From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV-1 replication

摘要

RNA interference (RNAi) has emerged as a powerful technique for studying loss-of-function phe-notypes by specific down-regulation of gene expression, allowing the investigation of virus-host interactions by large-scale high-throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single-cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two-stage procedure in which potential host factors are first identified in a primary screen and afterwards re-tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus-1 (HIV-1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus-host interactions. The ViroQuant-CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.