Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of MFN1 inhibits the redistribution of IPS-1 induced by NDV infection.A, HeLa cells were transfected with negative control (N.C.) or hMFN1-targeted siRNA (#1–#3) for 48 h, and the knockdown of endogenous MFN1 was analyzed by Western blotting using anti-MFN1 antibody. B, Cells transfected with siRNA as shown in a were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). C, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). D, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA#2 for 48 h. Cells were infected with NDV for 12 h and stained with anti-FLAG antibody (FLAG-IPS-1), anti-NP antibody (NDV NP), and MitoTracker (Mitochondria).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2908619&req=5

ppat-1001012-g011: Knockdown of MFN1 inhibits the redistribution of IPS-1 induced by NDV infection.A, HeLa cells were transfected with negative control (N.C.) or hMFN1-targeted siRNA (#1–#3) for 48 h, and the knockdown of endogenous MFN1 was analyzed by Western blotting using anti-MFN1 antibody. B, Cells transfected with siRNA as shown in a were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). C, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). D, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA#2 for 48 h. Cells were infected with NDV for 12 h and stained with anti-FLAG antibody (FLAG-IPS-1), anti-NP antibody (NDV NP), and MitoTracker (Mitochondria).

Mentions: Next, we examined what effect the knockdown of MFN1 would have on the virus-induced redistribution of IPS-1. Three independent siRNA efficiently knocked down MFN1 expression (Fig. 11A) resulting in a strong inhibition of the NDV-induced IFN-β gene expression in HeLa cells and IPS-1-HeLa cells (Fig. 11B and 11C). This once again suggests that IPS-1-HeLa cells tend to behave like normal cells. Upon NDV infection, IPS-I displayed a speckled staining pattern in control cells, but not in the MFN1-knockdown cells (Fig. 11D). Though the intensity of NP staining did not increase, MFN1-knockdown significantly inhibited IFN gene activation. This correlates with prior observations that although IFN production is inhibited by LGP2 overexpression, viral yield does not increase [22]. When control siRNA-treated cells were infected with NDV, a redistribution of IPS-1 was observed (69.3±15.7% of cells positive for NP). In MFN1-knockdown cells, although NDV infection resulted in the formation of NP foci, IPS-1 redistribution did not occur (4.5±1.3% of cells positive for NP). A similar effect was observed when MFN1-knockdown cells were infected with SeV (Fig. 12). These results strongly suggest that MFN1 is critical to the redistribution of IPS-1 triggered by RIG-I mediated sensing of viral RNA.


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)

Knockdown of MFN1 inhibits the redistribution of IPS-1 induced by NDV infection.A, HeLa cells were transfected with negative control (N.C.) or hMFN1-targeted siRNA (#1–#3) for 48 h, and the knockdown of endogenous MFN1 was analyzed by Western blotting using anti-MFN1 antibody. B, Cells transfected with siRNA as shown in a were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). C, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). D, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA#2 for 48 h. Cells were infected with NDV for 12 h and stained with anti-FLAG antibody (FLAG-IPS-1), anti-NP antibody (NDV NP), and MitoTracker (Mitochondria).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001012-g011: Knockdown of MFN1 inhibits the redistribution of IPS-1 induced by NDV infection.A, HeLa cells were transfected with negative control (N.C.) or hMFN1-targeted siRNA (#1–#3) for 48 h, and the knockdown of endogenous MFN1 was analyzed by Western blotting using anti-MFN1 antibody. B, Cells transfected with siRNA as shown in a were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). C, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA were infected with NDV for 12 h, and endogenous IFNB1 mRNA expression was quantified by qRT-PCR. Data represent means ± s.d. (n = 3). D, IPS-1-HeLa cells transfected with N.C. or hMFN1-targeted siRNA#2 for 48 h. Cells were infected with NDV for 12 h and stained with anti-FLAG antibody (FLAG-IPS-1), anti-NP antibody (NDV NP), and MitoTracker (Mitochondria).
Mentions: Next, we examined what effect the knockdown of MFN1 would have on the virus-induced redistribution of IPS-1. Three independent siRNA efficiently knocked down MFN1 expression (Fig. 11A) resulting in a strong inhibition of the NDV-induced IFN-β gene expression in HeLa cells and IPS-1-HeLa cells (Fig. 11B and 11C). This once again suggests that IPS-1-HeLa cells tend to behave like normal cells. Upon NDV infection, IPS-I displayed a speckled staining pattern in control cells, but not in the MFN1-knockdown cells (Fig. 11D). Though the intensity of NP staining did not increase, MFN1-knockdown significantly inhibited IFN gene activation. This correlates with prior observations that although IFN production is inhibited by LGP2 overexpression, viral yield does not increase [22]. When control siRNA-treated cells were infected with NDV, a redistribution of IPS-1 was observed (69.3±15.7% of cells positive for NP). In MFN1-knockdown cells, although NDV infection resulted in the formation of NP foci, IPS-1 redistribution did not occur (4.5±1.3% of cells positive for NP). A similar effect was observed when MFN1-knockdown cells were infected with SeV (Fig. 12). These results strongly suggest that MFN1 is critical to the redistribution of IPS-1 triggered by RIG-I mediated sensing of viral RNA.

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