Ryo Narita

Critical Role of an Antiviral Stress Granule Containing RIG-I and PKR in Viral Detection and Innate Immunity

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.

Functional IRF3 deficiency in a patient with herpes simplex encephalitis

Andersen et al. identify a novel genetic etiology of herpes encephalitis in an adult patient carrying a heterozygous loss-of-function mutation in the IRF3 gene. This mutation results in impaired INF production in response to viral infection

A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

Functional Characterization of Domains of IPS-1 Using an Inducible Oligomerization System.

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.

Animal components in the diet of Japanese black bears Ursus thibetanus japonicus in the Kyoto area, Japan

Abstract We measured stable carbon and nitrogen isotope ratios of hair, muscle and potential food items of Japanese black bears Ursus thibetanus japonicus in Kyoto Prefecture and its surrounding area in order to determine the contributions of animal and plant foods. The δ13C values of hair samples of bears were −22.7 ± 0.4‰ in early summer, −22.8 ± 0.3‰ in late summer and −22.7 ± 0.7‰ in autumn, while δ15N values were 3.5 ± 0.4‰ in early summer, 3.2 ± 0.4‰ in late summer and 3.6 ± 0.5‰ in autumn. The δ13C values of muscle samples of bears were −24.6‰ in early summer, −23.2‰ in late summer and −23.1 ± 0.3‰ in autumn, while δ15N values of bears captured were 3.7‰ in early summer, 5.0‰ in late summer and 4.5 ± 0.7‰ in autumn. We determined the isotopic endpoints of seven food groups from the isotope ratios of food groups and calculated the contribution of each food group using a stochastic method. Our results suggested that animal components were the major constituent of body tissue, contributing > 61% in all samples except for muscle samples collected in early summer. In muscle samples collected in early summer, none of the food items were estimated to be the major source. In cases in which the animal components were estimated to be major food sources, invertebrates were estimated to account for most of the animal components. It was concluded that animal components are an important source of tissue material in Japanese black bears in Kyoto and its surrounding area.

Variation in Maize Consumption by Brown Bears (Ursus arctos) in Two Coastal Areas of Hokkaido, Japan

Abstract. We measured carbon and nitrogen stable-isotope ratios in the livers of 130 brown bears (Ursus arctos) killed by hunters in two coastal areas of Hokkaido, Japan, from 2003 to 2004 to analyze the diets of individual bears, particularly maize. In both study areas, bears suspected of damaging maize, whose diets were higher in the C4 plant component, were mainly observed during late summer and autumn. Wide variation in the C4 plant component indicated that individual bears have differing degrees of dependence on maize, suggesting differential foraging behavior among the individual bears. We found that a stable-isotope ratio analysis of individual Hokkaido brown bears can serve as a tool to review the effectiveness of control killing and to monitor trends in maize consumption by local populations of bears.

Turnover of stable isotopes in Hokkaido brown bear (Ursus arctos yesoyensis)

Stable isotope analysis has been used in many studies on the nutritional ecology of mammalian species. One of the merits of using stable isotope ratios as a dietary indicator is that stable isotope ratios of body tissue reflect those of foods assimilated, not just ingested foods (Hobson and Clark 1992; Hilderbrand et al. 1996; Kelly 2000). Another merit of using stable isotope ratios is that isotopic signatures indicate integrated dietary information over different time periods (e.g., weeks, months, years) because of the different turnover rates of dietderived stable isotopes in consumer tissues (Tieszen et al. 1983; Hobson and Clark 1992; Hilderbrand et al. 1996). Results of analyses of fecal and stomach contents, which many wildlife researchers have relied on, have problems such as overestimation of hardly-digestible diets or diets consumed largely at some particular moment (Hewitt and Robbins 1996). These shortcomings of traditional techniques have been overcome by the development of stable isotope analysis methods, and stable isotope analysis has provided deeper insights for nutritional ecology of wildlife (Hilderbrand et al. 1996; Hilderbrand et al. 1999a; Hilderbrand et al. 1999b; Jacoby et al. 1999; Felicetti et al. 2003b). Studies on mammals in which stable isotopes are used as dietary tracers require estimation of how long it takes for stable isotopes in tissues to be replaced by isotopes derived from the diet. When we estimate food habits using stable isotopes, accuracy of estimation depends on how appropriately sources in the mass balance models are selected (Minagawa 1992; Phillips and Gregg 2003), and knowledge of the isotopic turnover rates is necessary for appropriate selection of the sources. When we conduct experiments to estimate isotopic discrimination values, information on isotopic turnover rates is essential to decide periods of feeding trials (Hilderbrand et al. 1996; Felicetti et al. 2003b). Food habits of the Hokkaido brown bear have been investigated by fecal or stomach contents analysis (Aoi 1985; Sato 2002). However, shortcomings in the analysis method mentioned earlier would be more significant for omnivorous species like brown bears. Introduction of a method for stable isotope analysis for this species is needed. Moreover, recent research revealed the importance of nutritional ecology of ursid (bear) species for ecosystem conservation (Hilderbrand et al. 2004; Robbins et al. 2004). Nutritional ecological research on bears in Japan would also be necessary for better ecosystem conservation in Japan. When we treat hibernating ursid species, physiological changes during hibernation should be considered. In this paper, we report the results of experiments estimating isotopic turnover rates as well as determining possible seasonal fluctuation in turnover rates of captive Hokkaido brown bears.

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.