Virus surface proteins mediate interactions with target cells during the initial events in the process of infection. Inhibiting these proteins is therefore a major target for the development of antiviral drugs. However, there are a very large number of different viruses, each with their own distinct surface proteins and, with just a few exceptions, it is not clear how to build novel molecules to inhibit them. Here we applied a computational binding optimization strategy to an atomic resolution structure of dengue virus serotype 2 envelope protein to generate peptide sequences that should interact strongly with this protein. We picked dengue virus as a target because it is the causative agent for the most important mosquito transmitted viral disease. Out of a small number of candidates designed and tested, we identified two different highly inhibitory peptides. To verify our results, we showed that these peptides block virus:cell binding, interfere with a step during viral entry, alter the surface structure of dengue viral particles, and that they interact directly with dengue virus envelope protein. We expect that our approach may be generally applicable to other viral surface proteins where a high resolution structure is available. Focus forming unit assay LLC-MK2 target cells were seeded at a density of 1610 5 cells in each well of a 6-well plate 24 h prior to infection. Approximately 200 focus forming units (FFU) of virus were incubated with or without peptide in serum-free DMEM for 1 h at rt. Virus/peptide or virus/ control mixtures were allowed to infect confluent target cell monolayers for 1 h at 37uC, with rocking every 15 m, after which time the medium was aspirated and overlaid with fresh DMEM/10% (v/v) FBS containing 0.85% (w/v) Sea-Plaque Agarose (Cambrex Bio Science, Rockland, ME) without rinsing. Cells with agar overlays were incubated at 4uC for 20 m to set the agar. Infected cells were then incubated at 37uC with 5% CO 2 for 5 days. Infected cultures were fixed with 10% formalin overnight at 4uC, permeablized with 70% (v/v) ethanol for 20 m, and rinsed with phosphate buffered saline, pH 7.4 (PBS) prior to immunostaining. Virus foci were detected using a specific mouse mAb from hybridoma E60 (obtained from M. Diamond at Washington University), followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Pierce, Rockford, IL), and developed using AEC chromogen substrate (Dako, Carpinteria, CA). Results are expressed as the average of at least two independent trials with three replicates each. IC 50 values were determined using variable slope sigmoidal doseresponse curve fits with GraphPad Prism 4.0 software (LaJolla, CA), except for DN81opt, which was determined graphically due to a lack of data points to produce a reasonable curve fit.