At the same time we added MTT reagents to prevent cellular loss during the high content screening inhibitor washing step. In general, insect and mammalian cell monolayers were intact after 30 min incubation with up to 16 or 24 mM of MbCD, respectively. Fig. 1B shows that insect cells were more affected by cholesterol depletion than mammalian cells. Indeed, 16 mM MbCD was able to decrease 50–60% of cholesterol in mammalian and insect cells but only affected the viability of insect cells. Thus, our results showed that some different MbCD concentrations can induce similar levels of cholesterol depletion but different responses in cellular viability. In our studies, low MbCD concentrations, which cause depletion of cholesterol but do not affect the cellular viability, were chosen to examine the role played by cholesterol during protein-membrane interaction. Recently, we have solved the NMR atomic structure of the Ebola fusion peptide in the presence of mimetic membranes, where a loop with a central 310-helix appears to be stabilized by aromatic-aromatic interaction. The ability of the Ebola peptide to induce membrane fusion has been related with the presence of phosphatidilinositol in the host cell membrane and Ca2+ during this process. 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.