Journal: Cellular and Molecular Immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans -transcriptional readthrough via SSU72

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: TRT enhanced by avian influenza A virus infection represses genes on the complementary DNA strand. A Gene profile of the averaged normalized expression levels of 5,052 genes in A549 cells at 24 h after H1N1/H5N1/H7N9 influenza virus infection or AF treatment. The gene bodies between the TSSs and TTSs were equally sized and scaled to 60 bins, and the gene flanking regions 4 kb upstream of the TSSs and 4 kb downstream of the TTSs were divided into 100-bp windows. B Numbers of TRT genes (FC in the expression level of the TRT region (H5N1/AF) > 5) at different times after H1N1/H5N1/H7N9 infection of A549 cells. C Spearman rank linear correlation coefficient between the upregulated expression levels of the TRT region and the downregulated expression levels of TRT-influenced genes following the trans -TRT and cis -TRT patterns, respectively, at different times after H5N1/H7N9 infection in A549 cells. D Numbers of trans -TRT-influenced genes (FC in the expression level of the trans -TRT gene (H5N1/AF) > 5 and FC in the expression level of the trans -TRT-influenced gene (H1N1/H5N1) > 1.5) at different times after H5N1/H7N9 infection in A549 cells. E Functional pathway enrichment analysis of trans -TRT-influenced genes in H5N1-infected A549 cells (two-tailed P < 0.05, Benjamini–Hochberg adjusted P < 0.05). Detection of F GLS -TRT or G IL23A -TRT in A549 cells by using fluorescence in situ hybridization (FISH). A549 cells were treated with AF/H1N1/H5N1 for 24 h [DAPI nuclear staining ( blue ) and FISH signals obtained using a Cy3-conjugated DNA probe ( red )]. The fluorescence intensity was semiquantitatively assessed using the mean fluorescence intensity (MFI) of each cell. The data are shown as the means ± SEMs. * P < 0.05, ** P < 0.01. RNA-seq coverage levels of H the GLS gene, trans -TRT region of GLS , and STAT1 gene, and I the IL23A gene, trans -TRT region of IL23A , and STAT2 gene 12 h after AF/H1N1/H5N1/H7N9 treatment of A549 cells. The gene bodies and intergenic regions, as well as the gene flanking regions 2 kb upstream of the TSSs, were divided into 50-bp windows. Only exon regions are shown in this graph. RNA-seq datasets were established in duplicate

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Virus, Infection, Expressing, Functional Assay, Two Tailed Test, Fluorescence, In Situ Hybridization, Staining, RNA Sequencing

Journal: Cellular and Molecular Immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans -transcriptional readthrough via SSU72

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: TRT inhibition by CRISPR interference enhances STAT1/STAT2 expression and cell viability. RT-qPCR analysis of the A GLS -TRT gRNA and B IL23A -TRT gRNA groups at different times after infection with H5N1 (MOI = 4). RT-qPCR analysis of C STAT1 mRNA expression in the Ctrl gRNA and GLS -TRT gRNA groups and D STAT2 mRNA expression in the Ctrl gRNA and IL23A -TRT gRNA groups at different times after infection with H5N1 (MOI = 4). Western blot analysis of E STAT1 protein expression in the Ctrl gRNA and GLS -TRT gRNA groups and F STAT2 protein expression in the Ctrl gRNA and IL23A -TRT gRNA groups at different times after infection with H5N1 (MOI = 4). β-Actin expression served as the reference control. MTS cell viability assay in the G GLS -TRT gRNA and H IL23A -TRT gRNA groups at 48 h after treatment with AF or infection with H5N1 (MOI = 4). RT-qPCR analysis of viral M2 expression levels in the I GLS -TRT gRNA and J IL23A -TRT gRNA groups at 24 h after infection with H5N1 (MOI = 4). The expression levels in I and J are normalized to the Ctrl gRNA group. Each experiment was repeated at least three times. The data are shown as the means ± SEMs. * P < 0.05, ** P < 0.01

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Inhibition, CRISPR, Expressing, Quantitative RT-PCR, Infection, Western Blot, Control, Viability Assay

Journal: Cellular and Molecular Immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans -transcriptional readthrough via SSU72

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: TRT is reduced and lung injury is ameliorated in SSU72 transgenic mice infected with the lethal H5N1 virus. A Western blot analysis of mouse SSU72 expression in mouse lung tissues at 3 days after treatment with AF/H1N1/H5N1. β-Actin expression served as an internal control. B Numbers of TRT genes (expression of the TRT region upregulated by more than 5 compared with the AF-treated condition) in lung tissues from control ( n = 5) and SSU72 transgenic mice ( n = 5) at 3 days after intratracheal infection with H5N1 (10 6 TCID 50 ). The relative mRNA expression ratios of C mouse STAT1 and D STAT2 in lung tissues from control ( n = 8) and SSU72 transgenic mice ( n = 4) at 3 days after intratracheal infection with H5N1 virus (10 6 TCID 50 ). Mouse β-actin expression served as the reference control. E Kaplan–Meier survival curves for control ( n = 8) and SSU72 transgenic mice ( n = 10) after intratracheal infection with H5N1 (10 6 TCID 50 ). F–H Control and SSU72 transgenic mice were infected with AF or H5N1 (10 6 TCID 50 ) via intratracheal instillation. F Viral titers in the lungs were assessed 4 days after infection with H5N1 in control (n = 7) and SSU72 transgenic mice (n = 3). G Wet-to-dry weight ratios of the lungs of control ( n = 4) and SSU72 transgenic mice ( n = 4) at 3 days after infection with H5N1. H Representative images of lung pathology in control and SSU72 transgenic mice at 3 days after H5N1 infection. The lung injury scores (means ± SEMs) and numbers of infiltrating cells per microscopic field (means ± SEMs) are shown in the bar graphs. N = 100 fields for control ( n = 15) and SSU72 transgenic ( n = 6) mice. Bar = 100 μm. * P < 0.05 and ** P < 0.01. Each experiment except for RNA-seq analysis of lungs from mice with or without H5N1 infection was repeated at least three times

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Transgenic Assay, Infection, Virus, Western Blot, Expressing, Control, RNA Sequencing