Knockout of IRF7 Highlights its Modulator Function of Host Response Against Avian Influenza Virus and the Involvement of MAPK and TOR Signaling Pathways in Chicken

Figures

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  Figure 1. The establishment of IRF7-/- and control (IRF7wt) DF-1 cell lines. (A) Left panel: a scheme outlining the IRF7 knockout by the mutagenic chain reaction (MCR). An MCR plasmid targeting the second exon of chicken IRF7 was designed. Three adjacent guide RNAs (gRNAs) targeting the IRF7 exon 2 locus were selected, and each gRNA was cloned into the designed MCR plasmid. The locations of the PCR primers used for the analysis of the genomic insertion site are indicated. Right panel: the SpCas9-puromycin resistant gene construct from the MCR plasmid was cloned into the piggyBac transposon expression vector to generate control (IRF7wt) DF-1 cells. (B) PCR analysis of bi-allelic MCR insertion cell clones for each gRNA is shown. The absence of a PCR band derived from the IRF7 exon2 locus (Exon2) and the presence of junctional bands corresponding to MCR insertion into the chromosomal IRF7 exon 2 locus (HA1, HA2) are shown for MCR cell clones. (C) RNA-seq alignment visualization of IRF7wt (upper panel) and IRF7-/- (lower panel) in non-infected cells (0 h). The genomic region of exons 1 and 2 of IRF7 is shown. The gray vertical dashed line indicates a putative double strand break and homology…

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  Figure 2. IRF7 deletion altered transcriptome regulation in DF-1 cells (A) Volcano plots of transcriptomic differences between IRF7-/- and IRF7wt cells at each time point; the colored dots correspond to significant differentially expressed genes (DEGs; False discovery rate (FDR) < 5 %). The numbers of DEGs are shown at the bottom of each plot, and the numbers of genes that have more than 2-fold changes are in the parentheses. (B) Gene ontology (GO) and pathway analysis by DAVID 6.8 using DEGs from each time point. The numbers of genes enriched in each biological process are in parentheses. (C–E) Heatmaps showing the expression fold change of DEGs in influenza A, MAPK (mitogen-activated protein kinase) signaling and the phagosome pathways at all time points (IRF7-/-/IRF7wt). Lists of DEGs from all time points in each pathway were combined to generate each heatmap.

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  Figure 3. Comparative analysis of IRF7 knockout and overexpression. The expression profile of 245 common DEGs between IRF7 knockout (IRF7-/-/IRF7wt) and IRF7 overexpression (CuO-IRF7/Control) [35] at 0 h (mock) and 6 hpi with H6N2 infection (heatmap). Each cluster’s representative genes are indicated on the left of the heatmap, and biological process gene ontology terms and their p-values are indicated on the right.

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  Figure 4. IRF7 deletion altered the host response against H6N2 infection (A) The number of differentially expressed genes (DEGs; FDR < 1%) for IRF7wt and IRF7-/- cell lines upon H6N2 infection at 2, 6 and 12 h post infections (hpi). Each horizontal comparison indicates the number of DEGs upon H6N2 infection at each time point compared to mock infection (0 h). The numbers of genes that have 2-fold expression changes are in the parentheses. (B) A Venn diagram of DEGs between IRF7wt and IRF7-/- cells upon H6N2 infection at each time point. (C) Gene ontology functional analysis was performed using each cell-specific DEG. The numbers of genes enriched in each biological process are in parentheses.

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  Figure 5. Gene expression profiles shifted as a result of IRF7 deletion in the host response. (A) The k-means clustering of differentially expressed genes (DEGs; n = 5091) and expression cluster shift of genes due to IRF7 knockout. Respective gene expression is shown in gray, and the representative expression pattern is highlighted in different colors. A chord diagram indicates the number of DEGs shifted clusters from IRF7wt to IRF7-/-. (B) The number of genes in each cluster. A complete list of genes in each cluster are in Table S4. (C) The biological process gene ontology terms of IRF7wt DEGs in each cluster. (D) DEGs in the influenza A pathway and their expression clusters between IRF7wt and IRF7-/- are shown on the left. The colors of the clusters correspond to the colors used in (A). A heatmap of gene expression of the pathway is in the right panel. See Figure S3 for each gene in the influenza A pathway.

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  Figure 6. A proposed model of the molecular mechanism of chicken IRF7 modulation in the host response to avian influenza virus (AIV) infection A schematic model of the early antiviral response in chickens regarding IRF7 is proposed based on the DEGs in this study. AIV infection is sensed by pattern recognition receptors. TLR3 and TLR7 recognize endosomal/phagosomal double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) respectively. MDA5 and PKR respond to cytosolic dsRNA. This leads to a subsequent signaling cascade via adaptor molecules and protein kinases that activate the key transcription factors AP1, NF-κB and IRF7. The MAPK and PI3K/Akt pathways are potentially involved in the activation process. Activated IRF7 translocates to the nucleus and activates the transcription of IFNβ. AP1 and NF-κB also translocate to the nucleus and induce type I IFNs and inflammatory cytokines. Type I IFNs activate ISGs via the Jak–STAT pathway in an autocrine/paracrine manner through their receptors (IFNAR). IFIT5, interferon induced protein with tetratricopeptide repeats 5; JAK1/2, Janus kinase 1/2; LY6E, lymphocyte antigen 6 family member E; MKK3/6, MAPK kinase 3/6; MYD88, myeloid…