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

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.

Role of Spike Protein Endodomains in Regulating Coronavirus Entry

“…1B). The COOH-terminal part of this region comprises the cysteine-rich motif, and if all cysteines are palmitoylated, as is strongly suggested by [ 3 H]palmitate labeling (28,30), then this region would be extraordinarily lipophilic. Indeed, each S trimer would add 27 16-carbon acyl chain lipids to the intravirion membrane leaflet.…”

“…Indeed, each S trimer would add 27 16-carbon acyl chain lipids to the intravirion membrane leaflet. Several reports evaluating truncated coronavirus S proteins missing part or all of these acylated tails have provided valuable data on the minimal tail lengths required to preserve biological function (28,30,67,68). We used a more subtle approach to evaluate tail activities by substituting one or more of the nine cysteines in the palmitoylation motif with alanines.…”

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry

“…The exact boundaries of transmembrane (TM) domains of coronaviruses have not yet been experimentally specified. Different predictions include or exclude part of the cysteine-rich domains as part of the TM domain (Bosch et al, 2005;Broer et al, 2006;Chang et al, 2000;Godeke et al, 2000;Petit et al, 2007;Thorp et al, 2006;Ye et al, 2004;Zheng et al, 2006). According to TMpred, the strongly preferred TM model of NL63 S protein predicts a TM to be located between residues 1299 and 1316 (VWLIISVVFVVLLSLLVF), which does not include any cysteine-rich domains.…”

Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

“…A short cytoplasmic tail or endodomain is located at the C-terminal end of S. It contains conserved stretches of cysteine residues, which can be palmitoylated. Palmitoylation of cysteine residues within the endodomain was found to be important for regulating fusogenicity of S (Petit et al, 2007; Shulla and Gallagher, 2009). Cryo-EM studies on single particles of the SARS-CoV have shed light on the overall architecture of S protein trimers and the conformational changes they undergo during fusion (Beniac et al, 2006; Beniac et al, 2007).…”