Model for the redistribution of IPS-1 mediated by mitochondrial organization.

<p><b>A</b>, Schematic representation of the redistribution of IPS-1 induced by viral infection. In uninfected cells, IPS-1 is evenly distributed in all mitochondria (left). In infected cells, foci of viral nucleoprotein form, which are surrounded by redistributed IPS-1 and mitochondria (right). <b>B</b>, A model for the redistribution of IPS-1 mediated by mitochondrial organization. Initially, IPS-1 is distributed evenly in mitochondria (left). Viruses replicate in restricted compartments in the cells, viral RNA accumulates, and then RIG-I re-localizes to these compartments. Viral RNA induces a conformational change of RIG-I, and results in mitochondria expressing accumulated IPS-1- around the RIG-I foci. IPS-1 may be redistributed, resulting in a local accumulation of IPS-1 on a mitochondrion (left). IPS-I may further segregate due to mitochondrial reorganization by fusion and fission (right). Local accumulation of IPS-1 may further recruit adaptors and protein kinases to activate antiviral signaling.</p

Knockdown of MFN1 inhibits the redistribution of IPS-1 induced by SeV infection.

<p>IPS-1-HeLa cells transfected with negative control (N.C.) or hMFN1-targeted siRNA#2 for 48 h. Cells were infected with SeV for 12 h and stained with anti-FLAG antibody (FLAG-IPS-1), MitoTracker (Mitochondria), and DAPI. The area enclosed by the red rectangle is enlarged at the right.</p

Redistribution of IPS-1 in SeV-infected cells.

<p><b>A</b>, The IPS-1-HeLa clone #2 was mock-treated or infected with SeV for 12 h and stained with MitoTracker (Mitochondria) and anti-FLAG antibody (FLAG-IPS-1). Nuclei were visualized by staining with DAPI throughout this study. The fluorescent image was quantified in the area indicated by blue line (right most panel). Quantification results from mock- or SeV-infected cells are shown at the bottom. Fluorescence of DAPI corresponds to area in the nucleus. The mitochondria heavily stained with MitoTracker but lightly stained with anti-FLAG are shown by arrows. <b>B</b>, IPS-1-HeLa cells were mock-treated or infected with SeV for 12 h. Cells were stained with anti-FLAG antibody (FLAG-IPS-1) and anti-ERAB antibody (ERAB).</p

IPS-1 interacts with MFN1 and MFN2.

<p>IPS-1-HeLa cells were infected with NDV for 12 h, and then FLAG-IPS-1 was immunoprecipitated with anti-FLAG antibody. Co-immunoprecipitated MFN1 and MFN2 were detected by anti-MFN1 antibody and anti-MFN2 antibody, respectively. Neither OPA1 nor DRP1 was co-immunoprecipitated with FLAG-IPS-1. Mitochondrial protein BCL-Xl was used as a control and was also examined by anti-BCLXl antibody.</p

Localization of viral nucleocapsid, RIG-I, and IPS-1.

<p><b>A</b>, HeLa cells were infected with NDV for 12 h and stained with anti-RIG-I antibody (RIG-I) and anti-NP antibody (NDV NP). <b>B</b> and <b>C</b>, IPS-1-HeLa cells were infected with NDV for 12 h and stained with anti-FLAG antibody and anti-RIG-I antibody or anti-NP antibody.</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

Localization of IPS-1 and mitochondria.

<p><b>A</b>, IPS-1-HeLa cells infected with NDV for 9 h were fixed, stained with anti-NP antibody, and subjected to ultrathin sectioning as shown in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001012#s4" target="_blank">Methods</a>. The area enclosed by a red rectangle is enlarged. NP: NP foci stained with the anti-NP antibody were visualized using gold particles. <b>B</b>, IPS-1-HeLa cells infected with NDV for 9 h were fixed, stained with anti-FLAG antibody, and subjected to ultra thin sectioning. The area enclosed by a red rectangle is enlarged. NP: morphologically similar structures are in <b>A</b>. IPS-1 was visualized using gold particles. The arrowheads indicate boundaries between IPS-1 and NP foci.</p

Redistribution of endogenous IPS-1 in virus-infected cells.

<p><b>A</b>, HeLa cells were infected with Mock or SeV for 12 h. The cells were stained with anti-IPS-1 antibody and MitoTracker (Mitochondria). <b>B</b>, SKHep1 cells were infected with NDV, SeV, Influenza virus, or Sindbis virus for 12 h. The cells were stained with anti-IPS-1 antibody and MitoTracker (Mitochondria).</p

MFN1 is involved in antiviral signaling.

<p><b>A</b>, Schematic representation of the MFN1 domain. <b>B</b>, L929 cells were transfected with a virus-responsive reporter gene (p-125 Luc) and either an empty vector (Empty), an expression vector for MFN1, or an expression vector for MFN2 as indicated. 48 h after the transfection, cells were mock-treated or infected with NDV. Luciferase activity was determined at 12 h after infection. <b>C</b> and <b>D</b>, L929 cells were transfected with a virus-responsive reporter gene (p-125 Luc) and either an empty vector (Empty) or an expression vector for MFN1 or its mutant (MFN1 T109A) as indicated. At 48 h after transfection, cells were mock-treated, infected with NDV, or transfected with 5′OH-RNA or 5′ppp-RNA. Luciferase activity was determined at 12 h (<b>C</b>) or 9 h (<b>D</b>) after induction. <b>E</b>, L929 cells were transfected with a virus-responsive reporter gene (p-125 Luc) and combinations of the indicated vectors. 48 h after the transfection, cells were mock-treated or infected with NDV. Luciferase activity was determined 12 h after infection.</p

MFN1 plays a critical role in RIG-I–induced signaling.

<p><b>A</b> and <b>B</b>, Wild-type (WT) MEFs and Mfn1 or Mfn2-knockout MEFs were infected with NDV for 9 h. The levels of endogenous <i>Ifna4</i> (<b>A</b>) and <i>Ifnb1</i> (<b>B</b>) mRNA were quantified by qRT-PCR. <b>C</b>, HeLa cells were transfected with negative control (N.C.) or hOPA1-targeted siRNA (#1–#3) for 72 h, and the expression of <i>OPA1</i> mRNA was analyzed by qRT-PCR. <b>D</b>, HeLa cells were transfected with N.C. siRNA or hOPA1-targeted siRNA. 72 h after transfection, cells were infected with NDV for 12 h. <i>IFNB1</i> mRNA expression was quantified by qRT-PCR. <b>E</b>, HeLa cells were transfected with N.C. siRNA or hDRP1-targeted siRNA (#1–#3) for 72 h, and the knockdown of endogenous DRP1 was analyzed by Western blotting using anti-DRP1 antibody. <b>F</b>, HeLa cells were transfected with N.C. siRNA or hDRP1-targeted siRNA. At 72 h after transfection, cells were infected with NDV for 12 h. <i>IFNB1</i> mRNA expression was quantified by qRT-PCR. <b>G</b>, WT and Mfn1 or Mfn2-knockout MEFs were transfected with a virus-responsive reporter gene (p-125 Luc) with either an empty vector (Empty) or an expression vector for RIG-I CARD or IPS-1. Luciferase activity was determined 48 h after transfection. Data represent means ± s.d. (n = 3).</p