Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion.
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.
Petit
Chad M
CM
Division of Biotechnology and Molecular Medicine (BIOMMED), USA.
Chouljenko
Vladimir N
VN
Iyer
Arun
A
Colgrove
Robin
R
Farzan
Michael
M
Knipe
David M
DM
Kousoulas
K G
KG
D000595:Amino Acid Sequence; D000818:Animals; D002459:Cell Fusion; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D007150:Immunohistochemistry; D007553:Isotope Labeling; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000032:analysis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000502:physiology*; D064370:Spike Glycoprotein, Coronavirus; D014316:Tritium / Q000378:metabolism; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; D014316:Tritium; D019308:Palmitic Acid; D003545:Cysteine