PLIC-1 Inhibits TLR3 Activation Figure 4. Interaction of PLIC-1 and Trif. A. 2 mg YFP-PLIC1or GFP and 2 mg 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 26 sample buffer and separated on a SDS-PAGE. Western blotting was performed using indicated antibodies. B. GST fusion proteins (around 2 mg) 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. doi:10.1371/journal.pone.0021153.g004 TRIF-dependent signaling from endosomes [38]. Because the latter event was more obvious in dendritic cells (DCs) and bone marrow derived macrophages, which were not employed in this study, the observed modest inhibition of PLIC-1 on TLR4-dependent ISG promoter activity could be more pronounced should DCs be examined. Nonetheless, we found that PLIC-1 inhibited the TLR3- Trif-dependent signaling both in reporter assays as well as during NDV infection. TLR3 detects double-stranded RNA (dsRNA), which is a common intermediate during virus replication, and signals exclusively via the adaptor protein Trif. The cell biology of TLR3 and Trif has been very interesting because TLR3 reportedly localizes to endosomes or phagolysosomes [39,40,41,42]. Trif, however, does not co-localize with TLR3 at resting state, but transiently associates with TLR3 upon ligand engagement and initiates the signaling [24]. The exact location of intracellular Trif remains mysterious as immunostaining of Trif did not co-localize with any known early or late endosomal marker [25,27]. Our finding that Trif co-localized with PLIC-1 and LC3 raises an interesting possibility that Trif may localize to autophagosome. Interestingly, results from the GST pull-down assay demonstrated that the Uba domain of PLIC-1 interacts with Trif, and this is also the domain that is reportedly required for PLIC-1 to target to autophagosomes [24]. Remarkably, ample evidence now highlights a connection between TLR-mediated innate immunity and autophagy. For instance, LPS increased autophagosomes and autophagy-mediated cell death of infected macrophages [43]. In addition, autophagy was shown to be essential for TLR7-mediated recognition of RNA viruses [43]. In future, it will be interesting to determine whether co-localization of Trif and PLIC-1 occurs constitutively or upon TLR3 activation. This likely requires imaging both proteins at endogenous level in cells or near endogenous level in a stable cell line that expresses tagged proteins. In our study, expression of PLIC-1 diminished the level of Trif when expressed from a transfected plasmid, suggesting PLIC-1 may accelerate the degradation of Trif. Cellular proteins can be degraded via two pathways: the ubiquitin-proteasomal pathway and the autophagic-lysosomal pathway. The ubiquitin-proteasome pathway is fundamental to protein degradation [44]. In brief, ubiquitin is initially activated by the ubiquitin-activating enzyme known as enzyme-1 (E1), and then transferred to the ubiquitin- conjugating enzyme (E2). Together with the ubiquitin-protein ligase (E3), E2 covalently attaches ubiquitins to the target protein at the e-amino group of a lysine residue. The polyubiquitin chain finally signals 26S proteasome to degrade the ubiquitylated proteins. The ubiquitin-like PLIC-1, however, has no enzymatic activity and is not known to be covalently linked to any protein. The autophagic pathway, on the other hand, offers an alternative mechanism to proteasomes for the degradation of cytoplasmic PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21153