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, 4 (3), e00385-13

A Short Hairpin RNA Screen of Interferon-Stimulated Genes Identifies a Novel Negative Regulator of the Cellular Antiviral Response

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A Short Hairpin RNA Screen of Interferon-Stimulated Genes Identifies a Novel Negative Regulator of the Cellular Antiviral Response

Jianqing Li et al. mBio.

Abstract

The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-β-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses. We also defined one ISG, activating signal cointegrator complex 3 (ASCC3), which functioned as a negative regulator of the host defense response. Silencing of ASCC3 resulted in upregulation of multiple antiviral ISGs, which correlated with inhibition of infection of several positive-strand RNA viruses. Reciprocally, ectopic expression of human ASCC3 or mouse Ascc3 resulted in downregulation of ISGs and increased viral infection. Mechanism-of-action and RNA sequencing studies revealed that ASCC3 functions to modulate ISG expression in an IRF-3- and IRF-7-dependent manner. Compared to prior ectopic ISG expression studies, our shRNA screen identified novel ISGs that restrict infection of WNV and other viruses and defined a new counterregulatory ISG, ASCC3, which tempers cell-intrinsic immunity.

Figures

FIG 1
FIG 1
Lentivirus-based shRNA screen for ISGs that affect WNV infection. (A) Schematic of pGIPZ lentivirus vector expressing shRNA and GFP. LTR, long terminal repeat of HIV; CMV, immediate early promoter from human cytomegalovirus; IRES, internal ribosome entry site; Puro, puromycin selection marker. (B) Cartoon of the shRNA screen. Packaged and pseudotyped lentiviruses expressing 813 shRNAs were transduced into HeLa cells (list in Table S1 in the supplemental material). Forty-eight hours later, cells were treated with human IFN-β (10 IU/ml for 6 h) and then infected with WNV at an MOI of 5. Forty-eight hours later, cells were processed by flow cytometry. (C) Representative flow cytometry contour plots (from two independent screens) showing cells transduced with lentiviral vectors containing a scrambled shRNA control or shRNA specific against ISGs with top Z scores from the primary screen. The x axis reports the lentivirus transduction efficiency as represented by GFP expression, and the y axis shows infectivity of WNV, as judged by anti-envelope protein staining.
FIG 2
FIG 2
Validation of ISGs with antiviral potential. (A) Distribution of WNV infection in cells transduced with shRNA (normalized to the scrambled shRNA). Selected ISGs targeted by shRNA hits are labeled in blue. (B) Pie chart showing classification of top ISG hits, grouped by their known or putative functional categories (see Table S2 in the supplemental material). (C) Pie charts showing the breakdown of the 47 ISGs that were validated by viral yield assays. The chart on the left shows the number of ISGs in which silencing resulted in enhanced WNV production (relative to cells transduced with the scrambled shRNA) at different time points. The chart on the right shows the relative fold differences in viral yield associated with silencing of these ISGs (see also Table S3). (D) mRNA was harvested from cells transduced with the indicated shRNA or the scrambled shRNA control, and relative expression of the corresponding ISGs was determined by qRT-PCR (see also Fig. S1). The results are the averages of three independent experiments and are shown as means ± standard deviations. Statistical significance was determined by Student’s t test (***, P < 0.001; **, P < 0.01; *, P < 0.05).
FIG 3
FIG 3
Antiviral genes against RNA viruses. (A to J) Multistep growth analysis of WNV infection on HeLa cells transduced with either scrambled shRNA or shRNA targeting the following top candidates: PKR, DDX24, IFI44L, IFI6, IFRD1, IL13RA1, MAFK, PAK3, SAMD9L, and SC4MOL. After transduction, cells were treated with 10 IU/ml of IFN-β for 6 h and then infected with WNV at an MOI of 0.05. The data are the averages of three independent experiments performed in triplicate with error bars indicating the standard deviations and asterisks marking values that are statistically different from the scrambled shRNA (***, P < 0.001; **, P < 0.01; *, P < 0.05). (K) HeLa cells were transfected with ISGs tagged or untagged with 3× Flag. One day later, cells were infected with WNV at an MOI of 0.3. One day after this, viral infectivity was measured as the percentage of infected cells and is represented as mean ± standard deviation. Statistical significance was determined by Student’s t test (***, P < 0.001; **, P < 0.01; *, P < 0.05). (L and M) Titers of DENV-2 and EMCV grown from HeLa cells transduced with scrambled shRNA and shRNA targeting IRF9, IFI6, IFI44L, DDX24, MAFK, PAK3, SC4MOL, and IFRD1. Data are shown as means ± standard deviations. Statistical significance was determined by Student’s t test (***, P < 0.001; **, P < 0.01; *, P < 0.05).
FIG 4
FIG 4
Silencing of ASCC3 restricts viral replication. (A) Expression of ASCC3 mRNA in HeLa cells expressing scrambled or ASCC3 shRNA. The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001). (B to D) Titers of WNV (B), Chikungunya virus (C), and EMCV (D) in the supernatants of HeLa cells transduced with either control or shRNA against ASCC3 at the indicated time points. The results are the averages of three independent experiments performed in triplicate, and asterisks indicate differences from the scrambled shRNA that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05). (E and F) HeLa cells were transfected with expression plasmids encoding GFP or human ASCC3 tagged at the C terminus with HA. (E) The transfection efficiency was measured by flow cytometry using an anti-HA tag antibody and an Alexa Fluor 647-conjugated secondary antibody. (F) Multistep growth analysis of WNV infection in GFP (vector) and human ASCC3-transfected cells. The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01). (G to I) NIH 3T3 cells were transduced with lentivirus carrying a control shRNA or shRNA against murine Ascc3. The mRNA levels of Ascc3 were measured under basal or IFN-β-induced conditions by qRT-PCR and are expressed relative to those after transduction with scrambled shRNA (G). Multistep growth analysis of WNV infection was performed in the corresponding cells without (H) or with (I) IFN-β pretreatment (50 IU/ml for 6 h). The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05).
FIG 5
FIG 5
Silencing ASCC3 expression upregulates cellular antiviral response. (A) HeLa cells were transduced with either scrambled shRNA or shRNA against ASCC3. Forty-eight hours later, cells were treated with 10 IU/ml of human IFN-β for 6 h. mRNAs were then harvested, purified, and sequenced. Ingenuity pathway analysis determined expression and interaction of ISGs and PRRs. Red and green colors indicate higher and lower gene expression levels, respectively, in ASCC3-silenced cells than in control cells. Fold differences in expression are listed in Table S4 in the supplemental material with a cutoff of a ≥2-fold change and an ANOVA P value with a Benjamini and Hochberg algorithm-corrected false discovery rate of <0.05. Shown are functional relationships that associate with antimicrobial pathway, inflammatory pathway, and posttranslational modification. (B and C) Gene expression profiles of 13 ISGs in HeLa cells that were transduced with an shRNA (scrambled or targeting ASCC3) under basal (B) or IFN-β treatment (50 IU/ml for 6 h) (C) conditions. mRNA abundance of ISGs was assayed using a commercial TaqMan array in 96-well plates and normalized to GAPDH. Statistical significance was determined by Student’s t test (**, P < 0.01; *, P < 0.05).
FIG 6
FIG 6
ASCC3 functions through an IRF-3- and IRF-7-dependent pathway. (A) Primary Ikkβ−/− MEFs were transfected with pCAGGS-GFP or pCAGGS-ASCC3-HA and then infected with WNV at an MOI of 0.05. Viral titers were monitored through a focus-forming assay at indicated time points. The results are the averages of three independent experiments performed in duplicate, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01). (B) Primary wild-type (WT) and Irf3−/− × Irf7−/− DKO MEFs were transduced with scrambled shRNA or shRNA against murine Ascc3. Cells were either untreated or treated with 10 IU/ml murine IFN-β for 6 h. Total RNA was harvested, and expression of Ascc3 was determined by qRT-PCR. (C) Multistep growth analysis of WNV infection was performed on the corresponding cells after IFN-β treatment. (D to F) Expression of three ISGs was assayed in shRNA-transduced WT and Irf3−/− × Irf7/− DKO MEFs at 72 h post-WNV infection. Relative expression levels of Ifi44 (D), Irf1 (E), and Rsad2 (F) were normalized to scrambled shRNA-transduced cells. The results are the averages of three independent experiments performed in triplicate, and asterisks indicate differences from the scrambled shRNA control that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant).

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