Intracellular pattern recognition receptors MDA5, RIG-I, andLGP2 are essential components of the cellular response to virus infection and are homologous to the DEXH box subfamily of RNA helicases. However, the relevance of helicase activity in the regulation of interferon production remains elusive. To examine the importance of the helicase domain function for these signaling proteins, a series of mutations targeting conserved helicase sequence motifs were analyzed for enzymatic activity, RNA binding, interferon induction, and antiviral signaling. Results indicate that all targeted motifs are required for ATP hydrolysis, but a subset is involved in RNA binding. The enzymatically inactive mutants differed in their signaling ability. Notably, mutations to MDA5 motifs I, III, and VI and RIG-I motif III produced helicase proteins with constitutive antiviral activity, whereas mutations in RIG-I motif V retained ATP hydrolysis but failed to mediate signal transduction. These findings demonstrate that type I interferon production mediated by full-length MDA5 and RIG-I is independent of the helicase domain catalytic activity. In addition, neither enzymatic activity nor RNA binding was required for negative regulation of antiviral signaling by LGP2, supporting an RNA-independent interference mechanism.The first line of defense against virus infection is provided by the cellular antiviral response and innate immune system. The immediate response includes the induction of type I interferon (IFN␣ and IFN␤, referred to herein as IFN 2 ) and other cytokines. Virus infections are sensed by cellular receptors that can recognize pathogen-associated molecular patterns such as viral nucleic acids (1). In addition to the transmembrane Toll-like receptors (2), a family of cytoplasmic RNA helicases has been identified that can detect cytosolic non-self RNA. Two members of this group are MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I) (3).These proteins are characterized by the combination of two caspase activation and recruitment domain (CARD) motifs linked to an RNA helicase domain.MDA5 and RIG-I can detect foreign RNAs and transmit a signal through the CARD-containing mitochondrial adaptor molecule IPS-1 (also named Cardif, MAVS, and VISA) (4 -7). IPS-1 apparently serves as a scaffold for propagation of the signaling cascade that leads to the activation of transcription factors, including IRF-3 and NFB (4, 7-9) which are responsible for transcriptional activation of a variety of antiviral effectors, including the IFN␤ gene that is fundamental to the antiviral response.Despite their similarities in domain structure and amino acid sequence, MDA5 and RIG-I are nonredundant and are involved in recognition of different types of non-self RNAs and therefore different viruses (10 -12). RIG-I exhibits ligand specificity for short (Ͻ2 kbp) double-stranded RNA (dsRNA) and 5Ј-triphosphorylated single-stranded RNA (11,(13)(14)(15)(16)(17) and has been demonstrated to specifically recognize homopolyme...

LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins.

The recent, rapid progress in our understanding of cytoplasmic RNA-mediated antiviral innate immune signaling was initiated by the discovery of retinoic acid-inducible gene I (RIG-I) as a sensor of viral RNA [1]. It is now widely recognized that RIG-I and related RNA helicases, melanoma differentiated-associated gene-5 (MDA5) and laboratory of genetics and physiology-2 (LGP2), can initiate and/or regulate RNA and virus -mediated type I IFN production and antiviral responses. As with other cytokine systems, production of type I IFN is a transient process, and can be hazardous to the host if unregulated, resulting in chronic cellular toxicity or inflammatory and autoimmune diseases [2][3][4][5][6][7][8][9]. In addition, the RIG-I-like receptor (RLR) system is a fundamental target for virusencoded immune suppression, with many indirect and direct examples of interference described. In this article, we review the current understanding of endogenous negative regulation in RLR signaling and explore direct inhibition of RLR signaling by viruses as a host immune evasion strategy. RLR signaling and its controlRIG-I and MDA5, two so-called RIG-I-like receptor (RLR) family proteins have been identified as cytoplasmic sensors of viral RNA [1,10]. RIG-I and MDA5 belong to the DExD/ H box RNA helicase family and also have two caspase activation and recruitment domains (CARD) N-terminal to the helicase region, implicated in relaying the signal downstream. Although similar, the two proteins differ in specificity of virus recognition as well as RNA binding specificity [11] as reviewed elsewhere [12]. For MDA5 neither biological substrate specificity nor exact RNA binding have been clearly specified experimentally, however MDA5 is thought to be the primary receptor for signaling initiated by cytoplasmic accumulation of the well-studied synthetic dsRNA analog, poly(I:C) [11,13]. While the helicase region represents one surface for interactions with dsRNAs, for the prototype, RIG-I, substrate recognition and binding specificity has been linked to a domain C-terminal to the helicase region. This RIG-I region has the ability to recognize 5′ tri-phosphorylated ends of doublestranded (ds) or single stranded (ss) RNA [14,15]. Structural analysis has determined that this regulatory domain contains an obligatory zinc binding module, which is conserved in both

Sainsbury et al.: Y2Y4 receptor double knockout protects against obesity due to a high--fat diet or Y1 receptor deficiency in mice Diabetes, 55(1): 19--26, 2006 Y2Y4 receptor double knockout protects against obesity due to a high-fat diet or Y1 receptor deficiency in mice Remarkably, the antiobesity effect of Y2Y4 deficiency was stronger than the obesogenic effect of Y1 deficiency, since Y1Y2Y4 triple knockouts did not develop obesity on the high-fat diet. Resistance to diet-induced obesity in Y2Y4 knockouts was associated with reduced food intake and improved glucose tolerance in the absence of changes in total physical activity. Fecal concentration of free fatty acids was significantly increased in Y2Y4 knockouts in association with a significantly reduced bile acid pool and marked alterations in intestinal morphology. In addition, hypothalamic proopiomelanocortin expression was decreased in diet-induced obesity (in both wild-type and Y1 receptor knockout mice) but not in obesity-resistant Y2Y4 receptor knockout mice fed a high-fat diet. Therefore, deletion of Y2 and Y4 receptors synergistically protects against diet-induced obesity, at least partially via changes in food intake and hypothalamic proopiomelanocortin expression.Members of the Y receptor family, notably Y1, Y2, Y4, and Y5 receptors, are implicated in energy homeostasis and the development of obesity and insulin resistance. These receptors are activated by three endogenous ligands: neuropeptide Y, the gut-derived hormones peptide YY, and pancreatic polypeptide. Recently, there has been renewed speculation that ligands for Y receptors, such as peptide YY3-36 and pancreatic polypeptide, may be of benefit for the treatment of obesity (1). There is considerable conflict in the literature about the role of Y receptors in the regulation of body weight. For instance, pharmacological studies suggested that Y1 receptors contribute to hyperphagia induced by increased hypothalamic neuropeptide Y secretion ( 2). Fasting-induced refeeding is reduced in Y1 knockout mice ( 3). Moreover, food intake and body weight of genetically obese ob/ob mice, in which hypothalamic neuropeptide Y-ergic activity is chronically increased, are significantly reduced by Y1 knockout ( 4). In contrast, Y1 knockouts develop significant increases in body weight, fat mass, and insulinemia in the absence of hyperphagia (3,5).Similar controversies prevail regarding the role of Y2 receptors in energy homeostasis. Hypothalamus-specific or germ-line deletion of Y2 receptors resulted in significant reductions in the body weight of lean mice (6) and significant reductions in adiposity or body weight and the type 2 diabetic syndrome of ob/ob mice in the absence of reductions in food intake (7,8). In contrast, another germline Y2 receptor knockout model was shown to develop increased body weight, fat deposition, and hyperphagia (9). Furthermore, intrahypothalamic administration of the Y2 agonist neuropeptide Y[13-36] to rats significantly decreased food intake (10). Moreover, circulating pep...