Characterization and functional analysis of chicken hnRNP protein

Figures

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  Fig. 1. Chicken hnRNPs interact with genomic dsRNA of IBDV. (A) DF-1 cells were infected with 1 MOI IBDV for 12 h and subsequently analyzed using immunofluorescence assay (IFA) with anti-VP1, VP3, and dsRNA antibodies, along with their respective secondary antibodies. Imaging was conducted using confocal microscopy, with a scale bar measuring 5 μm. (B) DF-1 cells were exposed to 1 MOI IBDV for different time. These cells underwent immunoprecipitation (IP) using anti-dsRNA antibodies and were then analyzed by western blot with the specified antibodies. (C) A schematic representation of the identification of dsRNA-binding proteins via LC-MS. (D) The list of the RNA-binding proteins identified by peptide analysis as shown in (C).

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  Fig. 2. IBDV infection recruits hnRNPs to co-localize with dsRNA. (A) Myc-tagged chicken hnRNPs expression plasmids were introduced into DF-1 cells and incubated for 24 h. Following this, the cells underwent IFA utilizing Myc antibodies and their respective secondary antibodies. The images obtained were analyzed using confocal microscopy, with a scale bar of 5 μm. (B) After being transfected with Myc-tagged chicken hnRNPs for 24 h, DF-1 cells were subsequently infected with 1 MOI IBDV for an additional 12 h. These cells were then subjected to IFA using Myc and dsRNA antibodies, along with the secondary antibodies. The resulting image was scanned via confocal microscopy, with the white arrow indicating the colocalization of dsRNA and chicken hnRNPs. The scale bar measures 5 μm.

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  Fig. 3. Chicken multiple hnRNPs regulate interferon signaling activation. (A-E) DF-1 cells were separately co-transfected with plasmids that encode chicken hnRNPs or Flag-chMDA5 (A), Flag-chMAVS (B), Flag-chTBK1 (C), Flag-chSTING (D), or Flag-chIRF7 (E), along with chIFN-β and pRL-TK for a duration of 36 h. The chIFN-β promoter activity was assessed using a dual-luciferase reporter assay. The results are expressed as means ± SDs (n = 3), with P > 0.05 regarded as not significant; ***P < 0.001. (F) Illustrates the mechanisms through which various chicken hnRNPs influence interferon activation.

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  Fig. 4. Chicken hnRNPA3 contributes IBDV replication. (A) DF-1 cells were infected with 1 MOI IBDV for different time to assess the protein levels of chicken hnRNPA3, with β-actin serving as the loading control. (B) Fresh DF-1 cells were transfected with Myc-chhnRNPA3 for 24 h before being infected with 1 MOI IBDV for different time. Whole-cell lysates were then obtained for Western blot analysis to evaluate the major IBDV proteins, with β-actin used as the loading control. (C) The Myc-chhnRNPA3 was transfected into DF-1 cells for 24 h. Following this, the transfected cells were exposed to 1 MOI IBDV for either 6 or 12 h. The levels of viral genomic dsRNA were quantified using qRT-PCR, with the chicken gapdh gene as a reference. (D) Fresh DF-1 cells were transfected with Myc-chhnRNPA3 for 24 h and subsequently infected with 1 MOI IBDV for different time. Whole-cell lysates were collected for the TCID50 assay to determine the viral titers. The data are shown as means ± SD (n = 3), with P > 0.05 indicating no significance; **P < 0.01; ***P < 0.001.

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  Fig. 5. Chicken hnRNPA3 silence disrupts IBDV replication. (A) DF-1 cells were individually transfected with 200 nM of siRNA (sictr), siRNA#1, siRNA#2, siRNA#3, and siRNA#4 for 36 h. The endogenous chhnRNPA3 levels were analyzed via Western blotting, with β-Actin serving as the loading control. Band densities were quantified using ImageJ software, and results are shown as relative band densities, representing the means from three independent experiments. (B) DF-1 cells were co-transfected with Myc-chhnRNPA3 or siRNA#1 alongside dsRNA, in addition to chIFN-β and pRL-TK, for 36 h. The activity of the chIFN-β promoter was evaluated using a dual-luciferase reporter assay. (C–E) DF-1 cells transfected with 200 nM siRNA#1 or a control siRNA for 36 h were subsequently infected with 1 MOI IBDV for an additional 6 or 12 h. (C) The expression levels of VP1, VP2, and VP3, were analyzed through western blotting, with β-actin as the loading control. (D) Total RNA was isolated for qRT-PCR to quantify viral RNA levels, using the chicken gapdh gene as a reference. (E) DF-1 cells were transfected with 200 mM siRNA#1 or control siRNA for 36 h, after which they were infected with 0.1 MOI IBDV for different…