Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: refinement of the requirements for SARS coronavirus cell entry.
BACKGROUND
The spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. Close to or in the viral membrane, the S protein contains three distinct motifs: a juxtamembrane aromatic part, a central highly hydrophobic stretch and a cysteine rich motif. Here, we investigate the role of aromatic and hydrophobic parts of S in the entry of SARS CoV and in cell-cell fusion. This was investigated using the previously described SARS pseudotyped particles system (SARSpp) and by fluorescence-based cell-cell fusion assays.


RESULTS
Mutagenesis showed that the aromatic domain was crucial for SARSpp entry into cells, with a likely role in pore enlargement.Introduction of lysine residues in the hydrophobic stretch of S also resulted in a block of entry, suggesting the borders of the actual transmembrane domain. Surprisingly, replacement of a glycine residue, situated close to the aromatic domain, with a lysine residue was tolerated, whereas the introduction of a lysine adjacent to the glycine, was not. In a model, we propose that during fusion, the lateral flexibility of the transmembrane domain plays a critical role, as do the tryptophans and the cysteines.


CONCLUSIONS
The aromatic domain plays a crucial role in the entry of SARS CoV into target cells. The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process.
Corver
Jeroen
J
Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, the Netherlands. j.corver@lumc.nl
Broer
Rene
R
van Kasteren
Puck
P
Spaan
Willy
W
D000595:Amino Acid Sequence; D002459:Cell Fusion; D002460:Cell Line; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000472:pathogenicity*; D064370:Spike Glycoprotein, Coronavirus; 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; C578553:spike protein, mouse hepatitis virus