Date Published: January 7, 2013
Publisher: Public Library of Science
Author(s): Shiori Takamatsu, Kazuhide Onoguchi, Koji Onomoto, Ryo Narita, Kiyohiro Takahasi, Fumiyoshi Ishidate, Takahiro K. Fujiwara, Mitsutoshi Yoneyama, Hiroki Kato, Takashi Fujita, Karin E. Peterson. http://doi.org/10.1371/journal.pone.0053578
The innate immune system recognizes viral nucleic acids and stimulates cellular antiviral responses. Intracellular detection of viral RNA is mediated by the Retinoic acid inducible gene (RIG)-I Like Receptor (RLR), leading to production of type I interferon (IFN) and pro-inflammatory cytokines. Once cells are infected with a virus, RIG-I and MDA5 bind to viral RNA and undergo conformational change to transmit a signal through direct interaction with downstream CARD-containing adaptor protein, IFN-β promoter stimulator-1 (IPS-1, also referred as MAVS/VISA/Cardif). IPS-1 is composed of N-terminal Caspase Activation and Recruitment Domain (CARD), proline-rich domain, intermediate domain, and C-terminal transmembrane (TM) domain. The TM domain of IPS-1 anchors it to the mitochondrial outer membrane. It has been hypothesized that activated RLR triggers the accumulation of IPS-1, which forms oligomer as a scaffold for downstream signal proteins. However, the exact mechanisms of IPS-1-mediated signaling remain controversial. In this study, to reveal the details of IPS-1 signaling, we used an artificial oligomerization system to induce oligomerization of IPS-1 in cells. Artificial oligomerization of IPS-1 activated antiviral signaling without a viral infection. Using this system, we investigated the domain-requirement of IPS-1 for its signaling. We discovered that artificial oligomerization of IPS-1 could overcome the requirement of CARD and the TM domain. Moreover, from deletion- and point-mutant analyses, the C-terminal Tumor necrosis factor Receptor-Associated Factor (TRAF) binding motif of IPS-1 (aa. 453–460) present in the intermediate domain is critical for downstream signal transduction. Our results suggest that IPS-1 oligomerization is essential for the formation of a multiprotein signaling complex and enables downstream activation of transcription factors, Interferon Regulatory Factor 3 (IRF3) and Nuclear Factor-κB (NF-κB), leading to type I IFN and pro-inflammatory cytokine production.
Viruses replicating within cells produce RNA with a non-self signature, such as a double stranded (ds) and 5′-triphosphate structure, which are recognized by sensor molecules Retinoic acid Inducible Gene-I (RIG-I), Melanoma Differentiation Associated gene 5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2), collectively known as RIG-I-Like Receptors (RLR) , , , . RLR elicits signals to activate a set of genes including those of type I and III interferon (IFN) to initiate innate antiviral responses . Several lines of evidence support a hypothesis that once RIG-I and MDA5 recognize non-self RNA, conformational changes are induced resulting in exposure of their CARD . The CARD of RIG-I and MDA5 transmits a signal to another CARD-containing adaptor, Interferon Promoter Stimulator-1 (IPS-1, also known as MAVS, VISA, and Cardif), which is anchored on the outer membrane of the mitochondrion , , , . Cells infected with a virus activate the RLR/IPS-1 signaling cascade and exhibit microscopic aggregation of IPS-1 . Activation of IPS-1 is reconstituted in vitro and the formation of detergent-insoluble IPS-1 aggregate has been reported . For intracellular aggregation of IPS-1, the involvement of mitofusin (MFN) 1, which is known to regulate mitochondrial fusion, has been reported , suggesting that IPS-1 aggregation is regulated through a complex mechanism of mitochondrial dynamics. There are several studies concerning how IPS-1 receives a signal from RLR and how it relays it downstream; however, some of the reports are not consistent with each other , , , . IPS-1 contains three potential TRAF binding motifs (TBMs) . To avoid confusion, we refer to them as TBM1 (aa. 143–147, human), TBM2 (aa. 154–159, human), and TBM3 (aa. 453–460, human). TBM1 and 2 are close to each other (5 amino acids apart) and reside within the proline-rich domain. TBM1 physically interacts with TRAF3  and a single amino acid substitution (T147I) abolishes binding. Early reports demonstrated that an artificial molecule essentially consisting of CARD and TM, therefore devoid of TBMs (termed mini MAVS), is sufficient for signaling , , . In particular, TM can be replaced with that of other mitochondrial proteins, suggesting the importance of its mitochondrial localization. Other reports have demonstrated that artificial oligomerization of CARD of IPS-1 in the cytosol is sufficient to activate the signal independent of the mitochondrion .
Signaling initiated by cytoplasmic viral RNA sensors involves a unique adaptor, IPS-1, which is specifically expressed on the outer membrane of the mitochondrion. IPS-1 is a problematic protein, since transient overexpression results in constitutive signaling, whereas endogenous IPS-1 is tightly regulated by post-translational mechanisms , . Here, we established a system to analyze the regulation of IPS-1 by its oligomerization. We obtained stable cell lines expressing FK-IPS fusion, which could be activated by a crosslinker. Upon oligomerization, IPS-1 rapidly elicited signaling leading to the activation of target genes including that of IFN-β, suggesting that IPS-1 aggregation is essential and precedes possible covalent modifications such as phosphorylation and ubiquitination , .