Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition - PubMed (original) (raw)

Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition

Sara Cherry et al. Genes Dev. 2005.

Abstract

The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.

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Figures

Figure 1.

Figure 1.

Genome-wide RNAi screen reveals that ribosomal protein levels are critical for DCV replication. (A) Schematic diagram of RNAi screen in pre-aliquoted 384-well plates. (B) Decreased viral antigen production post-dsRNA treatment with dsRNA against DCV, RpS6, or RpL19 as measured by the ratio of FITC-anti-DCV (green) versus Hoescht 33342 (red). (C) Frequency of encoded functional groups as curated by Gene Ontology (The FlyBase Consortium 2003) and manually assigned to representative categories for all verified candidates. The breakdown of the ribosomal components identified is shown.

Figure 2.

Figure 2.

RNAi against ribosomal proteins leads to a tolerated depletion in the ribosome and protection from DCV infection. (A) Comparison of the effect of dsRNA treatment on viral replication as measured by immunofluorescence or viral titers released at 24 h post-infection. Results for averaged triplicate experiment where the error bars represent one standard deviation and asterisks denote p < 0.05 by _t_-test. (_B_) Western blot analysis of dsRNA-treated cell lysates probed with anti-RpS6 (_top_ panel) or anti-tubulin (_bottom_ panel). (_C_) Host protein synthesis is reduced by less than twofold as observed by pulse-labeling cells with S35 methionine for 1 h post-dsRNA treatment with either gfp, RpS6, or RpL19 while cycloheximide (CHX) reduces incorporation by >10-fold. Extracts were analyzed by trichloroacetic acid precipitation and quantitation (results for averaged triplicate samples from three independent experiments; error bars represent one standard deviation). (D) Northern blot analysis of dsRNA-treated total RNA probed with RpS6 or RpL19, respectively. (E) Fluorescence microscopy of cells treated with dsRNA. No significant change in cell number or cell death is evident by comparing the number of nuclei (Hoechst 33342-red) or the fraction of Sytox (green)-labeled nuclei to total nuclei (red) after depletion of ribosomal proteins by RNAi. dsRNA treatment to thread results in a significant increase in cell death. Percent of dead cells is shown for each treatment.

Figure 3.

Figure 3.

Depletion of the ribosome affects IRES-dependent translation but not 5′-cap dependent translation. (A) Fluorescence microscopy of cells treated with dsRNA and subsequently infected with VSV-gfp. dsRNA against ribosomal proteins has no effect on the percent infection by comparing gfp expression (green) to total nuclei stained with Hoechst 33342 (red). dsRNA treatment against Rab5 results in a significant protection from infection. Percent infected cells are shown for each treatment. (B) Schematic diagram of the DCV genome describing the location of the two viral IRESs. (C) Schematic diagram of the bicistronic vector used to determine whether depletion of ribosomal proteins has an effect on translation from a 5′-capped message (Renilla luciferase) or a DCV IRES (firefly luciferase). (D) dsRNA treatment against ribosomal proteins RpS6 or RpL19 results in a significant effect on translation from DCV IRES1 and IRES2 as measured by firefly luciferase, but not on the translation of the 5′-capped message as measured by Renilla luciferase (results for averaged triplicate experiments where error bars represent one standard deviation and asterisks denote p < 0.05 by _t_-test). Cells treated with Gfp dsRNA are set to 1, and all other dsRNA treatments are normalized to that condition. (E) DCV continues to be translated while host synthesis is repressed as observed by pulse-labeling cells with S35 methionine for 2 h at the indicated times post-infection with DCV or mock infected (uninfected). Soluble extracts were analyzed by SDS–polyacrylamide gel electrophoresis followed by autoradiography.

Figure 4.

Figure 4.

Inhibition of ribosomal function protects from infection in vivo and from poliovirus infection. (A) Adult wild-type flies (male, Canton-S) fed serial dilutions of Hygromycin B (280–17.5 μM) and then challenged with DCV were monitored daily for mortality. Error bars represent one standard deviation; results from a triplicate representative experiment averaged. (B) Poliovirus infection of Hela cells pretransfected with siRNAs against RpS6 results in inhibition of viral replication. Control siRNA pretreatment is set to 1 and the relative production of poliovirus is shown for four independent experiments where error bars represent one standard deviation and asteriks denote p < 0.05. (C) Host protein synthesis is reduced twofold as observed by pulse-labeling cells with S35 methionine for 20 min post-siRNA treatment with RpS6. Extracts were analyzed by trichloroacetic acid precipitation and quantitation (results for two independent experiments averaged; error bars represent each value).

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