RIG-I (retinoic acid-inducible gene-I), a cytoplasmic RNA helicase, interacts with IPS-1/MAVS/Cardif/VISA, a protein on the outer membrane of mitochondria, to signal the presence of virus-derived RNA and induce type I interferon production. Activation of RIG-I requires the ubiquitin ligase, TRIM25, which mediates lysine 63-linked polyubiquitination of the RIG-I N-terminal CARD-like region. However, how this modification proceeds for activation of IPS-1 by RIG-I remains unclear. Here we identify an alternative factor, Riplet/RNF135, that promotes RIG-I activation independent of TRIM25. The Riplet/RNF135 protein consists of an N-terminal RING finger domain, C-terminal SPRY and PRY motifs, and shows sequence similarity to TRIM25. Immunoprecipitation analyses demonstrated that the C-terminal helicase and repressor domains of RIG-I interact with the Riplet/RNF135 C-terminal region, whereas the CARD-like region of RIG-I is dispensable for this interaction. Riplet/RNF135 promotes lysine 63-linked polyubiquitination of the C-terminal region of RIG-I, modification of which differs from the N-terminal ubiquitination by TRIM25. Overexpression and knockdown analyses revealed that Riplet/RNF135 promotes RIG-I-mediated interferon-β promoter activation and inhibits propagation of the negative-strand RNA virus, vesicular stomatitis virus. Our data suggest that Riplet/RNF135 is a novel factor of the RIG-I pathway that is involved in the evoking of human innate immunity against RNA virus infection, and activates RIG-I through ubiquitination of its C-terminal region. We infer that a variety of RIG-I-ubiquitinating molecular complexes sustain RIG-I activation to modulate RNA virus replication in the cytoplasm.

Smoldering inflammation often increases the risk of progression for malignant tumors and simultaneously matures myeloid dendritic cells (mDCs) for cell-mediated immunity. PolyI:C, a dsRNA analog, is reported to induce inflammation and potent antitumor immune responses via the Toll-like receptor 3/Toll–IL-1 receptor domain-containing adaptor molecule 1 (TICAM-1) and melanoma differentiation-associated protein 5/IFN-β promoter stimulator 1 (IPS-1) pathways in mDCs to drive activation of natural killer cells and cytotoxic T lymphocytes. Here, we found that i.p. or s.c. injection of polyI:C to Lewis lung carcinoma tumor-implant mice resulted in tumor regression by converting tumor-supporting macrophages (Mfs) to tumor suppressors. F4/80⁺/Gr1⁻Mfs infiltrating the tumor respond to polyI:C to rapidly produce inflammatory cytokines and thereafter accelerate M1 polarization. TNF-α was increased within 1 h in both tumor and serum upon polyI:C injection into tumor-bearing mice, followed by tumor hemorrhagic necrosis and growth suppression. These tumor responses were abolished in TNF-α^−/−mice. Furthermore, F4/80⁺Mfs in tumors extracted from polyI:C-injected mice sustained Lewis lung carcinoma cytotoxic activity, and this activity was partly abrogated by anti–TNF-α Ab. Genes for supporting M1 polarization were subsequently up-regulated in the tumor-infiltrating Mfs. These responses were completely abrogated in TICAM-1^−/−mice, and unaffected in myeloid differentiation factor 88^−/−and IPS-1^−/−mice. Thus, the TICAM-1 pathway is not only important to mature mDCs for cross-priming and natural killer cell activation in the induction of tumor immunity, but also critically engaged in tumor suppression by converting tumor-supporting Mfs to those with tumoricidal properties.

Retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLR) are members of the DEAD box helicases, and recognize viral RNA in the cytoplasm, leading to IFN‐β induction through the adaptor IFN‐β promoter stimulator‐1 (IPS‐1) (also known as Cardif, mitochondrial antiviral signaling protein or virus‐induced signaling adaptor). Since uninfected cells usually harbor a trace of RIG‐I, other RNA‐binding proteins may participate in assembling viral RNA into the IPS‐1 pathway during the initial response to infection. We searched for proteins coupling with human IPS‐1 by yeast two‐hybrid and identified another DEAD (Asp‐Glu‐Ala‐Asp) box helicase, DDX3 (DEAD/H BOX 3). DDX3 can bind viral RNA to join it in the IPS‐1 complex. Unlike RIG‐I, DDX3 was constitutively expressed in cells, and some fraction of DDX3 is colocalized with IPS‐1 around mitochondria. The 622‐662 a.a DDX3 C‐terminal region (DDX3‐C) directly bound to the IPS‐1 CARD‐like domain, and the whole DDX3 protein also associated with RLR. By reporter assay, DDX3 helped IPS‐1 up‐regulate IFN‐β promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN‐β induction. This activity was conserved on the DDX3‐C fragment. DDX3 only marginally enhanced IFN‐β promoter activation induced by transfected TANK‐binding kinase 1 (TBK1) or I‐kappa‐B kinase‐ε (IKKε). Forced expression of DDX3 augmented virus‐mediated IFN‐β induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS‐1 enhancer.

In myeloid dendritic cells (mDCs), TLR3 is expressed in the endosomal membrane and interacts with the adaptor toll/interleukin 1 receptor homology domain–containing adaptor molecule 1 (TICAM-1; TRIF). TICAM-1 signals culminate in interferon (IFN) regulatory factor (IRF) 3 activation. Co-culture of mDC pretreated with the TLR3 ligand polyI:C and natural killer (NK) cells resulted in NK cell activation. This activation was triggered by cell-to-cell contact but not cytokines. Using expression profiling and gain/loss-of-function analyses of mDC genes, we tried to identify a TICAM-1–inducing membrane protein that participates in mDC-mediated NK activation. Of the nine candidates screened, one contained a tetraspanin-like sequence and satisfied the screening criteria. The protein, referred to as IRF-3–dependent NK-activating molecule (INAM), functioned in both the mDC and NK cell to facilitate NK activation. In the mDC, TICAM-1, IFN promoter stimulator 1, and IRF-3, but not IRF-7, were required for mDC-mediated NK activation. INAM was minimally expressed on NK cells, was up-regulated in response to polyI:C, and contributed to mDC–NK reciprocal activation via its cytoplasmic tail, which was crucial for the activation signal in NK cells. Adoptive transfer of INAM-expressing mDCs into mice implanted with NK-sensitive tumors caused NK-mediated tumor regression. We identify a new pathway for mDC–NK contact-mediated NK activation that is governed by a TLR signal-derived membrane molecule.

Ligand stimulation of the Toll-like receptors (TLRs) triggers innate immune response, cytokine production and cellular immune activation in dendritic cells. However, most TLR ligands are microbial constituents, which cause inflammation and toxicity. Toxic response could be reduced for secure immunotherapy through the use of chemically synthesized ligands with defined functions. Here we create an RNA ligand for TLR3 with no ability to activate the RIG-I/MDA5 pathway. This TLR3 ligand is a chimeric molecule consisting of phosphorothioate ODN-guided dsRNA (sODN-dsRNA), which elicits far less cytokine production than poly(I:C) in vitro and in vivo. The activation of TLR3/TICAM-1 pathway by sODN-dsRNA effectively induces natural killer and cytotoxic T cells in tumour-loaded mice, thereby establishing antitumour immunity. Systemic cytokinemia does not occur following subcutaneous or even intraperitoneal administration of sODN-dsRNA, indicating that TICAM-1 signalling with minute local cytokines sufficiently activate dendritic cells to prime tumoricidal effectors in vivo.