Interferon inhibits a model RNA virus via a limited set of inducible effector genes

Abstract

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors-ZAP, IFIT3, and IFIT1-together constitute the majority of interferon-mediated restriction of VEEV, while accounting for < 0.5% of the interferon-induced transcriptome. Together, our data suggest a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

Keywords: CRISPR-Cas9 screening; Venezuelan equine encephalitis virus; alphavirus; interferon-stimulated genes; virus-host interactions.

Figure 1. CRISPR screening identifies a limited set of genes that restrict VEEV infection

1. A

   Schematic of genome‐wide and ISG‐targeted CRISPR screen to identify genes required for IFN‐mediated suppression of VEEV. Graphic generated with Biorender.

2. B

   Manhattan dot plots of genome‐wide CRISPR screen (left) or ISG‐targeted CRISPR screen (right) results. Significance was determined by MAGeCK analysis, and the dotted lines represent a cutoff of FDR < 0.25. Genome‐wide screen, n = 2 biological replicates. ISG‐targeted screen, n = 3 biological replicates. Genes are plotted in alphabetical order for both.

3. C

   Venn diagram comparing the CRISPR screening hits (FDR < 0.25) from the genome‐wide (blue) and ISG‐targeted (orange) screens.

4. D

   Validation of selected CRISPR screening hits by targeted gene inactivation. Genes are plotted on the x‐axis from lowest FDR value (IFIT1) to highest FDR value (CD163L1) as determined by MAGeCK analysis of the genome‐wide screen. All samples were normalized to non‐targeting controls (“Non‐Targ”) for graphing. Graphs represent the mean with error bars representing standard deviation. One‐way ANOVA with Dunnett's test comparing each group to the non‐targeting control was performed on non‐normalized data, n = 3 biological replicates.

Source data are available online for this figure.

Figure 2. RNA‐Seq reveals a limited set of IFN‐induced genes were identified as CRISPR screening hits

1. A

   Volcano plot of differentially expressed genes during RNA‐Seq of U‐2 OS cells treated with IFNβ for 6 h. Significance was determined by DESeq2, and red dots represent genes with a log2 fold‐change > 1 and a Padj < 0.05. Plots represent the mean of three biological replicates.

2. B

   Volcano plot of differentially expressed genes during RNA‐Seq of U‐2 OS cells treated with IFNβ for 24 h. Significance was determined by DESeq2, and red dots represent genes with a log2 fold‐change > 1 and a Padj < 0.05. Plots represent the mean of three biological replicates.

3. C

   Venn diagram of CRISPR screening hits (FDR < 0.25) and ISGs as determined by RNA‐Seq (log2 fold‐change > 1 and a Padj < 0.05), with overlapping genes listed.

4. D

   Heatmaps of FPKM (left) and log2FC (right) values (from RNA‐Seq) of IFN‐induced CRISPR screening hits at 6‐ and 24‐h post‐IFN treatment. Heatmap intensity represents the mean of three independent biological replicates.

5. E

   Western blots from lysates of U‐2 OS cells treated with IFNβ (20 U/ml) for 6 and 24 h. Antibodies were used to probe for IFN‐induced CRISPR screening hits.

Source data are available online for this figure.

Figure 3. ZAP, IFIT3, and IFIT1 are dominant contributors to the IFN‐mediated suppression of VEEV

1. A

   Effects of CRISPR‐based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 5 h) in U‐2 OS cells with IFNβ pretreatment (20 U/ml for 24 h).

2. B

   Effects of CRISPR‐based gene inactivation of IRF9, STAT1, and STAT2 or ZC3HAV1, IFIT3, and IFIT1 on VEEV infection (MOI of 10 for 4.5 h) in Huh7.5 cells with (+ symbols) and without (− symbols) IFNβ pretreatment (100 U/ml for 24 h).

3. C

   Effects of CRISPR‐based single‐, double‐, or triple‐gene targeting on VEEV infection (MOI of 5 for 5 h) in U‐2 OS cells with or without IFNβ pretreatment (20 U/ml for 24 h).

4. D

   Dose response curve of IFNβ pretreatment (left, two‐fold dilutions from 200 U/ml to 1.6 U/ml for 24 h) or IFNλ pretreatment (right, two‐fold dilutions from 500 to 3.9 ng/ml for 24 h) demonstrating inhibition of VEEV infection (MOI of 5 for 5 h) in control (circles), IRF9/STAT1/STAT2‐targeted (squares), or ZAP/IFIT3/IFIT1‐targeted (triangles) U‐2 OS cells. Graphs represent the mean of normalized and transformed (from %Infection to %Inhibition) data for three biological replicates with error bars representing standard deviation.

Data information: In (A–C), data are presented as mean ± standard deviation. n = 3 independent biological replicates, one‐way ANOVA with Dunnett's test. ^ns P > 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available online for this figure.

Figure EV1. CRISPR‐based gene inactivation efficiently reduces protein product abundance and demonstrates mostly IFN‐dependent antiviral activity of ZAP, IFIT3, and IFIT1 together

1. A

   Western blot showing reduced protein abundance in CRISPR‐targeted cells from Fig 3A. Red asterisks denote long (**) and short (*) isoforms of ZAP.

2. B

   Western blot showing reduced protein abundance in CRISPR‐targeted cells from Fig 3B. Red asterisks denote long (**) and short (*) isoforms of ZAP.

3. C

   Western blot showing reduced protein abundance in CRISPR‐targeted cells from Fig 3C. Red asterisks denote long (**) and short (*) isoforms of ZAP.

4. D

   (Top) Infection of CRISPR‐targeted U‐2 OS (left, top) or Huh7.5 cells (right, top) that were infected with an MOI of 1 VEEV‐GFP (5 h for U‐2 OS and 4.5 h for Huh7.5) with and without pretreatment of IFNβ (24‐h treatment, 20 U/ml for U‐2 OS and 100 U/ml for Huh7.5 cells). Graphs represent the mean with error bars representing standard deviation. n = 3 biological replicates, one‐way ANOVA with Dunnett's test. ^ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. (Bottom) Western blot of ISG20 from lysates of “quadruple‐targeted” U‐2 OS (left, bottom) or Huh7.5 cells (right, bottom) with and without pretreatment of IFNβ (24‐h treatment, 20 U/ml for U‐2 OS and 100 U/ml for Huh7.5 cells).

5. E

   Western blot from lysates of U‐2 OS cells after 5‐h infection with VEEV‐GFP or IFNβ treatment (20 U/ml for 24 h). Red asterisks denote long (**) and short (*) isoforms of ZAP.

6. F

   Western blot from lysates of U‐2 OS CRISPR‐targeted or control cells after 24‐h treatment 500 ng/ml of IFNλ. Red asterisks denote long (**) and short (*) isoforms of ZAP.

Source data are available online for this figure.

Figure 4. ZAP, IFIT3, and IFIT1 in combination exhibit differential antiviral activity toward diverse RNA viruses

1. A

   U‐2 OS control, IRF9/STAT1/STAT2‐targeted, and ZAP/IFIT3/IFIT1‐targeted cells were infected with GFP‐expressing ONNV virus (left) or ZIKV (right) after IFNβ pretreatment (20 and 100 U/ml). The percentage of infected (GFP⁺) cells was quantified by flow cytometry.

2. B

   Huh7.5 control, IRF9/STAT1/STAT2‐targeted, and ZAP/IFIT3/IFIT1‐targeted cells were infected with GFP‐expressing Sindbis virus (AR86 strain, left) or vesicular stomatitis virus (right) after IFNβ pretreatment (100 and 1000 U/ml). The percentage of infected (GFP⁺) cells was quantified by flow cytometry.

Data information: In (A, B), data are presented as mean ± standard deviation. n = 3 independent biological replicates, one‐way ANOVA with Dunnett's test. ^ns P > 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available online for this figure.

Figure 5. ZAP, IFIT3, and IFIT1, but not BST2, RSAD2, ISG20, or IFITM3 are required for IFN‐mediated suppression of VEEV production and spread

1. A

   Heatmaps showing the ratio of infected cells at 6, 9, and 12 h of infection with VEEV‐GFP. U‐2 OS control, IRF9/STAT1/STAT2‐targeted, or ZAP/IFIT3/IFIT1‐targeted cells were treated with or without IFNβ (20 U/ml for 24 h) and infected with an MOI of 0.5 (left), 1 (middle), or 5 (right) of VEEV‐GFP. Infection was quantified by flow cytometry. Heatmap intensity represents the mean of three independent biological replicates.

2. B

   (Top) Quantification of VEEV production (12‐h postinfection, MOI of 2, titered in BHK‐21J cells) by plaque assay of control (far left: without IFN pretreatment), IRF9/STAT1/STAT2‐targeted, or ZAP/IFIT3/IFIT1‐targeted cells after IFNβ pretreatment (IFNβ 20 U/ml for 24 h). (Bottom) Western blot shows IFN‐mediated induction and CRISPR‐based targeting of IRF9, STAT1, STAT2, ZAP, IFIT3, and IFIT1. Red asterisks denote long (**) and short (*) isoforms of ZAP.

3. C

   (Top) Quantification of VEEV production (12‐h postinfection, MOI of 2, titered in BHK‐21J cells) by plaque assay in control (far left: without IFN pretreatment), IRF9/STAT1/STAT2‐targeted, ZAP/IFIT3/IFIT1‐targeted, or ZAP/IFIT3/IFIT1‐targeted cells that were subsequently CRISPR targeted with a guide targeting BST2, RSAD2, ISG20, or IFITM3 after IFNβ pretreatment (IFNβ 20 U/ml for 24 h). (Bottom) Western blot shows IFN‐mediated induction and CRISPR‐based targeting of BST2, RSAD2, ISG20, and IFITM3. Black squares denote nonspecific bands.

Data information: In (B, C), data are presented as mean ± standard deviation. n = 3 independent biological replicates, one‐way ANOVA with Dunnett's test on log‐transformed data. ^ns P > 0.05, ***P < 0.001, and ****P < 0.0001. Source data are available online for this figure.

Figure EV2. VEEV infection alone does not induce selected ISGs

Western blot from lysates of U‐2 OS cells after 12 h infection with VEEV TC‐83 or IFNβ treatment (20 U/ml for 24 h). Red asterisks denote long (**) and short (*) isoforms of ZAP.Source data are available online for this figure.