation of [3H] palmitic acid into wild-type and single cysteine mutant forms mCL-I, mCL-II, mCL-III and mCL-IV of the SARS–CoV S glycoprotein. (A) Autoradiographic images of immunoprecipitates resolved by SDS–PAGE electrophoresis of cellular extracts obtained from transfected Vero cells labeled with [3H] palmitic acid. Apparent molecular mass controls are as shown. Sample from mock-transfected Vero cells was used as a negative protein control (protein control). The samples from transfected Vero cells with the wild-type {So–3xF (WT)} and each of the four mutated S genes are shown. (B) An estimation of the relative concentration of the palmitoylated S species in comparison to the wild-type S is shown. To determine the relative level of S palmitoylation, all S protein species film images obtained by autoradiography were scanned, digitally analyzed, and normalized to the total protein of the sample obtained by spectrophotometry (not shown). The amount of palmitoylation was then expressed as a ratio to the relative amount of wild-type S.
Fig. 7
Fig. 7
Incorporation of [3H] palmitic acid into wild-type and double cysteine mutant forms mCL-I + II of the SARS–CoV S glycoprotein. (A) Autoradiograms of immunoprecipitates as in Fig. 6A for the double-mutant mCL-I + II. (B) An estimation of the relative concentration of the palmitoylated S species in comparison to the wild-type S is shown as in Fig. 6B.
Fig. 8
Fig. 8
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 deviation calculated through comparison of the data from each of three experiments.
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