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The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, Fujita T, Wang T - PLoS ONE (2011)

Bottom Line: Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway.Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.

Methodology/principal findings: Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.

Conclusions: Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

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Related in: MedlinePlus

PLIC-1 co-localized with Trif.The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone (A) or with 0.1 µg Flag-Trif (B) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C. 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. D. 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.
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pone-0021153-g005: PLIC-1 co-localized with Trif.The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone (A) or with 0.1 µg Flag-Trif (B) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C. 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. D. 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.

Mentions: Endogenous PLIC-1 was previously documented to concentrate at perinuclear aggresomes [23]. A recent study, however, indicated both PLIC-1 and 2 associate with autophagosomes and depletion of PLICs inhibited autophagosome degradation during nutrient starvation [24]. Trif was found in some speckle-like structures in the cytosol and did not co-localize with TLR3, but transiently associated with TLR3 upon polyI∶C stimulation in the same speckle-like structures [25]. Overexpressed Trif reportedly multimerizes and localizes to punctate cytoplasmic structures referred to as the Trif “signalosome” [23], [24]. To investigate the subcellular distribution pattern of PLIC-1 and Trif, YFP-PLIC-1 and Flag-Trif were transfected into human hepatoma cell line Huh7.5.1 cells and analyzed by confocal microscopy. Huh7.5.1 cells were chosen for this part of the study because they produce very little TLR3 and Trif at endogenous level and have a relatively large volume of cytoplasm [26], which makes them ideal for imaging cytoplasmic proteins. To avoid potential artifact due to overexpression, we intentionally kept the amount of DNA plasmid very low in this study. It was found that YFP-PLIC-1 was concentrated into punctate ring-like structures (Fig. 5A), similar to what has been reported [24], [27]. Immunostaining revealed a rather punctate subcellular distribution pattern for Flag-Trif alone, which is also consistent with previous reports [25], [28]. Of note, we observed a pronounced co-localization of YFP-PLIC-1 and Flag-Trif in punctate cellular structures (Fig. 5B). Of note, GFP itself did not co-localize with Flag-Trif (Fig. 5C). By contrast, there was no co-localization of PLIC-1 and MAVS at all (Fig. 5D). Attempts to image ectopically expressed TLR3 in Huh7.5.1 cells were not successful due to the extremely low level of expression.


The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, Fujita T, Wang T - PLoS ONE (2011)

PLIC-1 co-localized with Trif.The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone (A) or with 0.1 µg Flag-Trif (B) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C. 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. D. 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.
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pone-0021153-g005: PLIC-1 co-localized with Trif.The human hepatoma cells Huh7.5.1, were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 alone (A) or with 0.1 µg Flag-Trif (B) in a 24-well plate format. 24 hours post-infection, cells were fixed, permeabilized and stained with anti-Flag M2 antibody. Confocal and differential interference contrast (DIC) images were taken with a Zeiss Meta LSM510 microscope. C. 0.1 µg of GFP expression plasmid was transfected with 0.1 µg Flag-Trif for confocal imaging. D. 0.1 µg of YFP-PLIC-1 expression plasmid was transfected with 0.1 µg Flag-MAVS into Huh7.5.1 cells for confocal imaging.
Mentions: Endogenous PLIC-1 was previously documented to concentrate at perinuclear aggresomes [23]. A recent study, however, indicated both PLIC-1 and 2 associate with autophagosomes and depletion of PLICs inhibited autophagosome degradation during nutrient starvation [24]. Trif was found in some speckle-like structures in the cytosol and did not co-localize with TLR3, but transiently associated with TLR3 upon polyI∶C stimulation in the same speckle-like structures [25]. Overexpressed Trif reportedly multimerizes and localizes to punctate cytoplasmic structures referred to as the Trif “signalosome” [23], [24]. To investigate the subcellular distribution pattern of PLIC-1 and Trif, YFP-PLIC-1 and Flag-Trif were transfected into human hepatoma cell line Huh7.5.1 cells and analyzed by confocal microscopy. Huh7.5.1 cells were chosen for this part of the study because they produce very little TLR3 and Trif at endogenous level and have a relatively large volume of cytoplasm [26], which makes them ideal for imaging cytoplasmic proteins. To avoid potential artifact due to overexpression, we intentionally kept the amount of DNA plasmid very low in this study. It was found that YFP-PLIC-1 was concentrated into punctate ring-like structures (Fig. 5A), similar to what has been reported [24], [27]. Immunostaining revealed a rather punctate subcellular distribution pattern for Flag-Trif alone, which is also consistent with previous reports [25], [28]. Of note, we observed a pronounced co-localization of YFP-PLIC-1 and Flag-Trif in punctate cellular structures (Fig. 5B). Of note, GFP itself did not co-localize with Flag-Trif (Fig. 5C). By contrast, there was no co-localization of PLIC-1 and MAVS at all (Fig. 5D). Attempts to image ectopically expressed TLR3 in Huh7.5.1 cells were not successful due to the extremely low level of expression.

Bottom Line: Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway.Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.

Methodology/principal findings: Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.

Conclusions: Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

Show MeSH
Related in: MedlinePlus