RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

Innate immune sensing of nucleic acids from pathogens

The innate immune system is important as the first line of defense to sense invading pathogens. Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response. Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures, however, several aspects of how cytosolic nucleic acids are sensed remain unclear. This special issue reviews how the host innate immune system senses nucleic acids from Brucella abortus, Mycobacterium sp and Legionella pneumophila, viral DNA, the role of STING in DNA sensing and inflammatory diseases and the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster.     

Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response

Nucleic acid–sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid–inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1^K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.     

Cell biology and immunology lessons taught by Legionella pneumophila

Legionella pneumophila is a facultative intracellular pathogen capable of replicating within a broad range of hosts. One unique feature of this pathogen is the cohort of ca. 300 virulence factors (effectors) delivered into host cells via its Dot/Icm type IV secretion system. Study of these proteins has produced novel insights into the mechanisms of host function modulation by pathogens, the regulation of essential processes of eukaryotic cells and of immunosurveillance. In this review, we will briefly discuss the roles of some of these effectors in the creation of a niche permissive for bacterial replication in phagocytes and recent advancements in the dissection of the innate immune detection mechanisms by challenging immune cells with L. pneumophila.     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N⁶-methyladenosine (m⁶A) and RSV grown in m⁶A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m⁶A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m⁶A methylation enhances innate immune responses which in turn promote adaptive immunity.     

Yersinia YopT inhibits RLH-mediated NF-κB and IRF3 signal transduction

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to block the host innate immune response. One of the effectors, YopT, is a potent cysteine protease that causes the disruption of the actin cytoskeleton to inhibit phagocytosis of the pathogen; however, its molecular mechanism and relevance to pathogenesis need further investigation. In this report, we show that RIG-I is a novel target of the YopT protein. Remarkably, YopT interacts with RIG-I and inhibits rat liver homogenate-mediated nuclear factor-κB and interferon regulatory factor-3 activation. Further studies revealed a YopT-dependent increase in the K48-polymerized ubiquitination of RIG-I. These findings suggest that YopT negatively regulates RIG-I-mediated cellular antibacterial response by targeting RIG-I.     

Uridine Composition of the Poly-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I

Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5′-triphosphate (5′-ppp) on the viral RNA in association with a 3′ poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine “core” of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5′-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5′-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5′-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.     

RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP

Triggering and propagating an intracellular innate immune response is essential for control of viral infections. RNase L is a host endoribonuclease and a pivotal component of innate immunity that cleaves viral and cellular RNA within single-stranded loops releasing small structured RNAs with 5′-hydroxyl (5′-OH) and 3′-monophosphoryl (3′-p) groups. In 2007, we reported that RNase L cleaves self RNA to produce small RNAs that function as pathogen-associated molecular patterns (PAMPs). However, the precise sequence and structure of PAMP RNAs produced by RNase L is unknown. Here we used hepatitis C virus RNA as substrate to characterize RNase L mediated cleavage products [named suppressor of virus RNA (svRNA)] for their ability to activate RIG-I like receptors (RLR). The NS5B region of HCV RNA was cleaved by RNase L to release an svRNA that bound to RIG-I, displacing its repressor domain and stimulating its ATPase activity while signaling to the IFN-β gene in intact cells. All three of these RIG-I functions were dependent on the presence in svRNA of the 3′-p. Furthermore, svRNA suppressed HCV replication in vitro through a mechanism involving IFN production and triggered a RIG-I-dependent hepatic innate immune response in mice. RNase L and OAS (required for its activation) were both expressed in hepatocytes from HCV-infected patients, raising the possibility that the OAS/RNase L pathway might suppress HCV replication in vivo. It is proposed that RNase L mediated cleavage of HCV RNA generates svRNA that activates RIG-I, thus propagating innate immune signaling to the IFN-β gene.     

RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.     

Interplay between influenza A virus and the innate immune signaling

Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response. In this article we will focus on new developments and discoveries concerning the interaction of IAV with the cellular innate immune signaling. We discuss new mechanisms of interference of IAV with the pathogen recognition receptor RIG-I and the type I IFN antagonist NS1 in the background of already known and established concepts. Further we summarize progress related to recently identified IFN induced proteins and the role of RNA interference in the context of IAV infection.     

Identification and Structural Characterization of a Legionella Phosphoinositide Phosphatase

Bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires'' disease, which is associated with intracellular replication of the bacteria in macrophages of human innate immune system. Recent studies indicate that pathogenic bacteria can subvert host cell phosphoinositide (PI) metabolism by translocated virulence effectors. However, in which manner Legionella actively exploits PI lipids to benefit its infection is not well characterized. Here we report that L. pneumophila encodes an effector protein, named SidP, that functions as a PI-3-phosphatase specifically hydrolyzing PI(3)P and PI(3,5)P₂ in vitro. This activity of SidP rescues the growth phenotype of a yeast strain defective in PI(3)P phosphatase activity. Crystal structure of SidP orthologue from Legionella longbeachae reveals that this unique PI-3-phosphatase is composed of three distinct domains: a large catalytic domain, an appendage domain that is inserted into the N-terminal portion of the catalytic domain, and a C-terminal α-helical domain. SidP has a small catalytic pocket that presumably provides substrate specificity by limiting the accessibility of bulky PIs with multiple phosphate groups. Together, our identification of a unique family of Legionella PI phosphatases highlights a common scheme of exploiting host PI lipids in many intracellular bacterial pathogen infections.     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.