Abstract
The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.
Highlights
The Filoviridae family contains the Ebola and Marburg viruses
Cholesterol has been proven to be essential for filovirus replication, and the entry of the Ebola and Marburg viruses is inhibited after cholesterol depletion of the target cells [25]
Since Vero and BHK-21 mammalian cells are permissive to infection mediated by the Ebola virus, initial attempts were performed by using those cells [28]. b-cyclodextrins were used to since they are very effective to selectively extract cholesterol from membranes of intact cells without binding or insertion into the plasma membrane [29,30]
Summary
The Filoviridae family contains the Ebola and Marburg viruses. These are enveloped viruses composed of seven genes which encode eight proteins in the Ebola virus and seven in the Marburg virus [1]. The single-stranded negative-sense RNA genome is encased in a nucleocapsid complex, which consists of the following four viral proteins: the nucleoprotein (NP), the viral proteins (VP35 and VP30) and the polymerase (L). This complex is surrounded by a matrix consisting of VP40 and VP24, which is packaged by a lipid membrane envelope obtained during budding from the host cell. GP1 is responsible for interaction with its cellular receptor, and GP2 is involved in the mechanism of membrane fusion [4,5]
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