Validation of synergy between ZAP and the top three ISGs in a knockdown system.

<p>A) Triplicate wells of Huh-7 cells were transfected with irrelevant siRNA, ZAP-specific siRNA, ISG-specific siRNA that targets IRF2, RIG-I or IL28RA, or siRNAs that target both ZAP and an ISG. ISG-specific siRNA was added to cells again on the second day after seeding. Forty-eight h after initial siRNA transfection, cells were infected with Toto1101/Luc (moi = 5). Viral replication was determined by firefly luciferase activity 4 h after infection. Huh-7 cells that were not transfected with siRNA were included as a negative control. Means and standard deviations of triplicate samples are shown. Asterisks indicate mean values statistically different between two siRNA treatments (unpaired t test, *, P<0.05; **, P<0.01; ***, P<0.001). B) Forty-eight h after initial siRNA transfection, total RNA was extracted from the cells and used to generate cDNA. RNA levels of IRF2, RIG-I, IL28RA and RPS11 were measured by real-time PCR. The ISG mRNA levels were normalized with that of RPS11, and the ISG mRNA levels in irrelevant siRNA-transfected cells were set as 1. Data are means +/− SD of one experiment in triplicate.</p

Estimated interaction coefficients from ANOVA of the 23 selected ISGs and corresponding P values.

1<p>The P values were adjusted using Hommel's adjustment for multiple comparisons.</p>2<p>Genes in bold font showed statistically significant synergy with ZAP (P<0.05).</p

Confirmatory testing of the top ISG hits synergizing with ZAP.

<p>Triplicate wells of BHK/HA-Zeo cells (Control cells, gray bars) or BHK/NZAP-Zeo cells expressing the amino terminal domain of rat ZAP (ZAP cells, blue bars) were transduced with lentiviruses co-expressing the indicated ISGs and the red fluorescent protein TagRFP. After 2 d, the cells were challenged with SINV expressing GFP (moi = 5). After 8 h, the cells were harvested and analyzed by flow cytometry to determine the percentage of infected cells (GFP+) within the transduced (RFP+) population. Mean values are plotted; error bars indicate the standard deviation. Dashed lines indicate the percentage of infection determined in control cells expressing Fluc (gray) or ZAP cells expressing Fluc (blue). For FLJ39739 transduction of ZAP cells, there was only one replicate for analysis. Asterisks indicate mean values statistically different than values obtained in Fluc-expressing cells for the corresponding cell type (unpaired <i>t</i> test, *, P<0.05; **, P<0.01; ***, P<0.001).</p

Reduction in the percentage of infected cells by ZAP.

<p>For each ISG the reduction in the percentage of infected cells due to ZAP co-expression was calculated by subtracting the percentage of infected cells in the ZAP cells from the percentage infected in the control cells. After sorting, the differences were plotted versus an arbitrary ISG number. The difference seen between the control and ZAP cells in the absence of ISG expression (Fluc) is shown by the red symbol. Gene symbols are shown for the 23 ISGs with the greatest difference in infection percentage (≥18) due to ZAP expression.</p

Summary of ISGs that showed significant synergy with ZAP in the top-23 screen and the larger confirmatory screens.

1<p>NT refers to a gene that was not tested in the particular screen.</p>2<p>A tick mark represents a gene that significantly synergized with ZAP (P<0.05).</p

Anti-SINV activity of a library of 383 ISGs in control and ZAP-expressing cells.

<p>BHK/HA-Zeo (Control) cells or BHK/NZAP-Zeo cells expressing the amino terminal domain of rat ZAP (ZAP cells) were transduced with lentiviruses co-expressing individual ISGs and the red fluorescent protein TagRFP. After 2 d, the cells were challenged with SINV expressing GFP (moi = 5). After 8 h, the cells were harvested and analyzed by flow cytometry to determine the percentage of infected cells (GFP+) within the transduced (RFP+) population. Red symbols indicate cells expressing the control protein, Fluc, while black open circles indicate cells expressing the individual ISGs. For each cell type, the line in the scatter plot indicates the mean value for the percentage of infected cells. Gene symbols are shown for ISGs resulting in infection rates below an arbitrary cutoff of 85% (dashed line).</p

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP)

<div><p>The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.</p></div

Both ZAPS and ZAPL are ubiquitinated by TRIM25.

<p>Cells were lysed in denaturing conditions to ensure pulldown of ZAP only and not ZAP-associated proteins. <b>(A)</b> WCL of 293T cells transfected with vector expressing HA-tagged ubiquitin (Ub), and mock infected or infected with the SINV Toto1101 strain (MOI = 1) for 18 hours were used for immunoprecipitation of endogenous ZAP with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(B)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing HA-tagged Ub, and ZAPS or ZAPL were used for immunoprecipitation of overexpressed ZAP with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(C)</b> WCL of wild type and TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing HA-tagged Ub, and ZAPS or ZAPL were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The data is representative of 2 independent experiments performed on both clones D and F. Only data for clone D is shown here. <b>(D)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vectors expressing ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of endogenous Ub in the ZAP pulldown is shown. The data is representative of 3 independent experiments. <b>(E)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vector expressing HA-tagged Ub, ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of HA-tagged Ub in the ZAP pulldown is shown. The data is representative of 2 independent experiments. <b>(F)</b> WCL of <i>ZC3HAV1</i>-knockout 293T cells transfected with vector expressing HA-tagged wild type (WT), K48 or K63 Ub, ZAPS or ZAPL, and/or V5-tagged TRIM25 were used for immunoprecipitation with an anti-ZAP antibody and immunoblotting. The level of HA-tagged WT or mutant Ub in the ZAP pulldown is shown. The data is representative of 2 independent experiments.</p

CRISPR targeting of <i>TRIM25</i> leads to increased virus replication and both the RING and CCD domains of TRIM25 are required for ZAP activation.

<p><b>(A)</b> Wild type (clone E) and TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells (clones D and F) were transfected with empty vector or vector expressing ZAPS or ZAPL and infected with Toto1101/Luc (MOI = 0.01) 2 days post-transfection. <b>(B)</b> TRIM25^lo <i>ZC3HAV1</i>-knockout 293T cells (clones D and F) were reconstituted with expression of FL or truncated TRIM25 (ΔRING, ΔCCD) and/or ZAPS or ZAPL, and infected with Toto1101/Luc (MOI = 10) 2 days post-transfection. <b>(A and B)</b> The data is representative of 2 independent experiments performed on both clones D and F. Cell lysates were harvested for measurement of luciferase activity at 24 h p.i. Relative luciferase units represent the level of SINV replication. Asterisks indicate statistically significant differences (Student’s t-test, *, p<0.05; **, p<0.005; ***, p<0.0005).</p

A loss-of-function RNAi screen uncovers many genes that significantly reduce the antiviral activity of ZAP when silenced.

<p><b>(A)</b> The experimental outline of the genome-wide siRNA screen is shown. T-REx-hZAP cells transfected with control or gene-specific siRNA were treated with doxycycline to induce ZAPS overexpression one day post-transfection and infected with SINV Toto1101/Luc two days post-transfection. Cell lysates were harvested for measurement of luciferase activity at 24 h post-infection (p.i.). Relative luciferase units represent the level of SINV replication. Cells treated with the control non-targeting (NT) pooled siRNA have low SINV replication while ZAP knockdown by <i>ZC3HAV1</i>-specific pooled siRNA rescues viral replication by 2 logs. The large dynamic range in which hypothetical hits (ZAP cofactors) were identified is plotted on the right side of the graph. <b>(B)</b> Pooled siRNAs targeting the entire human genome (Dharmacon) were tested in triplicate and genes with an average robust Z score of greater than 3 are plotted. <i>ZC3HAV1</i> is highlighted in red while the top hits immediately following <i>ZC3HAV1</i> are highlighted in blue.</p