Fig 1.
Sequence characteristics of chIRAP.
(A) Structure of chIRAP CDS. chIRAP CDS is 486 bp located in chicken chr5 from 1,779,250-1,779,735 bp (yellow). (B) Three-dimensional prediction of chIRAP by alpha fold. chIRAP contains 10 β-folds and 4 α-helices (shadow), and exists in position 108, 145 two glycosylation sites. The results of comparative analysis of chIRAP and other bird nucleotide sequences are shown in S1 Table. (C) Sequence alignment of chIRAP and different avian genes. The target gene exists in two forms in the chicken genome: one is the reference sequence of this study and the other has a GGA mutation at positions 352-354 compared to the reference sequence. (D) PCR amplified the chIRAP from chicken-derived cell cDNA (HD11, CEF, DF-1) (chIRAPF/chIRAPR). PCR amplification of target fragments in cDNAs and genomes of different chicken-derived cells. (E) PCR amplification of chIRAP in different tissues cDNA of chicken (chIRAPF/chIRAPR). Highest levels of target genes in liver (red).
Fig 2.
Viral infection and interferon stimulation upregulate chIRAP expression.
(A) qRT-PCR results showed that viral infection (NDVNa-EGFP, 1 MOI) promoted chIRAP expression at 12-48 h (qchIRAPF/qchIRAPR). (B-C) qRT-PCR analysis of the effect of IFN stimulation on chIRAP transcript levels. (B) I/II/III chIFN (chIFNβ, chIFNγ, chIFNλ3, 500 IU) stimulation up-regulated chIRAP expression in a time-dependent; (C) I/II/III chIFN (chIFNβ, chIFNγ, chIFNλ3, 250,500,1000 IU) stimulation up-regulated chIRAP expression in a dose-dependent manner (24 h). The primers used are shown in S1 Table (qchIRAPF/qchIRAPR). (D) Transcript levels of interferon pathway-related genes were analysed by qRT-PCR in DF-1 cells after overexpression of chIRAP. Fold change is the multiple of the background expression level of the target gene in blank cells.
Fig 3.
Analysis of antiviral effects of chIRAP.
(A) Schematic diagram of chIRAP recombinant expression plasmid construction. Expression of chIRAP identified by WB. (B) CCK8 analyses the effect of chIRAP overexpression on cell viability. (C) Cell localization of chIRAP was analyzed by laser confocal analysis. (D) Analysis of the antiviral activity of chIRAP by fluorescence observation and flow cytometry using 12 wells plate at 24 h after VSV-EGFP infection (0.01 MOI), 48 h after NDV-rL-EGFP infection (2 MOI), and 24 h after NDV-Na-EGFP infection (1 MOI). The scale bar corresponds to 150 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001. (E) Analysis of the antiviral activity of chIRAP by WB at 48 h after Influenza A virus strains H9N2 infection. Analysis of the level of H9N2 HA gene by qRT-PCR at 48 h after Influenza A virus strains H9N2 infection (qH9N2 HAF/qH9N2 HAR). (F) The antiviral effect of chIRAP is dose-dependent. (G-H) chIRAP can inhibit viral infections with different multiplicity of infections. (G) NDV-rL-EGFP and (H) NDV-Na-EGFP. mock: Untreated blank cell control group; Vector: Transient overexpression of pcDNA3.1(+) empty vector control group.
Fig 4.
Endogenous chIRAP -deficient cells are susceptible to viral infection.
(A) qRT-PCR screening for optimal siRNAs. The best interference is shown in red. (B) WB detection of siRNA interference effects. Polyclonal anti-chIRAP were used as primary antibodies and HRP labeled Goat anti-mouse IgG (H + L) as the secondary antibody. (C) CCK8 detects the effect of endogenous chIRAP deletion on cellular activity. **, P < 0.01. (D-E) Effects of endogenous chIRAP deletion on cellular resistance to viral infection analyzed by fluorescence observation(D) and flow cytometry(E) (NDV-Na-EGFP, 1 MOI, 24 h). The scale bar corresponds to 150 μm. *, P < 0.05. (F) WB analysis of the effect of endogenous chIRAP deletion on cellular resistance to viral infection at different time points of viral infection (NDV-Na-EGFP, 1 MOI, 24 h). (G) Effect of knock down chIRAP on the antiviral effects of chIFNλ3 (100 IU,12 h) by fluorescence observation. The scale bar corresponds to 150 μm. (H) Effect of knock down chIRAP on the antiviral effects of chIFNλ3 (100 IU,12 h) by flow cytometry. ns, no significant difference; ****, P < 0.0001. siNC: Negative control group for instant transfection of siRNA.
Fig 5.
chIRAP reduces the infection of influenza virus in chicken embryos.
(A) Schematic diagram of chicken embryo processing and detoxification. The 15-day-old chicken embryos were infected with the virus H1N1 (1 × 105 Pfu) 48 hours after transfection with chIRAP (n = 10), and samples were collected 48 hours after the virus infection. (B) The expression of chIRAP in chicken embryos was detected by WB (Five chicken embryos randomly selected from the overexpression group were used for the detection of chIRAP overexpression). Polyclonal antibodies anti-chIRAP were used as primary antibodies and HRP labeled Goat anti-mouse IgG (H + L) as the secondary antibody. (C) Determination of viral titer in allantoic fluid. ****, P < 0.0001. mock: Untreated blank cell control group; PC: Virus-infected untreated chicken embryos group.
Fig 6.
Construction and characterisation of inducible cell lines of chIRAP.
(A) Fluorescence observation analysis of the antiviral effects of cell lines with optimal induction conditions (NDV-Na-EGFP, 1 MOI, 24 h). DMSO was used as a negative control. The scale bar corresponds to 150 μm. (B) Flow cytometry analysis of the antiviral effects of cell lines with optimal induction conditions (NDV-Na-EGFP, 1 MOI, 24 h). DMSO was used as a negative control. ****, P < 0.0001. (C-D) Knockdown of chIRAP in overexpressing cell lines, after transfection of siRNA for 48h, cells were induced with Dox (2.5 μg for 24 hours) and infected with NDVNa-EGFP (1 MOI,24 h), fluorescence observation(C) and flow cytometry (D) were used to analyse the infection of NDVNa-EGFP.DMSO was used as a negative control. The scale bar corresponds to 150 μm. *, P < 0.05; **, P < 0.01; ****, P < 0.001.
Fig 7.
chIRAP may exert its antiviral effect by interacting with chPRDX1.
(A) Label-free for analyzing chIRAP interacting proteins. (B) Screened for highly expressed proteins. (C) GO analysis of up-regulated proteins. (D) KEGG analysis of the relevant pathways involved in the upregulation of proteins. (E) Flow cytometry was used to analyze the effects of 10 proteins screened by knockdown mass spectrometry on chIRAP’s anti-NDV-Na-EGFP. (NDV-Na-EGFP, 1 MOI, 24 h). ****, P < 0.0001. siRNAs are shown in S2 Table. PC: Virus-infected untreated cell group. (F) Co-IP was used to analyze the interaction between chIRAP and PRDX1, and PRDX1 specific antibody was used to coat immunomagnetic beads.Vector: Transient overexpression of pcDNA3.1(+) empty vector control group. (G) Molecular docking predicts potential interaction sites between chIRAP and PRDX1. chIRAP forms stable hydrogen bonds with the D79, T90, K93, Q94, R110 and R123 sites of PRDX1. (H) Flow cytometry was used to analyze the PRDX1 site that affects the effect of chIRAP (NDV-Na-EGFP, 1 MOI, 24 h). ****, P < 0.0001.
Fig 8.
chIRAP may affect intracellular ROS balance by regulating the PRDX1 D79 site.
(A) The determination of ROS levels analyzed the effect of site mutations on the ability of PRDX1 to clear ROS. mock: Untreated blank cell control group; PC: ROS-positive stimulant (Rosup) treated cell group. (B) Overexpression of chIRAP upregulates ROS levels. Vector: Transient overexpression of pcDNA3.1(+) empty vector control group. (C) Effect of endogenous PRDX1 deletion on chIRAP regulation of ROS levels. ns, no significant difference. ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. siNC: Negative control group for instant transfection of siRNA.