RIG-I-like Helicase Research Articles

PINK1 Inhibits Multimeric Aggregation and Signaling of MAVS and MAVS-Dependent Lung Pathology.

Mitochondria have emerged as important signaling organelles where intracellular perturbations are integrated and, consequently, intracellular signaling pathways are modulated to execute appropriate cellular functions. MAVS (mitochondrial antiviral signaling protein) represents such an example that functions as a platform molecule to mediate mitochondrial innate immune signaling. Recently, multimeric aggregation of MAVS has been identified as a key molecular process for its signaling. The underlying mechanisms to regulate this, however, are still incompletely understood. We hypothesized that PINK1 (PTEN-induced kinase 1) plays an important role in the regulation of multimeric MAVS aggregation and its consequent pathobiology. To test whether PINK1 interacts with MAVS, bimolecular fluorescence complementation analysis and IP were performed. RLH (RIG-I-like helicase) and NLRP3 inflammasome signaling were evaluated by in vitro assay. In vivo functional significance of PINK1 in the regulation of MAVS signaling was evaluated from both murine modeling of influenza viral infection and bleomycin-induced experimental pulmonary fibrosis, wherein MAVS plays important roles. Multimeric MAVS aggregation was induced by mitochondria dysfunction, and, during this event, the stabilized PINK1 interacted physically with MAVS and antagonized multimeric MAVS aggregation. Accordingly, the MAVS-mediated antiviral innate immune and NLRP3 inflammasome signaling were enhanced in PINK1 deficiency. In addition, in vivo studies revealed that MAVS-mediated pulmonary antiviral innate immune responses and fibrotic responses after bleomycin injury were enhanced in PINK1 deficiency. In conclusion, these results establish a new role of PINK1 in the regulation of MAVS signaling and the consequent pulmonary pathobiology.

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway

Mechanisms of hepatitis B virus (HBV) reactivation after hepatitis C virus (HCV) elimination by direct-acting antiviral (DAA) treatment in HBV/HCV-co-infected patients remain unclear. We examined RIG-I-like helicase (RLH) pathway activation by HBV mono-infection, HCV mono-infection or HBV/HCV co-infection and interference between HBV and HCV in primary human hepatocytes. Interference between HBV and HCV and HBV reactivation after DAA treatment in humanized-liver mice were assessed. HCV infection activated RLH pathway, as evidenced by RIG-I, ISG15 and ISG56 expression induction; HBV caused only RIG-I induction in vitro. RLH activation was also found in HBV/HCV-co-infected cells, and HBV replication were suppressed in HBV/HCV-co-infected than in HBV-mono-infected cells. siRNA-mediated double knockdown of ISG15 and ISG56 increased HBV replication in HBV/HCV-co-infected cells. HCV infection activated RLH pathway and suppressed HBV replication in humanized-liver mice. Subsequent elimination of HCV by DAA administration downregulated RLH pathway and upregulated HBV replication in mice. RLH pathway was activated in livers of chronic hepatitis C patients compared to those of chronic hepatitis B or non-B, non-C patients. The RLH pathway activation was downregulated by HCV elimination. In conclusion, HCV infection activated RLH pathway and suppressed HBV replication in human hepatocytes. HCV elimination upregulated HBV replication, probably through RLH pathway downregulation.

ID: 205: A new RNA-based adjuvant enhances virus-specific vaccine responses by locally activating DC and inducing IFN-I responses TLR- and RLH-dependently

Among new adjuvants conferring a TH1-shift, RNAdjuvant® consisting of non-coding RNA complexed with a cationic carrier peptide is a promising candidate. Upon RNAdjuvant® treatment, bone marrow-derived myeloid dendritic cells (BM-mDC) and plasmacytoid DC (BM-pDC) showed Toll-like receptor (TLR)7-dependent IFN-I induction and upregulation of CD69 and CD86. This result was verified by the ex vivo analysis of splenic DC. Immunization of mice with seasonal influenza vaccines plus RNAdjuvant® significantly enhanced vaccine-induced influenza-specific IgG responses. In particular, IgG2b and IgG2c antibody subclasses were elevated, indicating a TH1-biased immune response. RNAdjuvant® mediated enhancement of antibody responses was conferred by concomitant TLR- and RIG-I-like helicase (RLH)-dependent signaling. Thus, upon administration of RNAdjuvant® two different signaling pathways were induced, explaining the strong immunoenhancing effect of RNAdjuvant®. Notably, upon intramuscular (i.m.) injection of RNAdjuvant® IFN-I induction and CD69 upregulation on DC were only detectable in draining, but not in non-draining lymph nodes. Furthermore, transient lymphopenia as a sensitive readout of serum cytokine responses was only detected upon intravenous (i.v.), but not upon i.m., injection of RNAdjuvant®. These observations indicated that i.m. administration of RNAdjuvant® induced primarily local effects. In conclusion, RNAdjuvant® is a novel efficacious immune enhancer that augments vaccine-induced TH1 responses in a MyD88- and Cardif-dependent manner by triggering strictly local effects. Thus, RNAdjuvant® holds promise to be a particularly well tolerable new adjuvant also in humans.

M27 expressed by cytomegalovirus counteracts effective type I interferon induction of myeloid cells but not of plasmacytoid dendritic cells.

In healthy individuals, the functional immune system effectively confines human cytomegalovirus (CMV) replication, while viral immune evasion and persistence preclude sterile immunity. Mouse CMV (MCMV) is a well-established model to study the delicate CMV-host balance. Effective control of MCMV infection depends on the induction of protective type I interferon (IFN-I) responses. Nevertheless, it is unclear whether in professional antigen-presenting cell subsets MCMV-encoded evasins inhibit the induction of IFN-I responses. Upon MCMV treatment, enhanced expression of MCMV immediate-early and early proteins was detected in bone marrow cultures of macrophages and myeloid dendritic cells compared with plasmacytoid dendritic cell cultures, whereas plasmacytoid dendritic cells mounted more vigorous IFN-I responses. Experiments with Toll-like receptor (TLR)- and/or RIG-I like helicase (RLH)-deficient cell subsets revealed that upon MCMV treatment of myeloid cells, IFN-I responses were triggered independently of TLR and RLH signaling, whereas in plasmacytoid dendritic cells, IFN-I induction was strictly TLR dependent. Macrophages and myeloid dendritic cells treated with either UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist M27 mounted significantly higher IFN-I responses than cells treated with live wild-type MCMV. In contrast, plasmacytoid dendritic cells responded similarly to UV-inactivated and live MCMV. These experiments illustrated that M27 not only inhibited IFN-I-mediated receptor signaling, but also evaded the induction of IFN responses in myeloid dendritic cells. Furthermore, we found that additional MCMV-encoded evasins were needed to efficiently shut off IFN-I responses of macrophages, but not of myeloid dendritic cells, thus further elucidating the subtle adjustment of the host-pathogen balance. MCMV may induce IFN-I responses in fibroblasts and epithelial cells, as well as in antigen-presenting cell subsets. We focused on the analysis of IFN-I responses of antigen-presenting cell subsets, including plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, which are all triggered by MCMV to mount IFN-I responses. Interestingly, myeloid dendritic cells and macrophages, but not plasmacytoid dendritic cells, are readily MCMV infected and support viral gene expression. As expected from previous studies, plasmacytoid dendritic cells sense MCMV Toll-like receptor 9 (TLR9) dependently, whereas in myeloid cells, IFN-I induction is entirely TLR and RLH independent. MCMV-encoded M27 does not impair the IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells, it reduces IFN-I responses. In macrophages, M27 plus other, not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV has evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.

77: Kinetics of Type I interferon responses determine either establishment of antiviral memory or no induction of adaptive immunity

Many pathogens trigger type I interferon (IFN-I) responses early after infection that confer protection until adaptive immunity is induced. Upon infection with vesicular stomatitis virus (VSV) Toll-like receptor (TLR) and RIG-I-like helicase (RLH) signaling platforms are indispensable for the induction of protective IFN-I. Hence, TLR- and RLH-ablated MyTrCa −/− mice challenged with VSV do not mount IFN-I responses and are inevitably susceptible to lethal VSV infection. To study the impact of the kinetics of IFN-I responses on the induction of adaptive immunity, we infected MyTrCa −/− mice with VSV and treated them 4, 8, 16, and 24 h after infection with recombinant IFN- α (rIFN- α ) . Interestingly, under such conditions MyTrCa −/− mice mounted normal adaptive immune responses and approximately 76% of the mice survived. On the contrary, initiation of the rIFN- α treatment scheme 4 h before VSV infection significantly prolonged survival, whereas reduced VSV-specific cytotoxic T-lymphocytes and basically no VSV neutralizing antibody responses were induced and 100% of the mice finally died. Long-term rIFN- α treatment for 9 days initiated 4 h after VSV infection promoted 100% survival of MyTrCa −/− mice and protective immunity was induced normally as verified by 100% of the animals surviving even a VSV re-challenge 3 weeks after discontinuation of the rIFN- α treatment. Initiation of the 9 days rIFN- α regimen 4 h prior to infection also rescued 100% of the mice, however, no adaptive immunity developed and 100% of the animals succumbed to re-challenge. In conclusion, long-term rIFN- α treatment initiated before virus infection prevented induction of protective adaptive immunity, whereas initiation of rIFN- α treatment hours after infection supported development of normal adaptive immunity and protective memory response. These observations have implications for immunotherapies and vaccination strategies, in particular for patients under long-term IFN-I therapy who are treated with live attenuated vaccines.

Independent of plasmacytoid dendritic cell (pDC) infection, pDC triggered by virus-infected cells mount enhanced type I IFN responses of different composition as opposed to pDC stimulated with free virus.

Upon treatment with vesicular stomatitis virus (VSV) particles, plasmacytoid dendritic cells (pDC) are triggered to mount substantial type I IFN responses, whereas myeloid DC (mDC) are only minor producers. Interestingly, bone marrow-derived (BM-)mDC were more vulnerable to infection with enhanced GFP (eGFP)-expressing VSV (VSVeGFP) than BM-pDC. BM-pDC stimulated with wild-type VSV mounted TLR-dependent IFN responses that were independent of RIG-I-like helicase (RLH) signaling. In contrast, in BM-pDC the VSV variant M2 induced particularly high IFN responses triggered in a TLR- and RLH-dependent manner, whereas BM-mDC stimulation was solely RLH-dependent. Importantly, VSVeGFP treatment of BM-pDC derived from IFN-β yellow fluorescent protein (YFP) reporter mice (messenger of IFN-β) resulted in YFP(+) and eGFP(+) single-positive cells, whereas among messenger of IFN-β-BM-mDC most YFP(+) cells were also eGFP(+). This observation indicated that unlike mDC, direct virus infection was not required to trigger IFN responses of pDC. VSV-infected BM-mDC triggered BM-pDC to mount significantly higher IFN responses than free virus particles. Stimulation with infected cells enhanced the percentages of pDC subsets expressing either IFN-β(+) or IFN-α6(+) plus IFN-β(+). Irrespective of whether stimulated with free virus or infected cells, IFN induction was dependent on autophagy of pDC, whereas autophagy of the infected mDC was dispensable. Collectively, these results indicated that productive VSV infection was needed to trigger IFN responses of mDC, but not of pDC, and that IFN responses were primarily induced by virus-infected cells that stimulated pDC in a TLR-dependent manner.

RIG-I enhanced interferon independent apoptosis upon Junin virus infection.

Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death.

39: Differential post-translational modifications regulate pathway-specific functions of IRF-3

Interferon Regulatory Factor 3 (IRF-3) is a dual-function protein involved in transcription of antiviral genes e.g. interferons, and mediating apoptosis by RIPA [RIG-I-like helicase (RLH)-activated IRF-3-mediated Pathway of Apoptosis] (Chattopadhyay et al., EMBO J 2010, J. Virol 2011, 2013, mBio 2013). Triggering of both pathways requires signal-dependent activation of IRF-3 by post-translational modifications, which also lead to its proteasomal degradation. We hypothesized that differential functional consequences of IRF-3 activation are regulated by its differential phosphorylation and ubiquitination. Accordingly, we generated specific Ser to Ala mutants that could mediate either transcription or apoptosis, but not both. To examine the role of ubiquitination on the functional consequences of IRF-3 activation, we mutated its Lys residues that are the potential targets of ubiquitin conjugation. This mutational analysis revealed differential roles of specific Lys residues in activating IRF-3 for the three functional consequences – gene transcription, RIPA or proteasomal degradation. Only two, out of fourteen, Lys residues were required for triggering RIPA, a different one was needed for transcriptional activation and two others were required for proteasomal degradation. Using the RIPA-competent minimal mutant, we established that IRF-3 was indeed ubiquitinated upon RLH activation, which led to its mitochondrial translocation and apoptosis. Moreover, our results indicated that phosphorylation of the specific Ser residue, which was required for RIPA, was also required for its ubiquitination. These results strongly suggest that residue-specific phosphorylation, followed by residue-specific ubiquitination of IRF-3, activates RIPA. Current efforts are directed to elucidating the linkage of the responsible Ub modification and identifying the corresponding modifying enzymes.

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.

The STING pathway and regulation of innate immune signaling in response to DNA pathogens

The innate immune system has evolved a variety of sensing mechanisms to detect and counter microbial invasion. These include the Toll-like receptor (TLR), cytoplasmic, nucleotide binding oligomerization domain (NOD)-like receptor and RIG-I-like helicase (RLH) pathways. However, how the cell detects pathogen-associated DNA to trigger host defense, including the production of interferon, remains to be fully clarified. Understanding these processes could have profound implications into how we understand and treat a variety of microbial-related disease, including viral-associated cancers, as well as autoimmune disorders. Recently, an endoplasmic reticulum-associated molecule referred to as STING (for stimulator of interferon genes) was isolated and shown to be critical for regulating the production of IFN in response to cytoplasmic DNA. Here, we review recent discoveries relating to the detection of foreign DNA, including the importance of the STING and inflammasome pathways and the triggering of innate signaling processes.

Thogoto Virus Infection Induces Sustained Type I Interferon Responses That Depend on RIG-I-Like Helicase Signaling of Conventional Dendritic Cells

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.

Polymorphisms in RIG-I like helicases and their influence on the course of Hepatitis C Virus infection

Hepatitis C is a major cause of liver cirrhosis, liver failure and hepatocellular carcinoma in humans. Hepatitis C Virus (HCV) infects about 3% of the human population. Interestingly the course of infection varies widely among patients. Approximately 15–20% of the infected individuals clear the infection spontaneously, whereas in 80% the infection presides in a chronic state. Virus-related factors and clinical risk factors of the host such as sex, alcohol consumption and age at infection do not allow to predict the development of a chronic status. It is therefore suspected that the susceptibility to chronic HCV infection is determined by genetic polymorphisms in the host's immune system. The RIG-I like helicase (RLH) RIG-I was recently shown to be responsible for triggering the innate immune response after infection with HCV in cell culture systems. This led us to examine single nucleotide polymorphisms (SNPs) in RIG-I and other components of the RIG-I-like helicase pathway and correlate them with the outcome after HCV-infection. We focused our analysis on non-synonymous SNPs in coding regions and found in a european population that the SNP rs3747517 in Mda-5 (leading to mutation H843R) has a significantly higher prevalence in individuals that spontaneously resolved HCV infection as compared to chronically infected patients. In addition we made expression constructs of RLHs and analysed in complementary in vitro assays the functional consequences of the mutations caused by the analysed SNPs. Together these results suggest an important role of RLHs for the clinical outcome of an HCV infection.