Activation and regulation of pathogen sensor RIG-I

Cells are equipped with a large set of pattern recognition receptors or sensors that detect foreign molecules such as pathogenic nucleic acids and initiate proinflammatory and antimicrobial innate immune responses. RIG-I is a cytosolic sensor that detects 5′-triphosphate double-stranded RNAs produced during infection. RIG-I is responsible for mounting an antimicrobial response against a variety of viruses and intracellular bacteria. RIG-I contains an intricate structural architecture that allows for efficient signaling downstream in the pathway and autoregulation. The RIG-I-mediated antimicrobial pathway is highly regulated in cells requiring various cofactors, negative regulators, and posttranslational modifications. Modulation of RIG-I and RIG-I-mediated signaling in cells by pathogens to evade recognition and activation of the antimicrobial pathway highlights the essential nature of RIG-I in the innate immune response.     

Neutrophils express distinct RNA receptors in a non-canonical way

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.     

SUMOylation of RIG-I positively regulates the type I interferon signaling

Retinoic acid-inducible gene-I (RIG-I) functions as an intracellular pattern recognition receptor (PRR) that recognizes the 5’-triphosphate moiety of single-stranded RNA viruses to initiate the innate immune response. Previous studies have shown that Lys63-linked ubiquitylation is required for RIG-I activation and the downstream anti-viral type I interferon (IFN-I) induction. Herein we reported that, RIG-I was also modified by small ubiquitin-like modifier-1 (SUMO-1). Functional analysis showed that RIG-I SUMOylation enhanced IFN-I production through increased ubiquitylation and the interaction with its downstream adaptor molecule Cardif. Our results therefore suggested that SUMOylation might serve as an additional regulatory tier for RIG-I activation and IFN-I signaling.     

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2_∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2_∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2_∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.     

Mouse nucleolin binds to 4.5S RNAh, a small noncoding RNA

4.5S RNAh is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAh is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAh-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAhin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAh-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAh recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAh was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.     

Mechanism of STAT3 activation by insulin-like growth factor I receptor

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.     

A structure-based model of RIG-I activation

A series of high-resolution crystal structures of RIG-I and RIG-I:dsRNA cocrystals has recently been reported. Comparison of these structures provides considerable insight into how this innate immune pattern recognition receptor is activated upon detecting and binding a certain class of viral RNAs.     

Avian influenza virus H5N1 induces rapid interferon-beta production but shows more potent inhibition to retinoic acid-inducible gene I expression than H1N1 in vitro

ABSTRACT: BACKGROUND: The mechanisms through which the avian influenza virus H5N1 modulate the host's innate immune defense during invasion, remains incompletely understood. RIG-I as a pattern recognition receptor plays an important role in mediating innate immune response induced by influenza virus. So, modulating RIG-I might be adopted as a strategy by influenza virus to antagonize the host's innate immune defense. Methods: Here we chose an avian influenza virus A/tree sparrow/Henan/1/04 (H5N1) directly isolated from a free-living tree sparrow in Mainland China which is amplified in egg allantoic cavity, and researched its interferon induction and manipulation of RIG-I expression compared with influenza virus A/WSN/1933(H1N1), a well charaterized mouse adapted strain, in human lung epithelial A549 cells and human embryonic kidney 293T cells. Results: Although the avian influenza virus H5N1 infection initiated a rapid IFN-beta production early on, it eventually presented a more potent inhibition to IFN-beta production than H1N1. Correspondingly, the H5N1 infection induced low level expression of endogenous RIG-I, an Interferon Stimulating Gene (ISG), and showed more potent inhibition to the expression of endogenous RIG-I triggered by exogenous interferon than H1N1. Conclusions: Manipulating endogenous RIG-I expression might constitute one of the mechanisms through which avian influenza virus H5N1 control the host's innate immune response during infection.     

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay

Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.     

Adenovirus virus-associated RNAs induce type I interferon expression through a RIG-I-mediated pathway

The current study demonstrates that adenovirus virus-associated RNA (VA) is recognized by retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor, and activates RIG-I downstream signaling, leading to the induction of type I interferons (IFNs), similarly to Epstein-Barr virus-encoded small RNA. Further analysis revealed that adenovirus infection leads to biphasic type I IFN induction at 12 to 24 h and 48 to 60 h postinfection. The later induction coincided with VA expression and was reduced by virus UV inactivation or RIG-I silencing. These results suggest that VA-mediated RIG-I activation is involved in activating innate immune responses during adenovirus infection.     

Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis

The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.     

RIG-I mediates innate immune response in mouse neurons following Japanese encephalitis virus infection

Background

Neuroinflammation associated with Japanese encephalitis (JE) is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV) infections is yet to be elucidated.

Methodology/Principal Findings

Upon infecting murine neuroblastoma cells and primary cortical neurons with JEV the expression profile of key proinflammatory cyto/chemokines were analyzed by qRT-PCR and bead array, both before and after ablation of RIG-I. Immunoblotting was performed to evaluate the levels of key molecules downstream to RIG-I leading to production of proinflammatory mediators. Changes in the intracellular viral antigen expression were confirmed by intracellular staining and immunoblotting. JEV infection induced neuronal expression of IL-6, IL-12p70, MCP-1, IP-10 and TNF-α in a time-dependent manner, which showed significant reduction upon RIG-I ablation. Molecules downstream to RIG-I showed significant changes upon JEV-infection, that were modulated following RIG-I ablation. Ablation of RIG-I in neurons also increased their susceptibility to JEV.

Conclusions/Significance

In this study we propose that neurons are one of the potential sources of proinflammatory cyto/chemokines in JEV-infected brain that are produced via RIG-I dependent pathways. Ablation of RIG-I in neurons leads to increased viral load and reduced release of the cyto/chemokines.