鱼类和哺乳类RLR介导的抗病毒免疫反应的泛素化修饰调控

RLR[retinoic acid-inducible gene Ⅰ(RIG-Ⅰ)-like Receptors]是一类表达在胞浆中的模式识别受体, 在识别细胞质中经病毒复制产生的病毒RNA后, 启动一系列信号级联反应, 以诱导机体Ⅰ型干扰素及干扰素诱导的抗病毒基因的表达, 最后达到清除机体病毒感染的目的。由于在病毒感染时机体干扰素反应必须迅速启动, 当病毒清除后干扰素反应又需要立即恢复到正常本底水平, 因此RLR激活的信号转导途径受到了严格的调控, 其中就包括由E3泛素连接酶参与的泛素化修饰调控和由去泛素化酶参与的去泛素化修饰调控。自2003年成功鉴定出鱼类干扰素基因以来, 鱼类也被发现具有保守的RLR信号转导途径诱导干扰素抗病毒免疫反应, 该信号途径同样受到泛素化修饰的调控。文章总结了近年来泛素化修饰在哺乳类和鱼类RLR介导的抗病毒免疫应答通路中的调节机制。     

Study of Human RIG-I Polymorphisms Identifies Two Variants with an Opposite Impact on the Antiviral Immune Response

Background

RIG-I is a pivotal receptor that detects numerous RNA and DNA viruses. Thus, its defectiveness may strongly impair the host antiviral immunity. Remarkably, very little information is available on RIG-I single-nucleotide polymorphisms (SNPs) presenting a functional impact on the host response.

Methodology/Principal Findings

Here, we studied all non-synonymous SNPs of RIG-I using biochemical and structural modeling approaches. We identified two important variants: (i) a frameshift mutation (P₂₂₉fs) that generates a truncated, constitutively active receptor and (ii) a serine to isoleucine mutation (S₁₈₃I), which drastically inhibits antiviral signaling and exerts a down-regulatory effect, due to unintended stable complexes of RIG-I with itself and with MAVS, a key downstream adapter protein.

Conclusions/Significance

Hence, this study characterized P₂₂₉fs and S₁₈₃I SNPs as major functional RIG-I variants and potential genetic determinants of viral susceptibility. This work also demonstrated that serine 183 is a residue that critically regulates RIG-I-induced antiviral signaling.     

Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells

Background and Aim

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.

Methods

The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.

Results

HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.

Conclusion

These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.     

Morphine suppresses IFN signaling pathway and enhances AIDS virus infection

Background

Opioids exert a profound influence on immunomodulation and enhance HIV infection and replication. However, the mechanism(s) of their action remains to be determined. We thus investigated the impact of morphine on the intracellular innate antiviral immunity.

Methodology/Principal Findings

Seven-day-cultured macrophages were infected with equal amounts of cell-free HIV Bal or SIV Delta_B670 for 2 h at 37°C after 24 h of treatment with or without morphine. Effect of morphine on HIV/SIV infection and replication was evaluated by HIV/SIV RT activity assay and indirect immunofluorescence for HIV p24 or SIV p28 antigen. The mRNA expression of cellular factors suppressed or induced by morphine treatment was analyzed by the real-time RT-PCR. We demonstrated that morphine treatment of human blood monocyte-derived macrophages significantly inhibited the expression of interferons (IFN-α, IFN-β and IFN-λ) and IFN-inducible genes (APOBEC3C/3F/3G and 3H). The further experiments showed that morphine suppressed the expression of several key elements (RIG-I and IRF-7) in IFN signaling pathway. In addition, morphine treatment induced the expression of suppressor of cytokine signaling protein-1, 2, 3 (SOCS-1, 2, 3) and protein inhibitors of activated STAT-1, 3, X, Y (PIAS-1, 3, X, Y), the key negative regulators of IFN signaling pathway.

Conclusions

These findings indicate that morphine impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to AIDS virus infection.     

Green Tea Catechin,Epigallocatechin Gallate,Suppresses Signaling by the dsRNA Innate Immune Receptor RIG-I

Background

The Innate immune system constitutes the first line of defense against pathogen infections. The Retinoic acid-inducible gene I (RIG-I) receptor recognizes triphosphorylated ssRNAs and dsRNA to initiate downstream signaling of interferon response. However, unregulated activity of these receptors could lead to autoimmune diseases. We seek to identify small molecules that can specifically regulate RIG-I signaling.

Methodology/Principal Findings

Epigallocatechin gallate (EGCG), a polyphenolic catechin present in green tea, was identified in a small molecule screen. It was found to bind RIG-I and inhibits its signaling at low micromolar concentrations in HEK293T cells. Furthermore, EGCG dose-dependently inhibited the ATPase activity of recombinant RIG-I but did not compete with RIG-I interaction with RNA or with ATP. EGCG did not inhibit signaling by Toll-like receptors 3, 4, 9 or constitutive signaling by the adapter protein IPS-1. Structure activity relationship analysis showed that EGCG, its epimer GCG and a digallate-containing compound, theaflavin 3,3′ digallate (TFDG) were potent RIG-I inhibitors. EGCG also inhibited IL6 secretion and IFN- β mRNA synthesis in BEAS-2B cells, which harbors intact endogenous RIG-I signaling pathway.

Conclusions/Significance

EGCG and its derivatives could have potential therapeutic use as a modulator of RIG-I mediated immune responses.     

Innate host response in primary human hepatocytes with hepatitis C virus infection

Background and Aim

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.

Methods

An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.

Results

Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.

Conclusion

Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

Functional Characterizations of RIG-I to GCRV and Viral/Bacterial PAMPs in Grass Carp Ctenopharyngodon idella

Background

RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy.

Methodology/Principal Findings

To clarify the functional characterizations of RIG-I in grass carp Ctenopharyngodon idella (CiRIG-I), six representative overexpression plasmids were constructed and transfected into C. idella kidney (CIK) cell lines to obtain stably expressing recombinant proteins, respectively. A virus titer test and 96-well plate staining assay showed that all constructs exhibited the antiviral activity somewhat. The quantitative real-time RT-PCR (qRT-PCR) demonstrated that mRNA expressions of CiIPS-1, CiIFN-I and CiMx2 were regulated by not only virus (GCRV) or viral PAMP (poly(IC)) challenge but also bacterial PAMPs (LPS and PGN) stimulation in the steadily transfected cells. The results showed that the full-length CiRIG-I played a key role in RLR pathway. The repressor domain (RD) exerted an inhibitory function of the signaling channel under all utilized challenges. Caspase activation and recruitment domains (CARDs) showed a positive role in GCRV and poly(I:C) challenge. Helicase motifs were crucial for the signaling pathway upon LPS and PGN stimulation. Interestingly, ΔCARDs (CARDs deleted) showed postive modulation in RIG-I signal transduction.

Conclusions/Significance

The results provided some novel insights into RIG-I sensing with a strikingly broad regulation in teleosts, responding not only to the dsRNA virus or synthetic dsRNA but also bacterial PAMPs.     

Identification of Tyrosine-9 of MAVS as Critical Target for Inducible Phosphorylation That Determines Activation

Background

Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-β signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive.

Methodology/Principal Findings

Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9) is a phosphorylation site that is required for IFN-β signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property.

Conclusions/Significance

These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

IFN-λ3 inhibits HIV infection of macrophages through the JAK-STAT pathway

Background

Interferon lambda 3 (IFN-λ3) is a newly identified cytokine with antiviral activity, and its single nucleotide polymorphisms are strongly associated with the treatment effectiveness and development of chronic hepatitis C virus infection. We thus examined the potential of IFN-λ3 to inhibit HIV replication and the possible mechanisms of the anti-HIV action by IFN-λ3 in human macrophages.

Principal Findings

Under different conditions (before, during, and after HIV infection), IFN-λ3 significantly inhibited viral replication in macrophages, which was associated with the induction of multiple antiviral cellular factors (ISG56, MxA, OAS-1, A3G/F and tetherin) and IFN regulatory factors (IRF-1, 3, 5, 7 and 9). This anti-HIV action of IFN-λ3 could be compromised by the JAK-STAT inhibitor. In addition, IFN-λ3 treatment of macrophages induced the expression of toll-like receptor 3 (TLR3) and two key adaptors (MyD88 and TRIF) in type I IFN pathway activation. However, HIV infection compromised IFN-λ3-mediated induction of the key elements in JAK-STAT signaling pathway.

Conclusions

These data indicate that IFN-λ3 exerts its anti-HIV function by activating JAK-STAT pathway-mediated innate immunity in macrophages. Future in vivo studies are necessary in order to explore the potential for developing IFN-λ3-based therapy for HIV disease.     

Interferon Regulatory Factor 1 Polymorphisms Previously Associated with Reduced HIV Susceptibility Have No Effect on HIV Disease Progression

Introduction

Interferon regulatory factor 1 (IRF1) is induced by HIV early in the infection process and serves two functions: transactivation of the HIV-1 genome and thus replication, and eliciting antiviral innate immune responses. We previously described three IRF1 polymorphisms that correlate with reduced IRF1 expression and reduced HIV susceptibility.

Objective

To determine whether IRF1 polymorphisms previously associated with reduced HIV susceptibility play a role in HIV pathogenesis and disease progression in HIV-infected ART-naïve individuals.

Methods

IRF1 genotyping for polymorphisms (619, MS and 6516) was performed by PCR in 847 HIV positive participants from a sex worker cohort in Nairobi, Kenya. Rates of CD4+ T cell decline and viral loads (VL) were analyzed using linear mixed models.

Results

Three polymorphisms in the IRF1, located at 619, microsatellite region and 6516 of the gene, previously associated with decreased susceptibility to HIV infection show no effect on disease progression, either measured by HIV-1 RNA levels or the slopes of CD4 decline before treatment initiation.

Conclusion

Whereas these three polymorphisms in the IRF1 gene protect against HIV-1 acquisition, they appear to exert no discernable effects once infection is established.     

IPS-1 differentially induces TRAIL,BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners.The primary protection of the host from various pathogens is ensured by the innate immune system, which consists of families of sensors such as the Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors. These sensors recognize the diverse range of pathogens in various cellular compartments and lead to the activation of innate immunity, including the production of various cytokines that create an anti-pathogenic environment to limit the pathogen. RLRs are cytosolic sensors that recognize the viral RNA and recruit an adaptor, Interferon (IFN)-β promoter stimulator-1 (IPS-1), also known as CARDIF, MAVS or VISA. IPS-1, a protein that contains a caspase activation and -recruitment domain (CARD), is localized to the mitochondria for its antiviral function.^{1, 2, 3, 4} Mice lacking IPS-1 show severely impaired antiviral innate immunity.⁵ The RLRs/IPS-1 signaling axis activates a cascade of signals that predominantly induces the production of the type I IFN and pro-inflammatory cytokines through IRFs and NF-κB, respectively, to establish an antiviral state.In addition to the pivotal role that host immunity has against numerous pathogen challenges, it is crucial in immune surveillance against altered-self cells. Immune mediators such as cytokines, chemokines and type I IFN initiate a complex network of signals to induce an anti-tumor state by triggering various biochemical processes such as cell cycle arrest and apoptosis. Additionally, these immune mediators facilitate cytotoxicity to the tumor cells through the recruitment of immunocompetent cells. The cytotoxic activity is mediated through the upregulation of pro-apoptotic genes and the downregulation of anti-apoptotic genes. These changes are critical for cancer cell death.⁶ Various innate and adaptive cytokines are used for treatment of several types of cancer.^{7, 8} The type I IFN are essential for antiviral immunity and induce pleiotropic effects such as the inhibition of malignant growth and apoptosis of altered-self cells.In addition, pathogen-associated molecular patterns such as polyinosinic:polycytidylic acid (polyIC), a synthetic analog of double-stranded RNA and viruses known as oncolytic viruses such as Vesicular stomatitis virus, Newcastle disease virus (NDV) and Sendai virus induce anticancer activity.⁹ However, the molecular mechanisms for these agents are poorly understood.Here, we showed that treatment of cancer cells with polyIC transfection or NDV infection initiates RIG-I- and MDA5-dependent anticancer activity through recruitment of an adaptor, IPS-1. Using IFN α/β receptor1 (IFNAR1)-sufficient and IFNAR1-deficient cancer cells, we showed that these anticancer activities require the RLR signaling pathway. However, type I IFN are dispensable for the anticancer activity. The RLR pathway induces anticancer activity through the selective induction of cell death or apoptosis via upregulation of the pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE. These changes lead to post-translational activation of caspases −3 and −9 and PARP-1 in cancer cells. Furthermore, our study reveals that IFN regulatory factors (IRF)3 and IRF7 are indispensable for the RLR-mediated anticancer activity.