The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling Nabanita Biswas1., Shufeng Liu1., Tapani Ronni2¤, Steven E. Aussenberg1, Weiqun Liu1, Takashi Fujita3, 1 Tianyi Wang * 1 Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America, 3 Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan 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. 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-b 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. Citation: Biswas N, Liu S, Ronni T, Aussenberg SE, Liu W, et al. (2011) The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling. PLoS ONE 6(6): e21153. doi:10.1371/journal.pone.0021153 Editor: Roberto F. Speck, University Hospital Zurich, Switzerland Received December 1, 2010; Accepted May 21, 2011; Published June 17, 2011 Copyright: 2011 Biswas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health Grant R21AI068784. N.B. was supported by the Fogarty training grant 5D43TW001038. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tywang@pitt.edu . These authors contributed equally to this work. ¤ Current address: Polar Bear Communications, Medford, Massachusetts, United States of America Introduction Extensive studies in the past decade have now revealed a number of innate immune sensors including the Toll-like receptors, RIG-1, MDA-5, DAI, which recognize molecular patterns present on pathogens and rapidly alert the host to the presence of potentially dangers [1,2,3]. Specific to virus detection, cell surface or endosome-associated TLRs recognize viral DNA or RNA and initiate MyD88 or Trif-dependent signaling cascades that activate transcription factors NF-kappaB (NF-kB) and interferon regulatory factors (IRFs) and, ultimately, turn on the transcription of hundreds of cellular genes, of which type I interferons (IFNs) are best known for their antiviral effects. In addition, RNA helicase RIG-I or MDA5 can detect intracellular viral double-stranded RNA and activate NF-kB and IRFs via the adaptor protein MAVS/IPS-1/VISA/Cardif, a CARD domain containing protein that is anchored to the mitochondrial membrane [4,5,6,7]. Interestingly, viruses such as hepatitis C virus (HCV) have evolved mechanism to interfere with the critical innate signaling pathways, allowing escape from the host immune surveillance [5,8,9,10]. Ubiquitin is a 76-amino-acid globular protein that is nearly identical throughout eukaryotes [11]. Ubiquitylation, the process of conjugating ubiquitins to other proteins, plays a pivotal role in the physiological turnover or the degradation of proteins in response to environmental stimuli. Historically, ubiquitylation of a protein is known to target it to 26S proteasome for degradation. It is increasingly realized that ubiquitylation also plays a unique role in regulating multiple cell signaling pathways [12]. For example, the transcription factor NF-kB is normally held in cytoplasm in association with its inhibitor called I-kB. Upon cell stimulation with interleukin -1 (IL-1) or TLR ligands, I-kB is phosphorylated by an I-kB kinase (IKK) complex [12]. The phosphorylation of I- kB triggers its K48-linked polyubiquitylation and its subsequent degradation by the 26S proteasome, which then releases NF-kB for activation [12]. Although multiple studies revealed the involvement of ubiquitylation and de-ubiquitylation in regulating IL-1R/ PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21153