Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion.
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.
Petit
Chad M
CM
Division of Biotechnology and Molecular Medicine (BIOMMED), School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Melancon
Jeffrey M
JM
Chouljenko
Vladimir N
VN
Colgrove
Robin
R
Farzan
Michael
M
Knipe
David M
DM
Kousoulas
K G
KG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002459:Cell Fusion; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D007150:Immunohistochemistry; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000502:physiology; D017384:Sequence Deletion; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D014760:Viral Fusion Proteins / Q000032:analysis
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; C578557:spike glycoprotein, SARS-CoV