INCB18424 abmole bioscience fusion is mediated by the viral envelope protein that contains a nonpolar fusion peptide. In general, fusion peptides that belong to class I viral fusion proteins are located at the N-terminus, whereas in class II, they are in the internal region. However, in both cases they are typically rich in alanine and glycine residues and highly conserved within a virus family. The interaction of the fusion peptide with target membranes is critical for fusion. Therefore, this region has to be exposed at the proper place and time in order to trigger the interaction. Recent studies have suggested the critical role of lipid rafts in filovirus entry into the host cells. Lipid rafts are microdomains in biological membranes that are rich in cholesterol and sphingolipids and play an important role in many events including the endocytic, bio-synthetic and signal transduction pathways. The requirement of lipid rafts for the virus to enter host cells has been related with the localization of receptors and co-receptors in these microdomains. Many viruses use a specific interaction between their GPs and cell surface receptors to initiate the attachment to cells and subsequent fusion. Thus, lipid rafts may promote virus entry by concentrating the viral receptors and facilitating binding via an efficient interaction of these receptors with viral proteins. Interestingly, the filovirus co-factor folate receptor-a is a raft-associated glycophosphatidylinositol-anchored protein. However, the critical role of FRa has been questioned due to the fact that FRa ˜negative cells are fully infectible by GP pseudotypes. In order to determine the importance of cholesterol during membrane fusion and the real importance of the aromaticaromatic interaction in the peptide structure, we studied the interaction of the wild type fusion peptide and its mutant W8A peptide with either cholesterol-depleted cells or rafts isolated from Vero and BHK-21 cells. Our results show that the Ebola fusion peptide interacts with living cells, and its capacity to induce cellcell fusion is decreased in cholesterol-depleted cells. Force spectroscopy based on atomic force microscopy assays reveals a pattern of high affinity force when the Ebola fusion peptide interacts with membrane rafts. It is also observed that the peptide is able to induce aggregation of the lipid rafts, suggesting an important role for phosphatidylinositol and cholesterol during entry of the virus into the target cells. In addition, insect cells, previously grown in medium with cholesterol, were assayed as a cellular control of low cholesterol content cells. Insect cells are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. As observed in Fig. 1A, the cholesterol content of C6/36 cells was maintained after incubation with up to 12 mM MbCD. However, upon incubation with 20 and 24 mM MbCD, it was not possible to detect cholesterol due to the low cell adhesion induced by depletion.