Abstract
BackgroundThe 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.ResultsMutagenesis 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.ConclusionsThe 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.
Highlights
Introduction of lysines in the SARSS transmembrane domain (TMD) As shown in Figure 1A, the supposed TMD domain of SARS S is quite long, especially when the locations of charged residues, that usually define the borders of a TMD, are considered
The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process
By swapping the TMD of severe acute respiratory syndrome coronavirus (SARS CoV) spike for that of vesicular stomatitis virus glycoprotein (VSV-G), we have shown that both entry of SARS pseudoparticles (SARSpp) and SARS CoV spike protein mediated cell-cell fusion depends on the presence of the TMD of the spike [14]
Summary
Introduction of lysines in the SARSS TMD As shown in Figure 1A, the supposed TMD domain of SARS S is quite long, especially when the locations of charged residues, that usually define the borders of a TMD, are considered. The spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. The mechanism by which the viral spike proteins mediate the initial stages of membrane fusion is fairly well understood for a number of viruses. The viral fusion proteins of all classes refold to establish a conformation that brings the fusion peptide and the transmembrane domain (TMD) in close proximity, initializing membrane fusion [1]. Virology Journal 2009, 6:230 http://www.virologyj.com/content/6/1/230 domains (TMDs) or amino acid residues adjacent to the TMDs of viral fusion proteins, play a role in this process [2]. It has been shown that glycine residues of the TMD of the vesicular stomatitis virus glycoprotein (VSV-G), play a critical role in membrane fusion [6]. By swapping the TMD of severe acute respiratory syndrome coronavirus (SARS CoV) spike for that of VSV-G, we have shown that both entry of SARS pseudoparticles (SARSpp) and SARS CoV spike protein mediated cell-cell fusion depends on the presence of the TMD of the spike [14]
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