RNAi-mediated knockdown of Drosophila genes involved in glycerophospholipid metabolism modulate FHV replication in S2 cells. (A) Medium throughput RNAi-based functional screen of select glycerophospholipid metabolism genes. Drosophila S2 cells were cultured in 96-well plates, incubated with dsRNA targeting specific glycerophospholipid metabolism-related genes, positive control FHV RNA1, or negative control LacZ for 48 h, infected with FHV, and harvested 18 h after infection. As an additional separate control, selected wells were treated with 50 μM miltefosine at the time of infection. FHV replication was assayed by protein B2 (PtnB2)-specific capture ELISA. Parallel MTT viability assays demonstrated > 90% viability for all dsRNA- or miltefosine-treated samples compared to LacZ dsRNA-treated control (data not shown). Results are expressed as the percentage of PtnB2 accumulation relative to LacZ dsRNA-treated control (hatched line). P-value < 0.05* or 0.005**. (B) Schematic of eukaryotic glycerophospholipid synthesis pathways. The simplified biosynthetic pathways shown were adapted from the detailed and comprehensive KEGG glycerophospholipid metabolism pathway available at http://www.genome.jp/kegg/. Drosophila genes with known or hypothesized lipid biosynthetic functions are shown, and those identified as being functionally important for FHV RNA replication are boxed in grey. The major eukaryotic glycerophospholipids are indicated in bold type: CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine. The essential precursor glycerol-3-phosphate and important intermediates are shown in italics: DAG, diacylglycerol.