PLIC-1 Inhibits TLR3 Activation Figure 2. Reduction of PLIC-1 level enhanced TLR3-Trif-mediated signaling. A. Knocking down PLIC-1 by shRNA. 0.1 mg of HA-PLIC-1 was transfected with 0.1,1,2,4 mg of indicated shRNA constructs in 293T cells. Western blot was performed to detect HA-PLIC-1. 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. C. Indicated combinations of constructs were transfected into 293T cells. 48 hours following transfection, luciferase activity was determined and normalized against renilla luciferase. D. Reduction of PLIC-1 by shRNA 1602 enhanced the activation of IFN-b promoter activity by poly I:C stimulation. 293T cells were transfected with indicated plasmids and further stimulated with poly I:C (40 mg/ml) for 24 hours prior to lysis. Notice that transfection with shRNA construct itself did not activate the IFN-b promoter. doi:10.1371/journal.pone.0021153.g002 was compared by calculating the percentage of NDV infected cells, PLIC-1 knockdown A549 cells showed higher level of type I interferon activity. Altogether, these results reinforced that notion that PLIC-1 is an inhibitor of the TLR3 pathway. PLIC-1 interacts with Trif To explore the interaction between PLIC-1 and Trif, we first conducted Co-IP experiment. Fig. 4A showed that transiently expressed PLIC-1 was able to precipitate co-expressed Trif, but not the negative control protein. Moreover, in a separate experiment, we failed to pull down TLR3 and PLIC-1 together (data not shown). In order to determine which domain of PLIC-1 interacts with Trif, we produced GST fusion proteins containing PLIC-1 Uba domain alone, or PLIC-1 devoid of Uba or both Uba and Ubl domain and conducted a pull-down assay with Flag-Trif. It was found that the Uba domain of PLIC-1 alone is necessary and sufficient to bind Trif (Fig. 4B). Co-localization of PLIC-1, LC3, and Trif 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 multi- merizes 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 overex- pression, 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. To further explore the nature of the observed vesicular structures, we transfected cells with a RFP tagged LC3 to indicate the formation of autophagosomes. Remarkably, PLIC-1 largely co-localized with LC3 (Fig. 6A). Moreover, when similar PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21153