Figure 1.
Genome-wide RNAi screen in Drosophila for host factors involved in WNV infection.
A. Representative images of DL1 cells treated with the indicated dsRNAs and infected with WNV (nuclei, blue; WNV NS1, green). B. Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; ** p<0.01. C. Schematic of screening pipeline including the scatter plot of Robust Z-scores for each gene assayed in duplicate. VSFs (376) and VRFs (161) are noted. D. Bioinformatics show fraction of candidate genes that have human or mosquito orthologs. Significant enrichment of conserved genes (p<0.0001) and under-enrichment of Drosophila-specific genes (p<0.0001) as analyzed by chi-squared test. E. Pie chart of candidate genes and validation results (50 VRF, 96 VSF, 71 not validated). F–G. Gene ontology enrichment of validated genes with five or more members displayed (p<0.001). F. VSF categories enriched. G. VRF categories enriched.
Figure 2.
Comparisons of virus dependencies.
A. Table listing the vector-borne viruses tested and general classifications based on virus family, genome structure, and natural vector. B. A hierarchical heat map of six viruses screened displaying Robust Z-scores for each VRF against WNV, WNV-KUN, DEN, SINV, RVFV, and VSV. C. Percentage of WNV VRFs that also restricted the indicated virus. D. Table of the seven Drosophila genes that had antiviral activity against all six viruses tested. Human and mosquito orthologs are listed.
Figure 3.
dRUVBL1 is a broadly antiviral gene.
A. Representative images of Drosophila cells treated with control (β-gal) or dRUVBL1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). B, Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. C–D. Viral RNA levels measured using qRT-PCR in Drosophila cells treated with β-gal (control) or dRUVBL1 dsRNA infected with WNV (C) or VSV (D) Mean ± SD of fold change for 3 independent experiments; ** p<0.01. E–H. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (E–F) or VSV (G–H). Mortality was monitored as a function of time post-infection (E,G) (log rank: * p<0.05, ** p<0.01). (F,H) Groups of 15 flies of the indicated genotypes were challenged, and viral titers were assessed by plaque assay in 4–7 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean.
Figure 4.
Tip60 complex has antiviral activity.
A. Table of RUVBL1-associated complexes, whether the complex is dependent on RUVBL2, and other genes in the complexes tested for antiviral activity. Genes in red were found to be antiviral against both WNV and VSV. B–C. DL1 cells were treated with the indicated dsRNA and then infected with (B) WNV or (C) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01. D–E. Aag2 cells were treated with the indicated dsRNA and then infected with (D) WNV-KUN or (E) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.
Figure 5.
dXPO1 has antiviral activity in insects.
A. Representative images of Drosophila cells treated with control (β-gal) or dXPO1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). B. Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. C–D. Viral RNA levels measured using RT-qPCR in Drosophila cells treated with β-gal (control) or dXPO1 dsRNA and infected with WNV (C) or VSV (D). Mean ± SD of fold change for 3 independent experiments; * p<0.05. E–H. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (E, G) or VSV (F, H) and mortality (E, F) was monitored as a function of time post-infection (** p<0.01 log rank). (G, H) Groups of 15 flies of the indicated genotypes were challenged, and viral titer was assessed by plaque assay in 3 or 4 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean. I–J. Aag2 cells were treated with the indicated dsRNA and then infected with (I) WNV-KUN or (J) VSV. Mean ± SD of fold change in percent infection compared to control (β-gal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.
Figure 6.
dXPO1 targets dALDOA and restricts viral infection.
A. Table of genes whose mRNA export is LMB-dependent, their level of induction by VSV infection, and whether they have been identified as antiviral previously. B. Schematic overview of glycolysis pathway (red, Aldolase (Ald); blue, enzymes tested; black, enzymes not tested; green, inhibitors). C–D. DL1 cells were treated with the indicated dsRNA and then infected with (C) WNV-KUN or (D) VSV. Data is presented as Mean ± SD of fold change in percent infection compared to control (β-gal dsRNA) for 3 independent experiments; * p<0.05.
Figure 7.
RUVBL1 and XPO1 restrict viral infection in mammalian cells.
A–D. Human U2OS cells were transfected with siRNAs against a control, hRuvBL1, or hXPO1 and challenged 3 days post transfection with WNV-KUN for 20 hours (A–B) or VSV for 12 hours (C–D). Cells were fixed, processed for microscopy and quantified in A, C. Mean ± SD of fold change compared to control for 3 independent experiments; * p<0.05, **p<0.01. Cells were processed for northern blots and quantified displaying the mean for 3 independent experiments with control set to 1; * p<0.05, **p<0.01 in B, D. E. 293T cells were transfected with siRNAs against control or two independent siRNAs against hTIP60 and challenged 3 days post transfection with WNV for 24 hours and processed by flow cytometry. Three independent experiments were quantified; Mean ± SD of the fold change in infection is shown and normalized to the control; **p<0.01. F. Primary neurons transduced with lentiviruses expressing the indicated shRNAs were infected with WNV for 24 hours and processed for viral yield by focus forming assays. Mean ± SD for 3 independent experiments; * p<0.05, **p<0.01. G–H. U2OS cells were treated with vehicle or LMB and infected with (G) WNV-KUN or (H) VSV. Mean ± SD of fold change in percent infection compared to control (vehicle) for 3 independent experiments; * p<0.05, ** p<0.01.