Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection

Abstract

Bat cells and tissue have elevated basal expression levels of antiviral genes commonly associated with interferon alpha (IFNα) signaling. Here, we show Interferon Regulatory Factor 1 (IRF1), 3, and 7 levels are elevated in most bat tissues and that, basally, IRFs contribute to the expression of type I IFN ligands and high expression of interferon regulated genes (IRGs). CRISPR knockout (KO) of IRF 1/3/7 in cells reveals distinct subsets of genes affected by each IRF in an IFN-ligand signaling-dependent and largely independent manner. As the master regulators of innate immunity, the IRFs control the kinetics and maintenance of the IRG response and play essential roles in response to influenza A virus (IAV), herpes simplex virus 1 (HSV-1), Melaka virus/Pteropine orthoreovirus 3 Melaka (PRV3M), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) infection. With its differential expression in bats compared to that in humans, this highlights a critical role for basal IRF expression in viral responses and potentially immune cell development in bats with relevance for IRF function in human biology.

Keywords: IAV; IRF; IRG; Interferon Regulatory Factor; MERS; PRV3M; bat; innate immunity; interferon; virus.

Graphical abstract

Figure 1

IFN-Independent Regulation of Interferon Regulated Genes (A) Heatmap of ISGF3-regulated IRG in wild-type (WT) Paki cells (T03) or IFNAR2-KO clones (4A/9E) at basal state or treated with transfected polyIC (pIC) (1 mg/ml) or P. alecto IFNα3 (1,000 U/ml) for 3 h as measured by NGS. Orange box highlights the IFN-treated IRG response (expected), and green boxes highlight IRG-regulated controls still up/downregulated in the absence of IFNAR2 by polyIC. Scale is for average FPKM from blue-white-red for 0-10-70 (as indicated). Most genes are thought to be only responsive to ISGF3 and not to other elements like GAS or nuclear factor κB (NF-κB). (B) As per (A) for the unphosphorylated-ISGF3 (u-ISGF3) subset of IRGs. (C) As per (A) for the antiviral IRG-subset. (D) As per (A) for the human embryonic stem cells (hESCs) IRG subset. (E) As per (A) for all annotated IFN transcripts in the P. alecto genome, with both gene symbol and annotated gene name displayed. (F) Heatmap of IRG gene expression from spleen of P. alecto (n = 4), E. spelae (n = 2), Homo sapiens (n = 4), or Mus muscus (n = 3) shown as relative FPKM (FPKM/geometric mean of 13 housekeeping gene FPKMs) for hESC IRG subset as per (D); scale as indicated (based on min/med/max). (G) Cumulative graph of IRG relative FPKMs as per (F) for the ISGF3-IRG subset without E. spelaea due to multiple unannotated genes. Error bars are SEM. (H) As per (F) for the uISGF3-IRG -subset. Significance determined with unpaired t test; ^∗∗p < 0.02, ^∗p < 0.05. (I) Heatmap of relative FPKM (as per F) for components of the IFN signaling pathway.

Figure 2

The Role of IRFs in Intrinsic Innate Immunity (A) qPCR of IRF1 (log₁₀ of relative expression/geometric mean of housekeeping across all tissues) for various bat (P. alecto) and mouse (M. muscus) adult tissue cDNA panels (n = 3 each). (B) As per (A) for IRF3. (C) As per (A) for IRF7. Significance is as indicated (multi-row unpaired t test by tissue); ^∗∗∗p < 0.01. (D) qPCR of fold induction of IFNβ gene (relative to vector only control) for PAkiT03-4A IFNAR2 KO CRISPR cells transfected with control vector, IRF1, IRF3, or IRF7 with or without polyIC treatment (normalized to housekeeping, n = 4). (E) As per (D) for IFNα3 gene induction. (F) As per (D) for MX1 gene induction. (G) As per (D) for IFIT1 gene induction. Significance determined with unpaired t test with Welch’s correction; ^∗∗∗p < 0.01, ^∗∗p < 0.02, ^∗p < 0.05. (H) qPCR of IFIT1 expression (log₁₀, relative to housekeeping expression) in PakiT03 (WT) cells or IRF1/3/7 CRISPR KO clones treated with polyIC or IFNα3 for 3 h, as previously stated. (I) Titration on Vero cells with 2% methyl-cellulose of HSV-1 in the supernatant of WT or IRF1/3/7/ KO clones after infection with HSV at an MOI of 0.1 for 72 h. (J) Quantification of the titration in (I), as plaque-forming unit (PFU)/ml. (K) qPCR of HSV-1 ICP0 (relative to the geometric mean of WT) from RNA of infected cells as per (I) at an MOI of 0.1 or 1 for WT or IRF1/3/7 KO cells. (L) qPCR of IFIT1 (fold induction compared to uninfected) in PakiT03 WT or IRF1/3/7 KO cells following infection with MERS-CoV at an MOI of 0.1 for 48 h. (M) As per (L) for MX-2 gene induction. (N) As per (L) for MERS-CoV N gene induction (relative to WT infected). Significance determined with unpaired t test; ^∗∗∗p < 0.01, ^∗∗p < 0.02, ^∗p < 0.05 (n ≥ 3). All error bars are indicated as SEM.

Figure 3

IRF-Regulated IRGs without Induction (A) Volcano plot for genes differentially expressed (>2-fold change, p < 0.05) calculated by EdgeR in IRF1 CRISPR KO cells at the basal state compared to parental WT PakiT03 cells. Expressed as average −log₁₀ p value versus log₂ ratio. Intersecting lines indicate p = 0.05 and 2-fold change. Genes with a decreased expression in IRF1 KO cells (red) have expression driven by IRF1, and increased expression in KO (blue) indicates downregulation by IRF1. Data as per Table S2; n = 3 replicates each. (B) As per (A) for IRF3 CRISPR KO cells. (C) As per (A) for IRF7 CRISPR KO cells. (D) Venn diagram indicating IRF-specific and shared basally upregulated genes by IRF1/3/7 without induction (i.e., downregulated in KO cells), following the same cutoffs. (E) As per (D) for downregulated genes. (F) Example IRG geneset (antiviral IRGs, as previously mentioned) for IRGs regulated by IRF1/3/7 without induction. Scale as indicated (min/med/max) for average fold induction compared to WT, clustered based on Euclidian distance. (G) Ingenuity pathway analysis (IPA) of the genes in Figure S3A showing the significantly changed pathways with either IRF1, 3, or 7 CRISPR KO cells. Graph scale shows significance of the pathway (−log(p value)) and activation Z score, whereby a negative Z score in the KO indicates that IRF upregulates the pathway. Grey boxes indicate significantly divergent pathways with a mix of up/downregulation of the genes.

Figure 4

IRF-Regulated Genes Post-induction with polyIC or IFN (A) Venn diagram for significantly DEGs post-treatment with polyIC (in both time points) for IRF1/3/7 CRISPR KO cells or IFNAR2 and IFNAR2/IRF7 double KO cells. (B) Top 20 pathways that significantly affected post-polyIC induction at 6 h, compared to WT, calculated from DEG p value. Scale as indicated for −log(p value). (C) Top 20 pathways that significantly affected post-polyIC induction, compared to WT, calculated from the DEG coefficient of correlation. Scale as indicated for Z score (amplitude of response); gray indicates dysregulated genes (in the pathway up-and downregulated). (D) Gene set enrichment analysis (GSEA) score (k/K) for the hallmark response to IFNα-ligand geneset. (E) As per (D) for the IFN-induced antiviral BOSCO gene signature (GSEA c3). (F) As per (D) for the IFN-responsive gene signature post infection with (NS1-knockdown) RSV (ZHANG, GSEA c3). (G) qPCR validation of IFIT1 expression across time (as indicated) in PakiT03 WT, IRF1/3/7, or IFNAR2/IFNAR2/IRF7 (IFNAR2/I7) double KO cells, normalized to housekeeping and graphed as fold change relative to the geometric mean of WT for the same time point. All cells are significant by unpaired, two-tailed t test compared to WT unless indicated otherwise (ns). Error bars represent SEM. (H) DEG analysis as per (A) for IRF1/3/7 KO cells compared to WT for significant DEGs (>2-fold change, p < 0.05) in at least 2 time points post-IFNα3 treatment (3 h, 6 h, and 9 h). (I) Heatmap of significantly activated/suppressed pathways for cells as per (H) post-treatment with IFNα3 for 3 h, 6 h, and 9 h, as calculated by IPA for genes with >2-fold change and graphed as activation Z score (scale as indicated, min/0/max). Orange box indicates highlighted regions differently regulated by IRF KO compared to WT control. Less-red compared to WT indicates the pathway is upregulated by that IRF.

Figure 5

Regulation of the Antiviral Response to Melaka Virus by IRF1, 3, and 7 (A) PRV3M (Melaka virus) RNA load as measured by NGS transcriptome mean FPKM (all segments) at 9 h post-infection with an MOI of 1 (washed, 3 h post-infection). Cell lines as indicated including WT, IRF1/3/7, IFNAR2, and IFNAR2/IRF7 double CRISPR KO cells. (B) As per (A) at 24-h time point. (C) Venn diagram of overlapping and unique DEG analysis, compared to WT, from EdgeR in both time points after infection with PRV3M for cell lines as per (A). (D) IPA of significantly changed genes (>2-fold change, p < 0.05) compared to untreated for all cell lines as per (A) (scale as indicated, fold-change, min/max). Orange boxes highlight differences between WT and IRF clones. Blue boxes highlight similarities between WT and IFNAR2 KO cells (IFN independent). (E) Heatmap of fold-change post-infection compared to untreated cells for the antiviral IRG subset, normalized to geometric mean of 13 housekeeping genes. A 4-color non-linear scale is used from blue-white-red-black (−1, unchanged), 20, 300-fold induction), as indicated. (F) Viral titration on Vero E6 cells from supernatant 72 h post-infection with PRV3M at an MOI of 1; clonal cell line as indicated. Dilution series as indicated, in quadruplicate. Gaps in the monolayer occur from syncytia formation whereby syncytia are counted as a single plaque. (G) Quantitation of viral production from titrated supernatants as per (F), including IRF1/3/7 KO cells restored with IRF1/3/7-GFP fusions constructs, respectively. (H) IPA for the significant IP hits enriched above GFP control, expressed as p value for significance of the pathway; −log(p value), scale as indicated. Significance valeus for (A), (B), and (G) were determined by unpaired t test; ^∗∗∗p < 0.01, ^∗∗p < 0.02, ^∗p < 0.05 (n ≥ 3). All error bars are indicated as SEM.