The Aromatic Domain of the Coronavirus Class I Viral Fusion Protein Induces Membrane Permeabilization: Putative Role during Viral Entry †

Department of Biochemistry, Tulane University, New Orleans, Louisiana, United States
Biochemistry (Impact Factor: 3.02). 02/2005; 44(3):947-58. DOI: 10.1021/bi048515g
Source: PubMed


Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.

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    • "Several studies are in support of the lipid model. Sainz Jr. et al have suggested that during the conformational changes in the spike the aromatic domain might align the fusion peptide and the TMD, thereby functioning as a hydrophobic sheet to allow lipid flow between the two fusing membranes [22]. Other reports have shown that peptides, representing the aromatic domain (of SARS CoV or HIV) are membrane active and are capable of altering the biophysical properties of membranes [21,23-25]. "
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    ABSTRACT: 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. 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. 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.
    Full-text · Article · Dec 2009 · Virology Journal
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    • "Interestingly, the regions proximal to the TM domains, that are usually rich in aromatic amino acids, demonstrate strong tendencies to partition into membrane interfaces contributing to the destabilization of the membranes necessary for fusion [29] [30]. This has been widely reported in the literature for class I fusion proteins [31] [32] [33] [34]. "
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    ABSTRACT: A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314-342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1(314-342) peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319-323 and 329-338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide-bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.
    Full-text · Article · Nov 2009 · Biochimica et Biophysica Acta
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    • "Studies with a number of viral fusion proteins have showed that the region immediately adjacent to the membrane-spanning domain plays an essential role in the fusogenic activities of these proteins, being a common characteristic to viral fusion proteins of several virus families [31] [32] [33]. These pre-transmembrane (PTM) domains would function as promoters of membrane destabilization [34] [35] [36]. We have recently identified the membrane-active regions of the HCV E1 and E2 glycoproteins by observing the effect of E1 and E2 glycoprotein-derived peptide libraries on model membrane integrity [29]. "
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    ABSTRACT: The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1(PTM) strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.
    Full-text · Article · May 2008 · Biochimica et Biophysica Acta
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