In addition to evaluating the lipid composition of whole cells, we also explored the possibility of profiling the lipid repertoire of specific membranes induced by DENV. Given the extensive interconnectivity of the membranes induced in DENV infected cells, isolation of morphologically distinct membranes proved challenging. Therefore, we carried out subcellular fractionation of C6/36 cells and isolated post-nuclear supernatants that were further fractionated to provide a total membrane fraction (16K pellet) enriched in viral replication components and a remaining cytoplasmic extract (CE) [35] . To confirm that the 16K pellet was indeed the membrane fraction enriched in the replication complex components, we used quantitative RT-PCR to measure the ratio The fold changes represent DENV-infected cells or UV-DENV exposed cells compared to the mock control. A lack of cones indicates that the expression level of those specific lipids were not significant (p,0.05). Panels C-H are representative lipid molecular species from specific lipid classes significantly regulated at the two different time points. The data are plotted as the integrated LC-MS peak abundance, in log 2 scale with standard deviation. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; CER, ceramide; CER-PE, ceramide phosphoethanolamine; Lyso, lysophospholipids. See supplementary table S1 for a complete list of lipid features detected in this study. Four replicates were included in the lipidomic analyses. The error bars represent standard deviation of the mean. The blue dashed line separates species that remain elevated at both time points (36 and 60 hr) from species that are only elevated at the 36 hr time point. Infections were carried out using an MOI of 20 in C6/36 cells. Significantly expressed lipids species are shown denoted with an asterisk (*). doi:10.1371/journal.ppat.1002584.g003 of viral RNA to lipid in both 16K and CE fractions. Lipids were quantified by pulse-chase analysis using 14 C-acetate to label newly synthesized lipids and the results are shown in Figure 4 . In postnuclear supernatants, there was increase in the incorporation of 14 C-acetate into lipids in the DENV infected cells compared to the mock control ( Figure 4A ). This increase was observed at both 36 hr and 60 hr post-infection with the latter time point showing a greater difference between DENV and mock. A more detailed analysis of the lipid distribution in the subcellular fractions (16K and CE) is shown in Figure 4B . At the 36 hr time point, the CE fraction was enriched in newly synthesized lipids (compared to the 16K fraction) in both the DENV and mock samples, with DENV showing an almost 2-fold increase compared to mock. This distribution changed at the 60 hr time point where the 16K pellet was more enriched in newly synthesized lipids, however, the fold change (DENV compared to mock) was not as significant.