Cytoplasmic dsRNA induces the expression of OCT3/4 and NANOG mRNAs in differentiated human cells.

Cytoplasmic dsRNA is recognized by RNA helicase RIG-I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), triggering induction of the innate immune response via the mitochondrial antiviral signaling protein (MAVS). In contrast, extracellular dsRNA is internalized into endosomes and recognized by Toll-like receptor 3 (TLR3), which triggers signaling via the Toll-like receptor adaptor molecule 1 (TICAM-1). Poly(I:C) is a synthetic dsRNA analog and increases the expression of octamer-binding protein 3/4 (OCT3/4), NANOG, and SRY-box (SOX) mRNAs during pluripotency induction. However, the mechanism underlying this increase is unclear. Here, we focused on the mechanism of poly(I:C)-induced expression of stem cell-specific genes in human somatic cells. Addition of poly(I:C) to human fibroblast culture medium did not increase OCT3/4 mRNA expression, but poly(I:C) transfection markedly increased OCT3/4 expression and induced nuclear localization of the OCT3/4 protein, implying that not TLR3, but RIG-I and MDA5 are required for OCT3/4 expression. Moreover, although cytoplasmic dsRNA increased OCT3/4 mRNA, cytoplasmic dsDNAs, such as salmon sperm DNA and poly(dA:dT), did not. Interestingly, the expression of NANOG, SOX2, Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc was also increased by cytoplasmic dsRNA. Of note, siRNAs that silenced MAVS and interferon regulatory factor 1 (IRF1) expression reduced OCT3/4 levels after stimulation with poly(I:C); however, an NF-κB inhibitor and siRNA-mediated knockdown of proto-oncogene c-Jun did not significantly reduce the mRNA levels. We conclude that cytoplasmic dsRNA increases the expression of stem cell-specific genes in human somatic cells in a MAVS- and IRF1-dependent manner.

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type-Specific Manner.

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2-TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1-mediated signaling including IFN-ß production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-ß promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-ß production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein-2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein-2 in clathrin-mediated endocytosis of TLR3/4 ligands.

Functional interfaces between TICAM-2/TRAM and TICAM-1/TRIF in TLR4 signaling.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS), produces pro-inflammatory cytokines and type I interferons, and associates with a trigger of endotoxin shock. TLR4 is interacted with a TIR domain-containing adaptor molecule-2 (TICAM-2)/TRAM [TRIF (TIR domain-containing adaptor-inducing interferon-ß)-related adaptor molecule] via its Toll-interleukin-1 receptor homology (TIR) domain. TICAM-2 acts as a scaffold protein and activates TIR domain-containing adaptor molecule-1 (TICAM-1)/TRIF. According to the structural analysis by NMR, TICAM-2 interacts with TICAM-1 by the acidic amino acids motif, E87/D88/D89. The TIR domain of TICAM-2 couples with the dimer of TIR domain of TLR4 beneath the membrane, and TICAM-2 itself also forms dimer and constitutes a binding site with TICAM-1. Endosomal localization of TICAM-2 is essential for TLR4-mediated type I interferon-inducing signal from the endosome. N-terminal myristoylation allows TICAM-2 to anchor to the endosomal membrane. Additionally, we have identified two acidic amino acids, D91/E92, as a functional motif that cooperatively determines endosomal localization of TICAM-2. This structural information of TICAM-2 suggests that the specific structure is indispensable for the endosomal localization and type I interferon production of TICAM-2. Taken together with the knowledge on cytoplasmic sensors for LPS, TICAM-2/TICAM-1 may conform to a signal network on TLR4 to facilitate induction of cytokine disorders.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

BACKGROUND: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. METHODS: We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. RESULTS: We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. CONCLUSION: These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

LRRC59 Regulates Trafficking of Nucleic Acid-Sensing TLRs from the Endoplasmic Reticulum via Association with UNC93B1.

Compartmentalization of nucleic acid (NA)-sensing TLR3, 7, 8, and 9 is strictly regulated to direct optimal response against microbial infection and evade recognition of host-derived NAs. Uncoordinated 93 homolog B1 (UNC93B1) is indispensable for trafficking of NA-sensing TLRs from the endoplasmic reticulum (ER) to endosomes/lysosomes. UNC93B1 controls loading of the TLRs into COPII vesicles to exit from the ER and traffics with the TLRs in the steady state. Ligand-induced translocation also happens on NA-sensing TLRs. However, the molecular mechanism for ligand-dependent trafficking of TLRs from the ER to endosomes/lysosomes remains unclear. In this study, we demonstrated that leucine-rich repeat containing protein (LRRC) 59, an ER membrane protein, participated in trafficking of NA-sensing TLRs from the ER. Knockdown of LRRC59 reduced TLR3-, 8-, and 9-mediated, but not TLR4-mediated, signaling. Upon ligand stimulation, LRRC59 associated with UNC93B1 in a TLR-independent manner, which required signals induced by ligand internalization. Endosomal localization of endogenous TLR3 was decreased by silencing of LRRC59, suggesting that LRRC59 promotes UNC93B1-mediated translocation of NA-sensing TLRs from the ER upon infection. These findings help us understand how NA-sensing TLRs control their proper distribution in the infection/inflammatory state.

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.

TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain-containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain-containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.

Double-stranded RNA analog and type I interferon regulate expression of Trem paired receptors in murine myeloid cells.

BACKGROUND: Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors used to control innate immune responses such as proinflammatory cytokine production in mice. Trem genes belong to a rapidly expanding family of receptors that include activating and inhibitory paired-isoforms. RESULTS: By comparative genomic analysis, we found that Trem4, Trem5 and Trem-like transcript-6 (Treml6) genes typically paired receptors. These paired Trem genes were murine-specific and originated from an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing gene. Treml6 encoded ITIM, whereas Trem4 and Trem5 lacked the ITIM but possessed positively-charged residues to associate with DNAX activating protein of 12 kDa (DAP12). DAP12 was directly associated with Trem4 and Trem5, and DAP12 coupling was mandatory for their expression on the cell surface. In bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs), and splenic DC subsets, polyinosinic-polycytidylic acid (polyI:C) followed by type I interferon (IFN) production induced Trem4 and Treml6 whereas polyI:C or other TLR agonists failed to induce the expression of Trem5. PolyI:C induced Treml6 and Trem4 more efficiently in BMDMs than BMDCs. Treml6 was more potentially up-regulated in conventional DC (cDCs) and plasmacytoid DC (pDCs) than Trem4 in mice upon in vivo stimulation with polyI:C. DISCUSSION: Treml6-dependent inhibitory signal would be dominant in viral infection compared to resting state. Though no direct ligands of these Trem receptors have been determined, the results infer that a set of Trem receptors are up-regulated in response to viral RNA to regulate myeloid cell activation through modulation of DAP12-associated Trem4 and ITIM-containing Treml6.

Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

Nucleic acid-sensing TLRs are involved in both antimicrobial immune responses and autoimmune inflammation. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, which undergo proteolytic processing in the endolysosomes to generate functional receptors. Recent structural analyses of human TLR8 ectodomain and its liganded form demonstrated that TLR8 is also cleaved, and both the N- and C-terminal halves contribute to ligand binding. However, the structures and ssRNA recognition mode of endogenous TLR8 in human primary cells are largely unknown. In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. The insertion loop between leucine-rich repeats 14 and 15 in TLR8 is indispensable for the cleavage and stepwise processing that occurs in the N-terminal fragment. Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. TLR8 recognizes viral ssRNA and endogenous RNA, such as microRNAs, resulting in the production of proinflammatory cytokines. Hence, localization sites of the receptors are crucial for the nucleic acid-sensing mode and downstream signaling.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Cleaved/associated TLR3 represents the primary form of the signaling receptor.

TLR3 belongs to the family of intracellular TLRs that recognize nucleic acids. Endolysosomal localization and cleavage of intracellular TLRs play pivotal roles in signaling and represent fail-safe mechanisms to prevent self-nucleic acid recognition. Indeed, cleavage by cathepsins is required for native TLR3 to signal in response to dsRNA. Using novel Abs generated against TLR3, we show that the conserved loop exposed in LRR12 is the single cleavage site that lies between the two dsRNA binding sites required for TLR3 dimerization and signaling. Accordingly, we found that the cleavage does not dissociate the C- and N-terminal fragments, but it generates a very stable "cleaved/associated" TLR3 present in endolysosomes that recognizes dsRNA and signals. Moreover, comparison of wild-type, noncleavable, and C-terminal-only mutants of TLR3 demonstrates that efficient signaling requires cleavage of the LRR12 loop but not dissociation of the fragments. Thus, the proteolytic cleavage of TLR3 appears to fulfill function(s) other than separating the two fragments to generate a functional receptor.

Interferon (IFN) and Cellular Immune Response Evoked in RNA-Pattern Sensing During Infection with Hepatitis C Virus (HCV).

Hepatitis C virus (HCV) infects hepatocytes but not dendritic cells (DCs), but DCs effectively mature in response to HCV-infected hepatocytes. Using gene-disrupted mice and hydrodynamic injection strategy, we found the MAVS pathway to be crucial for induction of type III interferons (IFNs) in response to HCV in mouse. Human hepatocytes barely express TLR3 under non-infectious states, but frequently express it in HCV infection. Type I and III IFNs are induced upon stimulation with polyI:C, an analog of double-stranded (ds)RNA. Activation of TLR3 and the TICAM-1 pathway, followed by DC-mediated activation of cellular immunity, is augmented during exposure to viral RNA. Although type III IFNs are released from replication-competent human hepatocytes, DC-mediated CTL proliferation and NK cell activation hardly occur in response to the released type III IFNs. Yet, type I IFNs and HCV-infected hepatocytes can induce maturation of DCs in either human or mouse origin. In addition, mouse CD8+ DCs mature in response to HCV-infected hepatocytes unless the TLR3/TICAM-1 pathway is blocked. We found the exosomes containing HCV RNA in the supernatant of the HCV-infected hepatocytes act as a source of TLR3-mediated DC maturation. Here we summarize our view on the mechanism by which DCs mature to induce NK and CTL in a status of HCV infection.

Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells.

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1ß. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) ß mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPß target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPß in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense.

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.

Infectivity of hepatitis C virus is influenced by association with apolipoprotein E isoforms.

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5' untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-beta production.

TLR3 and cytoplasmic RIG-I-like receptor (RLR) recognize virus-derived dsRNA and induce type I IFN production in a distinct manner. Human TLR3 localizes to the endosomal compartments in myeloid dendritic cells (mDCs), while it localizes to both the cell surface and interior in fibroblasts and epithelial cells. TLR3 signaling arises in the intracellular compartment in both cell types and requires endosomal maturation. The mechanisms by which extracellular dsRNA is delivered to the TLR3-containing organelle remain largely unknown. Among various synthetic dsRNAs, poly(I:C) is preferentially internalized and activates TLR3 in mDCs. In vitro transcribed dsRNAs hardly induce IFN-beta production in mDCs. In this study, we demonstrate that the clathrin-dependent endocytic pathway mediates cell entry of poly(I:C) to induce IFN-beta gene transcription. Furthermore, poly(I:C)-induced IFN-beta production is inhibited by pretreatment of cells with B- and C-type oligodeoxynucleotides (ODNs) but not with TLR7/8 ligands. The binding and internalization of B-type ODNs by mDCs was reduced in the presence of poly(I:C), suggesting that poly(I:C) shares the uptake receptor with B- and C-type ODNs. Hence, foreign dsRNA is recognized by differently categorized receptors, cytoplasmic RIG-I-like receptor, membrane-bound TLR3 and cell-surface RNA capture. The endocytic pathway is critical for dsRNA-induced TLR3-mediated cell activation.

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction.

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.

Hepatitis C virus utilizes lipid droplet for production of infectious virus.

Hepatitis C virus (HCV) establishes a persistent infection and causes chronic hepatitis. Chronic hepatitis patients often develop hepatic cirrhosis and progress to liver cancer. The development of this pathological condition is linked to the persistent infection of the virus. In other words, viral replication/multiplication may contribute to disease pathology. Accumulating clinical studies suggest that HCV infection alters lipid metabolism, and thus causes fatty liver. It has been reported that this abnormal metabolism exacerbates hepatic diseases. Recently, we revealed that lipid droplets play a key role in HCV replication. Understanding the molecular mechanism of HCV replication will help elucidate the pathogenic mechanism and develop preventive measures that inhibit disease manifestation by blocking persistent infection. In this review, we outline recent findings on the function of lipid droplets in the HCV replication cycle and describe the relationship between the development of liver diseases and virus replication.

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily.

Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappaB reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pam3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappaB in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappaB. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events.