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Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1.

Onoguchi K, Onomoto K, Takamatsu S, Jogi M, Takemura A, Morimoto S, Julkunen I, Namiki H, Yoneyama M, Fujita T - PLoS Pathog. (2010)

Bottom Line: 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.Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan.

ABSTRACT
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-beta 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.

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A dominant negative mutant of RIG-I fails to induce IPS-1 redistribution.A, 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 IFNB1 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, 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. C, 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.
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ppat-1001012-g007: A dominant negative mutant of RIG-I fails to induce IPS-1 redistribution.A, 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 IFNB1 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, 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. C, 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.

Mentions: To determine if the observed redistribution of IPS-1 is functionally relevant, we used a point mutant of RIG-I (K270A), which normally recognizes ligand RNA but functions as a dominant negative inhibitor (Fig. 7A) [14]. It was observed that NDV infection induced foci of both wt and K270A RIG-I to form (Fig. 7B), however wt but not K270A promoted the speckled staining pattern of IPS-1 (Fig. 7C). The results indicate that the redistribution of IPS-1 is strongly correlated with the activation of antiviral signaling.


Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1.

Onoguchi K, Onomoto K, Takamatsu S, Jogi M, Takemura A, Morimoto S, Julkunen I, Namiki H, Yoneyama M, Fujita T - PLoS Pathog. (2010)

A dominant negative mutant of RIG-I fails to induce IPS-1 redistribution.A, 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 IFNB1 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, 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. C, 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908619&req=5

ppat-1001012-g007: A dominant negative mutant of RIG-I fails to induce IPS-1 redistribution.A, 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 IFNB1 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, 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. C, 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.
Mentions: To determine if the observed redistribution of IPS-1 is functionally relevant, we used a point mutant of RIG-I (K270A), which normally recognizes ligand RNA but functions as a dominant negative inhibitor (Fig. 7A) [14]. It was observed that NDV infection induced foci of both wt and K270A RIG-I to form (Fig. 7B), however wt but not K270A promoted the speckled staining pattern of IPS-1 (Fig. 7C). The results indicate that the redistribution of IPS-1 is strongly correlated with the activation of antiviral signaling.

Bottom Line: 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.Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan.

ABSTRACT
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-beta 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.

Show MeSH
Related in: MedlinePlus