Inhibition of avSG formation led to diminished <i>IFNB</i> gene expression.

<p><b>(A-C)</b> HeLa cells were transfected with siRNAs; siCtrl, siRIG-I, siPKR, or siG3BPs (for G3BP1 and G3BP2). After transfection, the cells were infected with NDV (MOI = 1) for the indicated time. (A) Expression levels of the indicated proteins were analyzed by western blotting. (B) <i>IFNB</i> mRNA expression was analyzed by RT-qPCR. Data are represented as means ±SD (t-test: ***p<0.01, **p<0.05, *p<0.1, NS = not significant). (C) Cells were immunostained for eIF3η (green) and N (red). <i>IFNB</i> mRNA (white) was detected by the RNA-FISH method. Nuclei were stained with DAPI (blue). Merge 1: nuclei, eIF3η, N. Merge 2: nuclei, eIF3η, N, and <i>IFNB</i> mRNA. The white scale bar corresponds to 20 μm.</p

RIG-I localized in NDV vRC and avSG.

<p><b>(A and B)</b> HeLa cells were either mock treated or infected with NDV (MOI = 1) for 6 and 12 hours. Cells were immunostained for RIG-I (green), N (red), and TIAR (white) (A), or RIG-I (green), TIAR (red), and IRF-3 (white) (B). <b>(C)</b> FLAG-IPS-1/HeLa cells were either mock treated or infected with NDV (MOI = 1) for 6 and 12 hours. The cells were immunostained for FLAG (green), L (red), and TIAR (white). Nuclei were stained with DAPI (blue). The white scale bar corresponds to 10 μm. The boxed area was enlarged and displayed on the right (zoom). Partial co-localization between IPS-1 and L (vRC) or TIAR (avSG) is shown by displaying them in green and red, respectively (zoom).</p

NDV infection induced formation of vRC independently of host avSG and vRNA(+) migrated from vRC to SG.

<p><b>(A)</b> Confocal micrographs of NDV-infected cells. HeLa cells were either mock treated or infected with NDV (MOI = 1) for 6 and 12 hours and immunostained for L (green), N (red), and TIAR (white). Nuclei were stained with DAPI (blue). The boxed area was enlarged and displayed on the upper left of the image. The white scale bar corresponds to 10 μm. <b>(B and C)</b> Distribution of NDV vRNAs in 6-h and 12-h NDV-infected (MOI = 1) HeLa cells. NDV vRNA(-) (red) and vRNA(+) (green) were detected by the RNA-FISH method. N (B) and TIAR (C) were immunostained with their respective antibodies (white). Nuclei were stained with DAPI (blue). A merged image at an original magnification was shown in the rightmost panel. The white scale bar corresponds to 10 μm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005444#ppat.1005444.s001" target="_blank">S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005444#ppat.1005444.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005444#ppat.1005444.s003" target="_blank">S3</a> Figs.</p

<i>In vitro</i> transcribed Le-N fusion RNA induced <i>IFNB</i> gene expression and SG formation.

<p><b>(A)</b> RNA corresponding to NDV Le RNA (Le(+)) and Le-N read-through RNA (Le-N(+)+poly(A)) were synthesized <i>in vitro</i>. These RNA preparations (200 ng) were tested for <i>IFNB</i> gene expression by transfecting to FLAG-RIG-I/HeLa cells (2×10⁵ cells). <i>IFNB</i> mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. <b>(B)</b> Cy3-labeled Le-N(+)+poly(A) (40 ng) was synthesized <i>in vitro</i> and transfected into EGFP-G3BP1/HeLa cells (0.25×10⁵ cells). After 6 hours, cells were treated with 10 μM chloroquine for 1 hour to enhance transfection efficiency. Cells were fixed and visualized for EGFP-G3BP1 (green) and Cy3-labelled RNA (red). Enlarged images of boxed areas are shown (zoom 1 and 2).</p

Inhibition of avSG formation led to diminished <i>IFNB</i> gene expression.

<p><b>(A-C)</b> HeLa cells were transfected with siRNAs; siCtrl, siRIG-I, siPKR, or siG3BPs (for G3BP1 and G3BP2). After transfection, the cells were infected with NDV (MOI = 1) for the indicated time. (A) Expression levels of the indicated proteins were analyzed by western blotting. (B) <i>IFNB</i> mRNA expression was analyzed by RT-qPCR. Data are represented as means ±SD (t-test: ***p<0.01, **p<0.05, *p<0.1, NS = not significant). (C) Cells were immunostained for eIF3η (green) and N (red). <i>IFNB</i> mRNA (white) was detected by the RNA-FISH method. Nuclei were stained with DAPI (blue). Merge 1: nuclei, eIF3η, N. Merge 2: nuclei, eIF3η, N, and <i>IFNB</i> mRNA. The white scale bar corresponds to 20 μm.</p

Uncapped, polyadenylated viral transcript induced <i>IFNB</i> gene expression.

<p><b>(A-C)</b> Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A)^- and poly(A)⁺ RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10⁵ cells were transfected with 200 ng RNA). <i>Ifnb</i> mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. <b>(D and E)</b> The Poly(A)⁺ RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10⁵ cells were transfected with 200 ng RNA). <i>Ifnb</i> mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p<0.05, NS = not significant). <b>(F)</b> NDV vRNA(+) detection by strand-specific northern blotting. Total, poly(A)^-, and poly(A)⁺ RNA (each 0250 ng) from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were separated on a denaturing agarose gel. An ethidium bromide (EtBr)-stained gel was shown in the bottom. vRNA(+) was detected by blotting with an N-specific RNA probe (N(-)). Positions of N vmRNA (N(+)) and N-P read-through RNA (N-P(+) RT) are shown. knt = kilo nucleotide. RNA probe and target vRNA(+) are illustrated alongside. <b>(G)</b> Poly(A)⁺ RNA from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were subjected to strand-specific northern blotting using a leader-specific RNA probe (Le(-)). The position of Le-N read-through RNA is shown (Le-N(+) RT). *non-specific band. RNA probe and target vRNA(+) are illustrated alongside. <b>(H and I)</b> Total, poly(A)^-, and poly(A)⁺ RNA (each 200 ng) from mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells were subjected to strand-specific RT-qPCR (ssRT-qPCR) targeting Le-N(+) read-through RNA (H) or targeting vRNA(-) (I). Primers used are listed in Table in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005444#ppat.1005444.s014" target="_blank">S1 Table</a>. Data are represented as means ±SD. Target vRNAs in the assay are illustrated below.</p

Formation of NDV vRC correlated with primary induction of <i>IFNB</i> mRNA.

<p><b>(A)</b> Time course of vRC, avSG, and <i>IFNB</i> mRNA in NDV-infected cells. HeLa cells were infected with NDV (MOI = 1) for the indicated time or treated with 0.5 mM sodium arsenite (ARS) for 30 minutes. After fixation, the cells were immunostained for TIAR (green) and N (red). <i>IFNB</i> mRNA (white) was detected by the RNA-FISH method. Nuclei were stained with DAPI (blue). Merge 1: nucleus, TIAR, N, merge 2: nucleus, TIAR, N and <i>IFNB</i> mRNA. The yellow arrowheads are cells double-positive for vRC and <i>IFNB</i> mRNA (without avSG). The white scale bar corresponds to 10 μm. <b>(B and C)</b> Quantitative analysis of vRC, avSG, and <i>IFNB</i> mRNA expression. Approximately 300 cells at the indicated time points of NDV infection (MOI = 1) or ARS treatment were counted by MetaMorph software. Cells were categorized according to the existence of vRC, avSG, and <i>IFNB</i> mRNA as shown on the top. Percentages were indicated inside the data bar. <b>(D)</b> HeLa cells were infected with NDV (MOI = 1) for the indicated time up to 12 hours. Expression levels of <i>IFNB</i> mRNA were measured by RT-qPCR. Data are represented as means ±SD.</p

Quantitative analysis of vRC, avSG, and <i>IFNB</i> mRNA expression in HeLa cells knocked down for RIG-I, PKR, and G3BPs.

<p><b>(A-H)</b> HeLa cells were transfected with siRNAs; siCtrl, siRIG-I, siPKR, or siG3BPs (siG3BP1 and siG3BP2). After transfection, the cells were infected with NDV (MOI = 1) for the indicated time up to 12 hours. Three hundred cells at each time point of infection were counted by MetaMorph software. Cells were categorized according to the existence of vRC, avSG, and <i>IFNB</i> mRNA, as shown on the top. Percentages were indicated inside the data bar.</p

Delimitation of critical domain in IPS-1 for IRF3 and NF-κB activation.

<p>A. Schematic representation of FK-IPS deletion mutants. B. HeLa cells stably expressing indicated FK-IPS deletion mutants were mock treated or treated with AP20187 for 3 h. Cell were fixed and stained for IRF-3 and NF-κB p65, respectively. Fluorescent microscopic images of IRF3 and NF-κB staining are shown (top). The percentage of cells with nuclear IRF-3 or NF-κB was determined by counting 100 cells (bottom). C, D. Cellular RNA was extracted and analyzed for IFN-β (C) or IL-6 (D) mRNA by qPCR. Representative data of at least two independent experiments are shown. Error bars: standard error of triplicated samples. Statistical analyses were conducted with an unpaired t test, with values of p<0.05 considered statistically significant. *p<0.05, **p<0.005.</p

Viral infection induces the molecular oligomer of IPS-1.

<p>A. Schematic representation of dimers detection by mKG-tagged IPS-1. B. Flow cytometry plots of control 293T cells and 2 clones stably expressing mKG-tagged IPS-1, #9 and #13. The cells were mock treated or infected with NDV for 9 h. Cells exhibiting fluorescent intensity >10¹ were quantified and expressed as % of total cell number.</p