This difference might be caused by the use of more than 1,000 times more virus in the genome degradation experiments, or having only partially released genomes after incubation with DN59. Although particles with partially released genomes are likely to be non-infectious, their genomes may still have been protected from degradation by RNase. This would cause the IC50 for the genome degradation assay to shift upwards in concentration compared to the FFU reduction assay. The separation of the genome from the virus particle would be expected to irreversibly destroy infectivity. Reversibility was tested directly by treating virus with peptide at a concentration expected to produce approximately 80% inhibition of infectivity, then diluting the virus:peptide mixture 10 fold to a peptide concentration expected to produce negligible inhibition. No reversibility of inhibition was observed in these experiments. The release of the virus RNA genome was confirmed by centrifuging peptide-treated, untreated, and triton detergenttreated virus particles BKM120 through a tartrate density gradient, and monitoring the amount of RNA genome and E protein in each fraction. The results showed that the genome and E protein comigrate in intact virus particles, but migrate to different fractions following peptide or detergent treatment, indicating that the genome and E protein are no longer associated after peptide treatment. To Rapamycin confirm that there were no other targets for the inhibitory activity of DN59, time of addition and infectivity assays in a different target cell line were conducted. There was no inhibition of infectivity when mammalian target cells were incubated with DN59 and then washed prior to the addition of virus. Nor was there inhibition of infectivity when DN59 was added after the cells had been infected. Furthermore, after coincubation of virus with DN59, infection was inhibited in both mammalian epithelial and mosquito cells, showing that changes of the host cell type and corresponding viral entry pathway did not result in changes of the neutralization profile. Therefore, it can be concluded that DN59 acts directly on the virus particle to release the RNA genome rather than on some other viral or cellular target. Based on these experiments, DN59 appears to induce formation of holes in the viral membrane. Thus, DN59 might be expected to interact with lipid membranes and form holes or otherwise disrupt membrane bilayer structures. Consistent with this expectation, a concentration-dependent increase in the fluorescence of the tryptophan residue at peptide position nine was observed when peptide was mixed with liposome vesicles composed of either 1palmitoyl-2-oleoyl-phosphatidylcholine, or a 9:1 molar ratio of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol, indicative of strong binding. Also, addition of DN59 peptide to either POPC or POPC/POPG vesicles containing a fluorescent dye and quencher caused extensive disruption of membrane integrity and leakage of contents to occur at concentrations as low as 2 mM. These observations confirm that DN59 interacts strongly with liposome vesicles and is capable of disrupting artificial lipid bilayers. The observed peptide-lipid membrane interactions are not merely charge based, as binding and disruption occurred with both zwitterionic POPC vesicles as well as negatively-charged 9:1 POPC/POPG vesicles. Supporting these observations, a recent study of the membrane disruption ability of overlapping peptides from dengue virus type 2 C and E proteins showed that E protein stem derived peptides were highly disruptive to liposomes prepared with a wide variety of lipid compositions.