Nor did DN59 induce substantial hemolysis of red blood cellsillustrating at concentrations as high as the 100 mM used for cryoEM. Additionally, DN59 does not inhibit the infectivity of other lipidenveloped viruses, including Sindbis virus or the negative-stranded RNA vesicular stomatitis virus. The lack of apparent disruption of cellular plasma membranes and other viral membranes may be due to lipid composition, protein incorporation, or active repair of cellular membranes. Dengue virus particles bud from 
internal endoplasmic reticulum membranes of infected cells and so likely have a AB1010 different composition from the plasma membrane, although the membrane disruption activity of stem region peptides is not strongly influenced by lipid membrane composition. Schmidt et al.studied a series of similar dengue E protein stem region peptides whose sequences extensively overlap the sequence of DN59. Consistent with our earlier work, they showed that their most active peptideinhibits dengue virus infection during an entry step and can bind to synthetic lipid vesicles. Furthermore, they reported that their peptide bound to the post-fusion trimeric form of recombinant dengue surface E proteinat low pH, but did not bind to the monomeric E protein at neutral pH. They therefore proposed that the peptide neutralizes the virus by first attaching to the viral membrane, and subsequently interacting with the E post-fusion Nutlin-3 trimers that form when the virus encounters the low pH environment of the endosome, thereby preventing fusion of the virus to the endosomal membrane. Here, however, we have shown that DN59 can induce the formation of holes in the viral membrane, release the genome, and causes the viral particles to become non-infectious even before interacting with cells. The discrepancy in the mechanism of neutralization detected by our group and Schmidt et al. could possibly be due to the differences in peptide concentration used in these assays. The most likely mechanism by which DN59 or other stem region peptides can penetrate the outer layer of E glycoproteins and gain access to the virus membrane is by way of dynamic “breathing” of the virus particle. The ease with which the virus can breathe will depend on the stability of the virus, which may account in part for the differing inhibitory activities against different flaviviruses. Once the DN59 peptide has inserted itself between the E ectodomain and the membrane, it likely competes with and displaces the virus E protein stem regionfor binding to the lipid membrane and the “underside” of the E protein. Formation of holes in the viral membrane large enough for the escape of the RNA genome may involve structural changes in the surface E and M proteins, or may be due to the action of the peptide alone, similar to what is observed for some anti-microbial peptidesand what we observed with liposome vesicles. The negative charge on the tightly packaged RNA may also help the RNA to exit the virus particle once the membrane has been destabilized. Our observations show that DN59, a 33 amino acid peptide mimicking a portion of the dengue virus E protein stem region, functions through an unexpected mechanism that involves disruption of the viral membrane and release of the viral genome. Caspases are a family of cysteinyl proteases that are key mediators of apoptosis and inflammation. The apoptotic “executioner” caspasesare translated as proenzymes containing a short pro-domain, a p20 subunit, a linker region, and p10 subunit. Their canonical activation mechanism involves proteolysis by “initiator” caspasesat three distinct sites to remove the prodomain and linker region.