18 research outputs found
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
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
A dominant negative mutant of RIG-I fails to induce IPS-1 redistribution.
<p><b>A</b>, IPS-1-HeLa cells stably expressing wild-type human RIG-I (RIG-I WT) or mutant RIG-I (RIG-I K270A) were mock-treated or infected with NDV for 12 h and expression of <i>IFNB1</i> mRNA was analyzed by qRT-PCR. Open and filled bars indicate RNA samples from mock-treated and NDV-infected cells, respectively. Data represent means ± s.d. (n = 3). <b>B</b>, IPS-1-HeLa cells expressing RIG-I WT or RIG-I K270A were infected with NDV for 12 h and stained with anti-RIG-I antibody and anti-NP antibody. RIG-I staining is diffuse in uninfected cells however infection by NDV produced RIG-I foci. Some RIG-I foci are co-localized with NDV NP foci. <b>C</b>, IPS-1-HeLa cells expressing RIG-I WT or RIG-I K270A were infected with NDV for 12 h and IPS-1 redistribution was examined. IPS-1 and NP were stained with anti-IPS-1 antibody and anti-NP antibody, respectively. The area enclosed by the red rectangle is enlarged at the right. Although the redistributed IPS-1 surrounds NP foci in RIG-I WT cells, K270A mutation of RIG-I failed to induce the redistribution of IPS-1, but not the formation of NP foci.</p