A comparative functional RNA-interference screen identifies factors differentially required for lipid droplet homeostasis and life cycle of Flaviviridae members

Lipid Droplets (LDs) play a central role in storage and mobilization of lipids and are involved in lipid metabolism-related diseases, as well as in the replication cycle of several viruses, including the hepatotropic hepatitis C virus (HCV) and the Dengue virus (DENV). However, in spite of their importance, the pathways and factors regulating LD homeostasis in human liver cells are poorly characterized, and their suitability as antiviral targets has not been explored. To overcome this limitation, we assembled a siRNA library targeting 230 genes potentially regulating LD homeostasis. This library was used to perform a comparative functional RNA-interference screen to identify key factors of LD homeostasis was well as factors promoting or restricting HCV and DENV replication. We could identify 59 genes as important factors for LD homeostasis, most of them also playing key roles for viral replication. Bioinformatic analysis of hits identified biological processes regulating both viral life cycle and LD homeostasis. These include COPI-coated vesicle budding, RNA splicing or proteasomal- and ubiquitin-dependent catabolic processes. Upon confirmation by a secondary deconvolution screen, the DDX3 dead box helicase and the calcium-independent phospholipase A2 iPLA2β were selected for follow-up studies. Our results suggest a novel role for DDX3 in HCV assembly/release, which appears to be independent from its interaction with core protein. Moreover, pharmacological ablation of iPLAβ activity selectively impaired HCV particle production, but did not affect the DENV replication cycle.