Bioactive sphingolipids such as SM and CER are also key lipids that were up regulated during DENV infection. In cells exposed to replication competent virus, CER was up regulated in both the whole cell lipidome as well as replication complex membranes. This is either a cellular response to virus infection, or a direct need for CER in the virus replication cycle. The latter hypothesis is particularly attractive considering the enrichment of specific molecular species of CER in the 16K pellet. CER is a cone-shaped Figure 6 . Bioactive sphingolipid species are differentially regulated in replication complex membranes isolated from DENVinfected mosquito cells. Multiple Reaction Monitoring (MRM) analysis of sphingolipids species differentially regulated in DENV-infected cells (MOI 20) or UV-DENV exposed cells compared to the mock control (see also supplementary table S3). The data represent fold changes observed in three subcellular fractions that were analyzed in this study; 16K, replication complex membranes; CE, cytoplasmic extracts following removal of replication complex membranes and nuclei; N, nuclear fraction. Panels A-C represent ceramide, sphingomyelin and monohexosylceramide species respectively. The dashed line highlights values equal to the mock. The data represent three independent experiments. The error bars represent standard deviation of the mean. doi:10.1371/journal.ppat.1002584.g006 molecule that promotes inward budding of membranes (negative curvature) [42] . Some DENV-induced membranes show a double membrane morphology indicating that negative curvature modifying lipids such as CER may be active in their formation.