WDFY1 mediates TLR3/4 signaling by recruiting TRIF

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR–TRIF interaction, which is necessary for TLR signaling.     

Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease

Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses. HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its proline-rich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-beta (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P(6) position in lieu of the acidic residue present in viral substrates. Circular dichroism (CD) spectroscopy confirmed that a substantial fraction of TRIF exists as polyproline II helices, and inclusion of the polyproline track increased affinity of P side TRIF peptides for the HCV-BK protease. A polyproline II peptide representing an SH3 binding motif (PPPVPPRRR, Sos) bound NS3 with moderate affinity, resulting in inhibition of proteolytic activity. Chemical shift perturbations in NMR spectra indicated that Sos binds a 3(10) helix close to the protease active site. Thus, a polyproline II interaction with the 3(10) helix likely facilitates NS3/4A recognition of TRIF, indicating a significant difference from NS3/4A recognition of viral substrates. Because SH3 binding motifs are also present in NS5A, a viral protein that interacts with NS3, we speculate that the NS3 3(10) helix may be a site of interaction with other viral proteins.     

TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.     

Differential requirements of NS4A for internal NS3 cleavage and polyprotein processing of hepatitis C virus

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.     

Specific inhibition of MyD88-independent signaling pathways of TLR3 and TLR4 by resveratrol: molecular targets are TBK1 and RIP1 in TRIF complex

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-kappaB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-kappaB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-kappaB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-beta induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-kappaB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IkappaBalpha and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-kappaB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.     

The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF

Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.     

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.     

丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus,HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)来逃逸天然免疫识别,但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程,探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein,GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462),用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示,病毒复制早期MAVS切割效率较低;NS3/4A高效切割MAVS发生于HCV复制晚期,且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞,在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况,发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现,不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示,HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积,对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。     

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95

Activation of phosphatidylinositol 3'-kinase (PI 3'-K) after ligation of CD3 protects Th2 cells from CD95-mediated apoptosis. Here we show that protection is achieved by inhibition of the formation of CD95 aggregates and consequent activation of caspase-8. Inhibition of aggregate formation is mediated by changes in the actin cytoskeleton, which in turn inhibit lateral diffusion of CD95, reducing its diffusion coefficient, D, 10-fold. After cytochalasin D treatment of stimulated cells, the lateral diffusion of CD95 increases to the value measured on unstimulated cells, and CD95 molecules aggregate to process caspase-8 and mediate apoptosis. Regulation of functional receptor formation by modulating lateral diffusion is a novel mechanism for controlling receptor activity.     

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.