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

Kathleen Börner (1) , Johannes Hermle (1) , Christoph Sommer (2) , Nigel P Brown (3) , Bettina Knapp (4) , Glass Bärbel (1) , Julian Kunkel (5) , Gloria Torralba (6) , Jürgen Reymann (7) , Nina Beil (1) , Jürgen Beneke (1) , Rainer Pepperkok (8) , Reinhard Schneider (1) , Thomas Ludwig (1) , Michael Hausmann (1) , Fred Hamprecht (1) , Holger Erfle (1) , Lars Kaderali (1) , Hans-Georg Kräusslich (1) , Maik Joerg Lehmann (1)

1 Department of Virology,
2 HCI - Heidelberg Collaboratory for Image Processing
3 Structural and Computational Biology Unit, European Molecular Biology Laboratory,
4 Viroquant Research Group Modeling, Bioquant Center, University of Heidelberg,
5 IPVS - Institute of Parallel and Distributed Systems [Stuttgart]
6 Kirchhoff Institut für Physik
7 Viroquant-CellNetworks RNAi Screening Facility, Bioquant Center, University of Heidelberg,
8 Cell Biology and Cell Biophysics Unit, European Molecular Biology Laboratory,

RNA interference (RNAi) has emerged as a powerful technique for studying loss of function phenotypes 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 siRNA 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 the description of the platform will guide further screening approaches for virus host interactions. Moreover, 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 providing service to external users in the next future.

Mots clés: Life Sciences

Kathleen Börner, Johannes Hermle, Christoph Sommer, Nigel P Brown, Bettina Knapp, et al.. From experimental setup to bioinformatics: an RNAi screening platform to identify host factors involved in HIV-1 replication. Biotechnology Journal, 2009, 5 (1), pp.39. ⟨10.1002/biot.200900226⟩. ⟨hal-00545273⟩