Drosophila RNAi screen identifies host genes important for influenza virus replication - PubMed (original) (raw)

Drosophila RNAi screen identifies host genes important for influenza virus replication

Linhui Hao et al. Nature. 2008.

Abstract

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.

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Figures

Fig. 1

Fig. 1. Overview of genome-wide RNAi screen to identify host factors involved in influenza virus replication in Drosophila cells

Schematic diagrams showing recombinant influenza viruses, (A) FVG-G, in which genes encoding the HA and NA proteins were replaced with the VSV-G and eGFP genes, respectively, and (B) FVG-R, in which the genes encoding the HA and NA were replaced with the VSV-G and Renilla luciferase genes, respectively. (C) Schematic diagram of systematic analysis of host genes affecting influenza virus replication and gene expression in Drosophila cells. Experimental details are given in Methods.

Fig. 2

Fig. 2. Effect of selected siRNAs and inhibitors on Renilla luciferase expression in FVG-R-infected human cells

Renilla luciferase activity was measured in FVG-R-infected 293 cells treated with siRNAs against (A) ATP6V0D1, COX6A1, (B) NXF1, or (C) the indicated mitochondrial electron transport chain inhibitors. Inhibitors of complexes III, IV and V inhibited FVG-R-directed Renilla luciferase expression significantly, while complex I and II inhibitors had little or no effect. In contrast, the inhibitors had no significant effects on cell viability and Gaussia luciferase expression of a murine leukemia virus derivative (MLV-GL) that, like FVG-R, depended on VSV G-mediated entry. All experiments (A-C) were conducted three times in duplicate, with the results reported as means ± SD.

Fig. 3

Fig. 3. Effect of siRNAs against selected genes on the replication of influenza viruses, VSV or vaccinia virus in human 293 cells

The titers of influenza viruses (WSN and Indonesia 7), VSV and vaccinia virus in 293 cells treated with siRNA against (A) ATP6V0D1, COX6A1 or (B) NXF1 are shown. Experimental details are given in Supplementary Methods. All experiments were conducted three times, with the results reported as means ± SD.

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