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

Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs

Nerezi, Sveti Panteleimon

Diabeschriftung Hallensleben: "Nerezi, Nordarm / Nordwand/ [Bild] / Zone I-III"Bestand Hallensleben Digitales Forschungsarchiv Byzan

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease

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

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

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