Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB

Abstract

The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape we conducted a gain of function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including JADE3 a protein involved in directing the histone acetyltransferase HBO1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Interestingly, expression of the closely related paralogues JADE1 and JADE2 are unable to restrict influenza A virus infection, suggesting a distinct function of JADE3. We identify both shared and unique transcriptional signatures between uninfected cells expressing JADE3 and JADE2. These data provide a framework for understanding the overlapping and distinct functions of the JADE family of paralogues. Specifically, we find that JADE3 expression activates the NF-kB signaling pathway, consistent with an antiviral function. Therefore, we propose JADE3, but not JADE1 or JADE2, activates an antiviral genetic program involving the NF-kB pathway to restrict influenza A virus infection.

Figure 1:. Genome-wide CRISPR activation screen identifies antiviral proteins against influenza A virus

A. Schematic of genome wide CRISPRa screening strategy to identify antiviral genes for influenza a virus (IAV). Sequences of the sgRNAs from cells surviving IAV challenge were quantified and compared to the relative abundance of a mock-infected sample. B. Table of significantly enriched genes. STARS algorithm was used to calculate STARS score, p-value, and False Discovery Rate (FDR). Genes colored in blue have previously been shown to possess anti-IAV activity. FDR scores are shown and color coded for <0.01 (yellow), <0.05 (orange), and <0.25 (gold). JADE3 (bolded) was chosen for additional validation.

Figure 2:. Validation of CRISPR activation screen

A. Table showing mRNA fold change of indicated gene in HeLa-dCas9 VP64 cells expressing guides A or B to the indicated gene. Data is shown as the average of three independent experiments. B. Cartoon schematic of the validation assay in which HeLa-dCas9 VP64 cells were infected with PR8 mNeon reporter virus and mNeon fluorescence was monitored every 3 hours via IncuCyte. C. Heatmap displaying the number of infected cells per well over a 48 hour time course for each cell line expressing the indicated sgRNA construct.. Data is the average from three independent experiments. D. Number of cells infected with influenza PR8 mNeon at 24 hours post infection from (C). The data are shown as means ± the SEM from three independent experiments, and data were analyzed for statistical differences for each cell line compared to the vector control by one-way ANOVA with Tukey’s multiple-comparison test. *, p<0.05; ** p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant

Figure 3:. JADE3 is sufficient and necessary to restrict influenza A infection

A. Maximum likelihood phylogenetic tree of JADE3 orthologs. 100 bootstrap replicates were performed and bootstrap values are indicated. Branch length represents amino acid changes per site. GenBank common names are used for species. Ortholog gene groups for JADE3 (red), JADE1 (purple), and JADE2 (green) are colored. Potential JADE orthologs that do not branch with one of the three JADE proteins are in black. B. Representative western blot from three experiments of JADE3 Flag expression from control and JADE3 Flag transduced A549 cells. C. Vector control or JADE3 Flag A549 cells were challenged with WT PR8 at a MOI of 0.01. Infectious virus titer was determined by TCID50 24 hours post infection. Data was analyzed using a paired two-tail t-test. D. Representative western blot from three experiments for JADE3 expression in indicated cell lines. Blue arrow indicates expected molecular weight of JADE3. E. A549 WT or A549ΔJADE3 cells were challenged with WT PR8 at a MOI of 0.01. Infectious virus titer was determined by TCID50 24 hours post infection. Data was analyzed using a paired two-tail t-test. F. Representative western blot from three experiments for JADE3 expression in indicated cell lines. Blue arrow indicates expected molecular weight of JADE3. G. A549 control or A549ΔJADE3 cells complemented with either a vector control or Jade3 were challenged with WT PR8 at a MOI of 0.01. Infectious virus titer was determined by TCID50 24 hours post infection. Data was analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. All data shown is mean +/− SD from three independent experiments. *, p < 0.05; **, p < 0.01; ns, not significant

Figure 4:. Analysis of JADE3 domains and paralogs antiviral function

A. Schematic of JADE3 and its PHDs. Representative western blot from three experiments of A549 cells transduced with a vector control, JADE3 Flag or JADE3 lacking both or either PHDs. B. A549 cells expressing either a vector control or the indicated PHD truncations were challenged with WT PR8 at a MOI of 0.01. Infectious virus titer was determined by TCID50 24 hours post infection. C. Representative western blot from three experiments of expression of flag tagged JADE1, JADE2, JADE3 in A549 cells. D. A549 cells expressing different Jade family proteins were challenged with WT PR8 at a MOI of 0.01. Infectious virus titer was determined by TCID50 24 hours post infection. All data shown is mean +/− SD from three independent experiments. Data was analyzed using a one-way ANOVA with Dunnett’s multiple comparisons test against the vector control. *, p < 0.05; **, p < 0.01; ns, not significant

Figure 5:. Transcriptional profiling reveals that JADE3 activates the TNF/NF-kB signaling pathway

A. Principal component analysis scatter plot showing differential gene expression signatures of control, JADE3 or JADE2 expressing cells. PC1 and PC2 corresponding to the principal component 1 and principal component 2. B. Volcano plot of RNA-seq of A549 JADE3 Flag cells vs vector control cells. An FDR-adjusted p-value < 0.01 (dotted line) was considered significant. Data is from three biological replicates. C. Venn diagram of differentially expressed genes (DEGs) significantly upregulated by JADE3 Flag vs vector control and JADE2 Flag vs vector control. D. Gene set enrichment analysis of the 500 most significantly upregulated genes in A549 cells expressing either JADE3 Flag or a vector control. The 10 most significantly enriched gene sets are shown

Figure 6:. JADE3 activates NF-kB p65

A. Heatmap of RNA-sequencing normalized read count for select genes involved in canonical TNF-α/NF-kB signaling pathway. Coloring is done relative to each row with higher normalized read counts in red and lower normalized read counts in blue. Each box is one biological replicate. B. Vector control or JADE3 Flag A549 cells were lysed and nuclei were fractionated. Whole cell lysis and nuclear fractions were resolved using SDS-PAGE and probed for NF-kB p65. Image was spliced at blue dashed line from a single blot. Western blot shown is representative of three experiments. C. Vector control or JADE3 Flag A549 cells were treated with 20 ng/mL of TNF-α for 15 minutes, then lysed. Whole cell lysis were resolved using SDS-PAGE and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments. D. Whole cell lysis from A549 cells expressing either vector control, JADE3 Flag or JADE3 Flag lacking the indicated PHDs were resolved using SDS-PAGE and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments. E. Whole cell lysis from A549 cells expressing either a vector control or indicated JADE family members were resolved using SDS-page and probed for phosphorylated NF-kB p65 (Serine 536). Western blot shown is representative of three experiments.