TLR pathways, only one recent report by Chuang and Ulevitch described that the ubiquitin-mediated receptor degradation negatively regulated TLR signaling [13]. In that study, a novel TLR interacting protein termed Triad 3A was demonstrated to be an E3 ligase which interacts with several TLRs. Triad3A induces ubiquitylation of TLR9 and TLR4 but not TLR2, which then targets them for proteasomal degradation. As a result of this, the TLR-mediated activation of NF-kB was suppressed. Most recently, it is shown that Triad3A promoted down-regulation of two TIR domain-containing adapter proteins, TIRAP and Trif, RIP1 which is the essential downstream protein of TNF-a signaling [14], as well as TRAF3 which mediates the RIG-I/MAVS signaling pathway [15]. Thus, ubiquitylation and degradation of innate signaling molecules by Triad3A represents a novel pathway by which the host limits the intensity and duration of innate signaling pathways. In a yeast two-hybrid screening study, we identified an ubiquitin-like protein PLIC-1 as a potential interacting partner of the TIR domain-containing proteins. Using luciferase reporter assays, we found that PLIC-1potently suppressed the TLR3-Trif-dependent IFN-b promoter activity while exerted minimal effect on TLR4-mediated NF-kB activation or TLR7- or MAVS-dependent IFN-b promoter activity. When endoge- nous PLIC-1 was reduced by short-hairpin based interfering RNA (shRNA), new castle diseases virus (NDV) became hypersensitive to poly I:C treatment, reinforcing the idea that PLIC-1 negatively regulates TLR3-pathway. Coimmunopreci- pitation (Co-IP) studies revealed that PLIC-1 coprecipitated with Trif, but not TLR3. A GST-pull down further showed that the ubiquitin associated (Uba) domain of PLIC-1 directly pulled down Trif. Subsequent confocal microscopy revealed that PLIC-1 co-localized both with Trif and LC3, an autophago- some marker, in punctate structures within the cytoplasm. Furthermore, overexpression of PLIC-1 decreased the cellular abundance of Trif via a pathway that can be blocked by Nocodazole, but not MG132. Altogether, these data suggest that PLIC-1 is a novel inhibitor of TLR3-Trif signaling pathway by decreasing the abundance of the innate signaling adaptor molecule Trif via the autophagic pathway. Results Identification of PLIC-1 as a TIR-interacting protein To screen for novel regulators of TLR-pathway, we conducted a yeast-two hybrid assay using the Clontech Matchmaker III system (Palo Alto, CA) according to the manufacturer’s instruction. The intracellular domain (TIR domain) of TLR4 was chosen as the bait. 36 million clones from a human leukocyte cDNA library HL4021B were screened and 6 clones showed strong interaction with bait. After sequencing, all six clones were found to contain partial fragments matching a gene named PLIC-1 in the NCBI database. PLIC-1 contains an amino terminal ubiquitin-like domain (Ubl) and carboxyl terminal ubiquitin associated domain (Uba) as well as two internal repeats of ,85 amino acids that contain Sti I motifs. PLIC-1 is known to bind to membrane proteins including presenilins and GABA receptors [16,17]. PLIC-1 was reported to bind ubiquitylated proteins and ubiquitin ligases such as E6AP and bTRCP, and proteasome subunits [18]. These interactions are thought to interfere with normal targeting of proteins for proteasome-dependent-degradation, resulting in enhanced stabil- ity potential affecting the signaling pathways in which these proteins are involved. PLIC-1 Inhibits TLR3 Activation PLIC-1 exerted a strong inhibitory effect on Trif-dependent signaling in reporter assays We first investigated the role of PLIC-1 in regulating TLR4 signaling using luciferase reporter assays. To our surprise, overexpression of PLIC-1 had no effect on TLR4-dependent NF-kB activation (Fig. 1A), while modestly suppressed the activation of an ISG promoter driven luciferase reporter by a constitutive TLR4 activator, CD4-TLR4 [19] (Fig. 1B). We then sought to explore its effect on other TLR signalings. To this end, we performed a series of reporter assays and found that PLIC-1 is a potent inhibitor of TLR3-Trif-pathway. First, overexpression of PLIC-1 inhibited the Trif-dependent activation of NF-kB- dependent reporter (Fig. 1C) as well as p125-Luc whose transcription is under the control of IFN-b promoter [20] (Fig. 1D), but had negligible effect on the basal transcription of the reporter gene (data not shown). To rule out the possibility that this inhibition was associated with a specific tagged PLIC-1, YFP- or flag-tagged PLIC-1 constructs were included in the transfection and similar dose-dependent inhibition was observed (Fig. 1E and data not shown). Of note, expression of PLIC-1 in 293T cells did not inhibit the activation of p125-Luc by overexpressing IRF-3, suggesting that PLIC-1 exerts its inhibitory effect at a step that is upstream of IRF-3 (Fig. 1F). To investigate whether PLIC-1 exerts effects on other innate immune signaling, similar studies were repeated on HEK-TLR7 stable cell line and cells transfected with MAVS. It was observed that PLIC-1 failed to have any effect in either case (Fig. 1G&H). Thus, the inhibitory effect of PLIC-1 appeared to be limited to Trif signaling. Because Trif is absolutely required in TLR3 pathway whose activation increases transcrip- tion of IFN-b gene [21], we assessed the effect of PLIC-1 on TLR3 activation. In mouse macrophage cells J774A.1 that were stimulated with poly I:C (TLR3 ligand), the activation of IFN-b- driven luciferase decreased as the amount of PLIC-1 increased (Fig. 1I). Together, these data suggest that PLIC-1 negatively modulates the TLR3-Trif pathway. Reduction of endogenous PLIC-1 enhanced TLR3-Trif signaling Subsequently, we designed short hairpin interfering RNA (shRNA) knocking down the expression of PLIC-1. The shRNA is expressed from a retroviral vector under the control of human H1 promoter [22]. Two sequences were selected to target three different regions of hPLIC-1. The shRNA harboring a scrambled sequence and the one targeting mouse PLIC-1(LMP) were included as controls for specificity. To monitor the knockdown effect, shRNA vectors were first transfected along with HA- hPLIC-1 construct in 293T cells. Two shRNA constructs targeting to human PLIC-1 specifically suppressed the expression of HA- hPLIC-1 expression in a dose-dependent manner (Fig. 2A). The shRNA construct (#1602) was subsequently employed to reduce the endogenous PLIC-1 (Fig. 2B) and this led to enhanced activation of Trif-dependent IFN-b promoter activity (Fig. 2C). Moreover, transfection with this construct enhanced the activation of the IFN-b-promoter activity (p125-Luc) upon poly I:C stimulation (Fig. 2D). Taken together, these results reinforced the finding that PLIC-1 inhibits the TLR3/Trif-mediated innate immune signaling activation. Reduction of endogenous PLIC-1 rendered NDV hypersensitive to Poly I:C treatment The above experiments were carried out using luciferase reporter assays. In order to validate the observations in an infection system, we generated PLIC-1 stable knockdown cells in PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21153