The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection [1][2][3][4][5][6] . MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)-and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH-and MAVS-mediated interferon-b promoter activity and in the disruption of virus-induced RLH-MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.Mammalian members of the nucleotide-binding domain (NBD) and leucine-rich-repeat-containing (LRR) (known as NLR, see http://www.genenames.org/genefamily/nacht.html) family of proteins are indispensable for cellular responses to pathogens. This NBD-LRR protein structure is ancient and highly conserved, as shown by its initial identification among plant disease-resistance proteins 7-12 . Current dogma posits that NLRs function as cytoplasmic surveillance molecules that sense intracellular pathogen-associated molecular patterns (PAMPs), or as regulators of pathogen-initiated signalling cascades 13,14 . Viral PAMPs are detected by the cytoplasmic RLH receptors RIG-I (also known as DDX58) and MDA-5 (also known as IFIH1), which signal through the mitochondrial protein MAVS, resulting in the activation of interferon regulatory factor 3 (IRF3) and NF-kB and type-1 interferon transcription [1][2][3][4][5][6] . Abrogation of MAVS expression or function leads to reduced type 1 interferon production and antiviral protection 15 .To study the potential role of NLR proteins in regulating mitochondrial antiviral signalling, we used bioinformatics to identify NLRs localized to the mitochondria. We identified one putative mitochondrial NLR called NLRX1 (previously known as CLR11.3 and NOD9) 9,16 (Fig. 1a). The predicted peptide sequence and distinct domains of NLRX1 are shown in Supplementary Fig. 1. Consistent with the conserved motif structure of the NLR family, NLRX1 contains a central putative NBD and carboxy-terminal LRRs. The assignment of the amino-terminal effector domain to a subclass i...

Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus.MiRNAs constitute a large family of small noncoding RNAs that regulate gene expression posttranscriptionally, affecting mRNA degradation and translation by base-pairing with the 3′ untranslated regions (3′UTRs) (1). The recent discovery of virally encoded miRNAs, mostly in herpesviruses, intriguingly suggests that miRNAs may function in interspecies Copyright © 2007 We applied RepTar and subsequently cRepTar to all human 3′UTRs, searching for potential binding sites of the 11 HCMV miRNAs listed in miRBase 7.0 (9). MICB, an immunorelated gene, was among the highest ranking predicted targets and the top prediction for hcmv-miR-UL112 (Fig. 1A). MICB is a stress-induced ligand of NKG2D, a natural killer (NK) activating receptor expressed on almost all human NK cells and activated cytotoxic T lymphocytes (CTLs) (10). The importance of MICB in the immune response against HCMV infection is substantiated by the specific down-regulation of MICB surface expression via the UL16 protein of HCMV (11,12). MICA, another stress-induced ligand of NKG2D, was also ranked among the top predicted targets of hcmv-miR-UL112 (Fig. 1A). The hcmv-miR-UL112 putative binding sites of both genes are almost identical and are located within a highly similar but not evolutionarily conserved (7) 150-nucleotide (nt) region of their 3′UTRs.To assess the function of hcmv-miR-UL112, we expressed this miRNA in various human tumor cell lines that endogenously express MICA and MICB with the use of recombinant lentiviral vectors: hcmv-miR-UL112 and two control vectors, a non-miRNA sequence (miRcontrol) and hcmv-miR-US5-1. The expression of hcmv-miR-UL112 was confirmed by quantative real-time polymerase chain reaction (qPCR) ( fig. S1). The vectors contained green fluorescent protein (GFP) for monitoring the infection efficiency (7). No difference in the transduction efficiency of the different lentiviral vectors was measured ( fig. S2). Analysis of the various tumor cells transduced with hcmv-miR-UL112 revealed a specific and extensive reduction of MICB and little or no reduction of MICA (Fig. 1B). The downregulation was specific to MICB and to hcmv-miR-UL112, because no change in the level of major histocompati...