NK cells control tumor and virus-infected cells through releasing cytotoxic granules and proinflammatory cytokines. IFN-γ and TNF-α secretions and cytotoxicity are regarded as two distinct functions of NK cells with little synergy in between as results of early association of the two functions with distinct subsets of NK populations and of the studies showing target cells developing NK resistance upon IFN-γ treatment. Here, we show that IFN-γ and TNF-α synergistically enhance NK cell cytotoxicity through NF-κB-dependent up-regulation of ICAM-1 expression in target cells, thereby promoting their conjugate formation with NK cells. Neutralizing IFN-γ and TNF-α during cytolysis significantly impaired NK cell lysis of the target cells. Further, tumor cells exhibiting IFN-γ-inducible lysis are generally less-sensitive NK target cells but express inducible levels of ICAM-1. In contrast, sensitive NK targets tend to express higher but less-inducible ICAM-1. Their preferential induction in the lysis of insensitive NK target cells suggests that IFN-γ and TNF-α are functionally linked to and should be regarded as an integral part of NK cytolytic function.

RIG-I and MDA5 are cytoplasmic sensors that recognize different species of viral RNAs, leads to activation of the transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons. In this study, we identified REUL, a RING-finger protein, as a specific RIG-I-interacting protein. REUL was associated with RIG-I, but not MDA5, through its PRY and SPRY domains. Overexpression of REUL potently potentiated RIG-I-, but not MDA5-mediated downstream signalling and antiviral activity. In contrast, the RING domain deletion mutant of REUL suppressed Sendai virus (SV)-induced, but not cytoplasmic polyI:C-induced activation of IFN-β promoter. Knockdown of endogenous REUL by RNAi inhibited SV-triggered IFN-β expression, and also increased VSV replication. Full-length RIG-I, but not the CARD domain deletion mutant of RIG-I, underwent ubiquitination induced by REUL. The Lys 154, 164, and 172 residues of the RIG-I CARD domain were critical for efficient REUL-mediated ubiquitination, as well as the ability of RIG-I to induce activation of IFN-β promoter. These findings suggest that REUL is an E3 ubiquitin ligase of RIG-I and specifically stimulates RIG-I-mediated innate antiviral activity.

Viral infection leads to activation of the transcription factors interferon regulatory factor-3 and NF-B, which collaborate to induce type I IFNs. The RNA helicase proteins RIG-I and MDA5 were recently identified as two cytoplasmic viral RNA sensors that recognize different species of viral RNAs produced during viral replication. In this study, we identified DAK, a functionally unknown dihydroacetone kinase, as a specific MDA5-interacting protein. DAK was associated with MDA5, but not RIG-I, under physiological conditions. Overexpression of DAK inhibited MDA5-but not RIG-I-or TLR3-mediated IFN-␤ induction. Overexpression of DAK also inhibited cytoplasmic dsRNA and SeV-induced activation of the IFN-␤ promoter, whereas knockdown of endogenous DAK by RNAi activated the IFN-␤ promoter, and increased cytoplasmic dsRNA-or SeV-triggered activation of the IFN-␤ promoter. In addition, overexpression of DAK inhibited MDA5-but not RIG-Imediated antiviral activity, whereas DAK RNAi increased cytoplasmic dsRNA-triggered antiviral activity. These findings suggest that DAK is a physiological suppressor of MDA5 and specifically inhibits MDA5-but not RIG-I-mediated innate antiviral signaling. The innate immune system has developed at least two distinct mechanisms for the recognition of viral RNAs (2, 3, 9). One is mediated by Toll-like receptor 3 (TLR3), which recognizes viral dsRNA released by infected cells (10). Engagement of TLR3 by dsRNA triggers TRIF-mediated signaling pathways, leading to IRF-3 and NF-B activation (11-16). The second mechanism involves two RNA helicase proteins, RIG-I and MDA5, which function as cytoplasmic viral RNA sensors (17, 18). Both RIG-I and MDA5 contain two CARD modules at their N terminus and a DexD/H-box helicase domain at their C terminus. The helicase domains of RIG-I and MDA5 serve as intracellular viral RNA receptors, whereas the CARD modules are responsible for transmitting signals to downstream CARD-containing adaptor VISA/ MAVS/IPS-1/Cardif, which in turn activates TAK1-IKK␤ and TBK1/IKK kinases, leading to activation of NF-B and IRF-3 and induction of type I IFNs (19)(20)(21)(22)(23)(24).Although MDA5 and RIG-I share a similar structural architecture, and their signaling pathways converge at the adaptor level, gene-knockout studies indicate that the two proteins are required for responding to distinct species of RNA viruses. RIG-I responds to in vitro-transcribed dsRNA, vesicular stomatitis virus (VSV), Newcastle disease virus, and influenza virus in mice. In contrast, MDA5 recognizes poly(I:C) and is essential for the antiviral response to the picornavirus encephalomyocarditis virus (25,26). Recently, it was demonstrated that RIG-I, but not MDA5, recognizes single-strand RNA bearing 5Ј phosphate (27,28). In addition, it has been shown that signaling mediated by RIG-I-and MDA5-mediated signaling is differentially regulated. For example, the V protein of paramyxovirus appears to selectively target MDA5-but not RIG-I-mediated IFN response (29)(30)(31).In the present study, we identified D...

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