PLIC-1 Inhibits TLR3 Activation Figure 3. PLIC-1 blocked TLR3-induced production of IFN-a during NDV infection. 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 mg/ 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-a 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. doi:10.1371/journal.pone.0021153.g003 experiment was carried out using a YFP-Trif and RFP-LC3, at least a portion of Trif co-localized with LC3 (Fig. 6B&C). These results pointed out that autophagosomes may be where the PLIC- 1 and Trif complex locates. Downregulation of Trif by PLIC-1 In order to gain mechanistic insight into the action of PLIC-1- dependent inhibition, we transfected 293T cells with a plasmid encoding Trif along with specified amounts of PLIC-1 plasmid. After 18 hours, cell lysates were prepared and analyzed by immunoblot of Trif expression. PLIC-1 decreased the abundance of Trif but not an irrelevant protein named Flap (a negative control) in a dose- dependent manner (Fig. 7A). Notably, Flag-Flap was transcribed by a heterologous promoter (CMV promoter) from the same vector in which Trif was cloned. Therefore, PLIC-1 appears to decrease Trif abundance at protein level, presumably via protein degradation. There are two known pathways for protein degradation: ubiquitin-proteasomal pathway as well as the autophagy-depen- dent pathways. Since PLIC-1 has been implicated in both pathways, we added the proteasome inhibitor MG132 and the microtubule inhibitor Nocodazle, which is known to block the autophagy-lysosome pathway [29], to the cell cultures. Shown in Figure 7B&C, addition of Nocodazole significantly reversed the PLIC-1-dependent Trif degradation, whereas the effect of MG132 on this process was nearly negligible. Discussion Innate immune signalings mediated by TLRs, RIG-1, and MDA5 are critical in recognizing microbial pathogens and triggering the first line of host defense. Unchecked signaling activation, however, can lead to excessive production of inflammatory mediators that cause tissue damages. Studies in recent years have now revealed a number of negative regulators of innate immune signaling pathways, such as IL-1R-associated kinase M (IRAK-M) [30], CYLD [31], SIGIRR [32], suppression of cytokine signaling 1 (SOCS1) [33], etc. Most recently, Triad3A was demonstrated to act as an E3 ubiquitin-protein ligase and enhance ubiquitylation and proteolytic degradation of several TLRs and RIP1, resulting in the attenuation of TLR signalings [13,14]. The identification of PLIC-1 now adds a new member to this long list of negative regulators. PLIC-1 is a type 2 ubiquitin-like (ubl) proteins. In contract to the small-sized type 1 ubl proteins that are covalently linked to target proteins in a fashion similar to ubiquitin, type 2 ubl proteins are not ligated to other proteins and their functions are poorly defined. There are four members of PLIC in mouse and human [18]. Data from functional analyses suggest a role for PLICs in the in vivo degradation of several proteins known to be ubiquitylation- dependent substrates of the proteasome [18,34,35]. Interestingly, in transfection studies, PLICs either inhibit or promote ubiquitin- dependent proteasome degradation [16,18,36]. Moreover, inhibi- tion of G protein signaling by PLIC-1 is independent of proteasome activity [37]. Like ubiquitin, the expression of PLICs is ubiquitous in almost all cell types [17,31]. Particularly interesting, it is recently shown that PLIC-1 and 2 locate to autophagosomes [23]. Despite that our study initially identified PLIC-1 as a TLR4 interacting partner, we failed to observe any noticeable effect of PLIC-1 on TLR4-dependent NF-kB activation in reporter assays. It must be pointed out, however, that TLR4 signals through MyD88 and Trif sequentially [38] to activate a second-phase signaling. LPS stimulation first triggers TLR4-MyD88-depdent signaling from plasma membrane and activates NF-kB; endocytosis of TLR4 then quenches MyD88-dependent signaling and starts a second phase of PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21153