Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-β production was enhanced in AHR- IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.AHR was originally discovered as a xenobiotic sensor that mediates the toxicity of the persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), more commonly known as dioxin [1][2][3][4] . Activation of AHR induces its target genes, including those encoding cytochrome P4501A1, cytochrome P4501B1, AHR repressor, TCDD-inducible poly(ADP-ribose)polymerase (TIPARP) and aldehyde dehydrogenase 1A3 (refs. 1,2,5-9), which are involved in the adaptive metabolism of xenobiotic compounds. This property of AHR has been implicated in host defense against bacterial infection, as certain bacterial pigmented virulence factors are AHR agonists that are subsequently metabolized by AHR-regulated drug-metabolizing enzymes 10 . Studies of AHR-deficient mice have identified important physiological roles for AHR in response to endogenous ligands in cell cycle regulation, cell differentiation and immune responses 8,[11][12][13][14] . In relation to this, several putative endogenous ligands for the AHR have also been reported, including heme metabolites, arachidonic acids or leukotrienes and tryptophan metabolites, such as 6-formylindolo(3,2-b)carbazole (FICZ) and kynurenine (Kyn) 2,8,15 .There has been increased interest in understanding the role of AHR in immunity.Several reports, most of which are based mainly on experiments with dioxin treatment, have shown that the AHR is involved in the differentiation and/or function of T cells, macrophages and dendritic cells 7,9,11,[16][17][18][19][20][21] . AHR has been implicated in the control of acute graft-versus-host disease and autoimmunity 11,12,21 . Dioxin-activated AHR also reduces the survival rate of mice infected with influenza A virus 22,23 and indirectly suppresses the proliferation and differentiation of virus-specific CD8 + T cells via its regulatory role in dendritic cells 24 . FICZ and dioxin diminish CD8 + T cell responsiveness, whereas dioxin, but not FICZ, affects neutrophil recruitment or pulmonary inducible nitric oxide synthase (iNOS) induction in response to influenza virus infection 25 .Tryptophan metabolites such as Kyn are upregulated during inflammation and/or tumor progression in several types of immune and tumor cells through the catalytic activity of tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which catalyze the first step in the formation of Kyn from tryptophan 2,9 . This increase in Kyn leads to an increase in regulatory T c...

Japan. 8 These authors contributed equally to this work. Correspondence should be addressed to A.T. (takaoka@igm.hokudai.ac.jp). At least three DExD/H-box RNA helicases 4,5 , RIG-I, MDA5 and LGP2 are included in the RLR family. RIG-I is a key PRR for the detection of positive-and negative-stranded RNA viruses in the cytoplasm of cells 6,7 , and plays an important role in triggering responses against many viruses, such as orthomyxovirus (influenza A virus) and paramyxovirus (measles, mumps, and Sendai virus (SeV)) families, hepatitis C virus (HCV), and Japanese encephalitis virus (JEV) 8 . 5'-triphosphate modifications of RNA (3pRNA) are essential for RIG-I recognition and activation 9,10 . Ligand-binding activates the ATPase activity of RIG-I to change its structural conformation 6 , which in turn enables RIG-I to interact through its N-terminal tandem caspase recruitment domain (CARD) with the adaptor protein MAVS (for mitochondrial antiviral signaling protein, also known as IPS-1, VISA, or Cardif) [11][12][13][14] . MAVS then initiates the activation of interferon (IFN)-regulatory factor (IRF) 3, IRF7 and NF-κB transcriptional pathways for the subsequent production of type I IFNs and inflammatory cytokines, which are crucial for activating innate immune responses to viral infection 6,15 . Given the important role of the RIG-I pathway in the antiviral innate response, the mechanisms regulating RIG-I activation represent a topic of intense research [16][17][18] .Poly(ADP-ribose) polymerases (PARPs), a superfamily with least 17 members, are known to regulate not only cell survival and cell death programs triggered by DNA 3 damage, but also other biological functions as well as pathological processes, such as inflammatory and degenerative diseases, in a manner dependent or independent of their PARP activity [19][20][21][22] . Several PARP-superfamily members have a direct regulatory effect on replication of certain viruses 19,20,22,23 RESULTS 4PARPs contribute to the IFN response To investigate the role of the PARP-superfamily members in nucleic acid induced innate immune responses, we selected some of the PARP-superfamily members known to be involved in microbial infection, inflammation and immunity: PARP-1, PARP-2, PARP-7, PARP-9, 23,[31][32][33][34]. We then examined whether they have the ability to enhance the induction of IFNB mRNA in HEK293T cells in response to stimulation with three different types of nucleic acids-3pRNA, poly(rI:rC) and poly(dA-dT)•poly(dA-dT) (named poly(dA:dT) hereafter).Among the protein tested, PARP-13 uniquely showed a marked enhancing effect on IFNB mRNA expression induced by stimulation with 3pRNA, poly(rI:rC) and poly(dA:dT) ( Fig. 1a), all of which are known to activate the RIG-I-mediated pathway in HEK293T cells 9,10,[35][36][37] . A weak increase in IFNB mRNA expression was also detected in cells expressing PARP-1, PARP-2 and PARP-9. PARP-13 exists in at least two isoforms 22,26,38 . The amino-terminal 254-amino acid fragment of the rat homologue, which corresponds to the N...

MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid-inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with increased amounts of the H5N1 virus. These findings highlight the dual role of miR-485 in preventing spurious activation of antiviral signaling and restricting influenza virus infection.

More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q-and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutantions occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.TP53 tumor suppressor gene encodes a transcription factor (p53), which forms homotetramer and binds to DNA in a sequence-specific manner to transcribe target genes. It is well known that genotoxic stress induces the stabilization and activation of the p53 protein, resulting in apoptosis, inhibition of cellcycle progression, differentiation, senescence, or accelerated rates of DNA repair (18). More than half of all human cancers are associated with alterations of the TP53 gene (7). Most TP53 alterations are missense mutations, localized in the DNA-binding domain, and abolish the transcriptional activity via p53-responsive element. The residues such as R175, G245, R248, R249, R273 and R282 in the p53 protein are frequently mutated, which called "hot spots" (7). In their functional interactions with the remain-

Normal epithelial cells have an ability to sense and actively eliminate neighboring transformed cells, a process called epithelial defense against cancer (EDAC). Exogenous S1P plays a crucial role in EDAC; the S1P–S1PR2 pathway regulates Rho–Rho kinase–filamin in the surrounding normal cells, promoting apical extrusion of RasV12-transformed cells from epithelia.

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Background & AimsThe interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV).MethodsThis study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice.ResultsFollowing treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways.ConclusionsThese findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.