Virus-Infection or 5′ppp-RNA Activates Antiviral Signal through Redistribution of IPS-1 Mediated by MFN1

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-β promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5′ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes

Encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses.

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses

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

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