The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

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. © 2011 Biswas et al

The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

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

Overexpression of PLIC-1 decreased Trif abundance in a Nocodazole-sensitive manner.

<p><b>A</b>. 293T cells were transfected with indicated plasmids. 48 hours post-transfection, lysates were prepared and subjected to western blot analysis. HCV NS3/4A was known to cleave Trif and was included as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021153#pone.0021153-Li1" target="_blank">[8]</a>. However, it only marginally reduced the level of Trif. PLIC-1 has no effect on the expression of a protein named Flap, which was included as a negative control here. <b>B</b>. 293T cells were transfected with 0.2 µg Flag-Trif and indicated amount of HA-PLIC-1 plasmid for 24 hours. Cells were then treated with 30 µM Nocodazole or MG132 (50 µM) for 6 hours prior to cell lysis. Western blotting was performed to quantitate the level of Flag-Trif, HA-PLIC-1. A non-specific band was indicated as the loading control.</p

Reduction of PLIC-1 level enhanced TLR3-Trif-mediated signaling.

<p><b>A</b>. Knocking down PLIC-1 by shRNA. 0.1 µg of HA-PLIC-1 was transfected with 0.1,1,2,4 µg of indicated shRNA constructs in 293T cells. Western blot was performed to detect HA-PLIC-1. <b>B</b>. shRNA 1602, 733, the scramble construct, and LMP (targeting mouse PLIC-1) were expressed in 293T cells. Knockdown of endogenous PLIC-1 was verified by western blotting using an anti-PLIC-1 antibody. <b>C</b>. Indicated combinations of constructs were transfected into 293T cells. 48 hours following transfection, luciferase activity was determined and normalized against renilla luciferase. <b>D</b>. Reduction of PLIC-1 by shRNA 1602 enhanced the activation of IFN-β promoter activity by poly I∶C stimulation. 293T cells were transfected with indicated plasmids and further stimulated with poly I∶C (40 µg/ml) for 24 hours prior to lysis. Notice that transfection with shRNA construct itself did not activate the IFN-β promoter.</p

Interaction of PLIC-1 and Trif.

<p><b>A</b>. 2 µg YFP-PLIC1or GFP and 2 µg Flag-Trif were cotransfected into 293T cells at 70% confluence in a 60-mm plate. 48 hours post-transfection, cell lysates were prepared and subjected to M2 anti-Flag affinity resin. After extensive wash with PBS, proteins were eluted by boiling the beads in 2× sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. <b>B</b>. GST fusion proteins (around 2 µg) were mixed with cell lysates containing Flag-Trif for 2 hours and bound proteins were eluted from the GST column and analyzed for the presence of Flag-Trif. One tenth of the input protein was loaded. A Coomassie stained gel image was included showing the expression of each GST fusion protein.</p

PLIC-1 and Trif co-localized with LC3.

<p>The human hepatoma cell line Huh7.5.1 cells were seeded on glass cover slip and then transfected with 0.1 µg of YFP-PLIC-1 and RFP-LC3 (<b>A</b>), or 0.1 µg YFP-Trif and RFP-LC3 in HEK (<b>B</b>) or Huh7.5.1 (<b>C</b>) cells in a 24-well plate format. 24 hours post-infection, cells were fixed and imaged using a Zeiss Meta LSM510 microscope. In all figures, co-localization was indicated as yellow dots in the merged images.</p

PLIC-1 blocked TLR3-induced production of IFN-α during NDV infection.

<p>The parental human lung cancer cell line A549 or the A549 stably expressing shRNA #1602 and 733 (specifically target hPLIC-1) or the negative control shRNA (LMP) were stimulated with poly I∶C (100 µg/ml) for 24 hours and then infected by NDV-GFP (MOI 1). 12 hours post-infection, images were taken to visualize cells that had been productively infected by the green virus. Recombinant IFN-α was added as a positive control to suppress NDV infection. Percentages of positive infection were calculated by averaging the number of green cells over that of blue cells (DAPI staining of nuclei) in 4 fields. The numbers were indicated in the upper right corner of each merged image.</p