Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.
Human metapneumovirus (hMPV) is a recently discovered pathogen causing a significant portion of respiratory infections in young infants, the elderly and immunocompromised patients. Very little is known regarding the cellular signaling elicited by this virus in airway epithelial cells, the target of hMPV infection. In this study, we investigated the role of the RNA helicases RIG-I (retinoic acid inducible gene-I) and MDA-5 (melanoma differentiation-associated gene-5) as the main pattern recognition receptors (PRRs) involved in viral detection and subsequent expression of proinflammatory and antiviral genes. HMPV infection readily induced RIG-I and MDA-5 gene and protein expression in A549 cells, a type II-like alveolar epithelial cell line. Expression of dominant negative (DN) RIG-I or downregulation of RIG-I gene expression using small interfering (si)RNA significantly decreased hMPV-induced Interferon (IFN)-β, Interleukin (IL)-8, and RANTES gene transcription, by inhibiting viral-induced activation of Nuclear Factor (NF)-kB and Interferon Regulatory Factor (IRF), leading to enhanced viral replication. On the other hand, MDA-5 did not seem to play a significant role in hMPV-induced cellular responses. MAVS (mitochondrial antiviral signaling protein), an adaptor protein linking both RIG-I and MDA-5 to downstream activation of IRF-3 and NF-kB, was also necessary for hMPV-induced cellular signaling. Expression of a MAVS DN significantly reduced IFN-β and chemokine gene transcription, by inhibiting NF-kB and IRF-dependent gene transcription, in response to hMPV infection. Our results show that hMPV activates the RIG-I-MAVS signaling pathway in airway epithelial cells, leading to the expression of important proinflammatory and antiviral molecules involved in the innate immune response to viruses.
Measles virus (MV), a member of the family Paramyxoviridae, is a nonsegmented negative-strand RNA virus. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are differentially involved in the detection of cytoplasmic viral RNAs and induction of alpha/beta interferon (IFN-α/β). RIG-I is generally believed to play a major role in the recognition of paramyxoviruses, whereas many viruses of this family produce V proteins that can inhibit MDA5. To determine the individual roles of MDA5 and RIG-I in IFN induction after MV infection, small interfering RNA-mediated knockdown of MDA5 or RIG-I was performed in the human epithelial cell line H358, which is susceptible to wild-type MV isolates. The production of IFN-β mRNA in response to MV infection was greatly reduced in RIG-I knockdown clones compared to that in H358 cells, confirming the importance of RIG-I in the detection of MV. The IFN-β mRNA levels were also moderately reduced in MDA5 knockdown clones, even though these clones retained fully functional RIG-I. A V protein-deficient recombinant MV (MVΔV) induced higher amounts of IFN-β mRNA at the early stage of infection in H358 cells compared to the parental virus. The reductions in the IFN-β mRNA levels in RIG-I knockdown clones were less pronounced after infection with MVΔV than after infection with the parental virus. Taken together, the present results indicate that RIG-I and MDA5 both contribute to the recognition of MV and that the V protein promotes MV growth at least partly by inhibiting the MDA5-mediated IFN responses.
RIG-I is important in recognizing viral RNA and stimulating the immune system. Human RIG-I protein was crystallized in complex with 18-mer double-stranded RNA to study the recognition mechanism.
Retinoic acid inducible gene-I (RIG-I) is an essential component of the innate immune system that is responsible for the detection and elimination of invading viruses. RIG-I recognizes viral RNAs inside the cell and then initiates downstream signalling to activate the IRF-3 and NF-κB genes, which results in the production of type I interferons. RIG-I is composed of an N-terminal CARD domain for signalling and C-terminal helicase and repressor domains for RNA recognition. A RIG-I–RNA binding assay was performed to investigate the in vitro RIG-I–RNA binding properties. Selenomethionine-incorporated RIG-I was expressed using Escherichia coli and purified for crystallization. X-ray data were collected from RIG-I–dsRNA complex crystals to 2.8 Å resolution using synchrotron radiation.
retinoic acid inducible gene-I
Retinoic acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) responsible for detection of nucleic acids from pathogens in the cytoplasm of infected cells and induction of type I interferon (IFN). RIG-I-specific pathogen associated molecular patterns (PAMPs) are characterized by RNA molecules with a 5′-triphosphate (5′-ppp) group and partial double-stranded composition. Although many RNA molecules capable of activating RIG-I have been described, the exact nature of viral RNAs that are responsible for triggering RIG-I activity during the course of an infection has not been extensively explored and the specificity of RIG-I for various viral RNA molecules remains largely unknown. By examining endogenous RIG-I/RNA complexes in influenza virus- and Sendai virus-infected cells we were able to identify viral RNA molecules that specifically associated with RIG-I during infection. We showed that in Sendai virus-infected cells, RIG-I specifically and preferentially associated with the copy-back defective interfering (DI) particle RNA and not with the full-length Sendai virus genome or Sendai virus encoded mRNAs. In influenza virus-infected cells RIG-I also preferentially associated with DI RNAs as well as with the shorter genomic segments.
RIG-I; Sendai; influenza; PAMP; PRR
Virus infection triggers interferon (IFN)-mediated innate immune defenses in part through viral nucleic acid interactions. However, the immune recognition mechanisms by which the host identifies incoming DNA viruses are still elusive. Here, we show that increased levels of Kaposi's sarcoma-associated herpesvirus (KSHV) persistency are observed in retinoic acid-inducible gene I (RIG-I)-deficient cells and that KSHV ORF64, a tegument protein with deubiqutinase (DUB) activity, suppresses RIG-I-mediated IFN signaling by reducing the ubiquitination of RIG-I, crucial for its activation. This study suggests that RIG-I plays a potential role in sensing KSHV infection and that KSHV ORF64 DUB counteracts RIG-I signaling.
Human metapneumovirus (hMPV) is a respiratory paramyxovirus of global clinical relevance. Despite the substantial knowledge generated during the last 10 years about hMPV infection, information regarding the activation of the immune response against this virus remains largely unknown. In this study, we demonstrated that the helicase melanoma differentiation-associated gene 5 (MDA5) is essential to induce the interferon response after hMPV infection in human and mouse dendritic cells as well as in an experimental mouse model of infection. Our findings in vitro and in vivo showed that MDA5 is required for the expression and activation of interferon (IFN) regulatory factors (IRFs). hMPV infection induces activation of IRF-3, and it regulates the expression of IRF-7. However, both IRF-3 and IRF-7 are critical for the production of type I and type III IFNs. In addition, our in vivo studies in hMPV-infected mice indicated that MDA5 alters viral clearance, enhances disease severity and pulmonary inflammation, and regulates the production of cytokines and chemokines in response to hMPV. These findings are relevant for a better understanding of the pathogenesis of hMPV infection.
Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I “knockdown” significantly inhibited early nuclear factor-κB (NF-κB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-β), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-κB binding but did inhibit IFN-β, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-κB/RelA transcription by its ability to block the activating phosphorylation of NF-κB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-β secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-κB activation.
Recognition of pathogens by the innate immune system is mediated by pattern recognition receptors (PRRs), which recognize specific molecular structures of the infectious agents and subsequently trigger expression of genes involved in host defense. Toll-like receptors (TLRs) represent a well-characterized class of membrane-bound PRRs, and the RNA helicase retinoic acid inducible gene I (RIG-I) has recently been described as a novel cytoplasmic PRR recognizing double-stranded RNA (dsRNA). Here we show that activation of signal transduction and induction of cytokine expression by the paramyxovirus Sendai virus is dependent on virus replication and involves PRRs in a cell-type-dependent manner. While nonimmune cells relied entirely on recognition of dsRNA through RIG-I for activation of an antiviral response, myeloid cells utilized both the single-stranded RNA sensing TLR7 and TLR8 and dsRNA-dependent mechanisms independent of RIG-I, TLR3, and dsRNA-activated protein kinase R to trigger this response. Therefore, there appears to be a large degree of cell-type specificity in the mechanisms used by the host to recognize infecting viruses.
Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K63-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I's ability to induce an antiviral IFN response, phosphorylation of RIG-I at S8 or T170 suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S8 and T170 phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-α (PKC-α) and PKC-β as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S8 or T170 potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-α/β are the primary kinases responsible for RIG-I S8 and T170 phosphorylation. Coimmunoprecipitation showed that PKC-α/β interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-α/β double-knockdown cells exhibited markedly decreased S8/T170 phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/β-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.
Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.
The cytosolic retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that senses HCV double-stranded RNA and triggers type I interferon pathways. The clone Huh7.5 of human hepatoma Huh7 cells contains a mutation in RIG-I that is believed to be responsible for the improved replication of HCV in these cells relative to the parental strain. We hypothesized that, in addition to RIG-I, other determinant(s) outside the RIG-I coding sequence are involved in limiting HCV replication in cell culture. To test our hypothesis, we analyzed Huh7 cell clones that support the efficient replication of HCV and analyzed the RIG-I gene.
One clone, termed Huh7D, was more permissive for HCV replication and more efficient for HCV-neomycin and HCV-hygromycin based replicon colony formation than parental Huh7 cells. Nucleotide sequence analysis of the RIG-I mRNA coding region from Huh7D cells showed no mutations relative to Huh7 parental cells.
We derived a new Huh7 cell line, Huh7D, which is more permissive for HCV replication than parental Huh7 cells. The higher permissiveness of Huh7D cells is not due to mutations in the RIG-I protein, indicating that cellular determinants other than the RIG-I amino-acid sequence are responsible for controlling HCV replication. In addition, we have selected Huh7 cells resistant to hygromycin via newly generated HCV-replicons carrying the hygromycin resistant gene. Further studies on Huh7D cells will allow the identification of cellular factors that increased the susceptibility to HCV infection, which could be targeted for anti-HCV therapies.
Antiviral innate immunity is triggered by sensing viral nucleic acids. RIG-I (retinoic acid-inducible gene-I) is an intracellular molecule that responds to viral nucleic acids and activates downstream signaling, resulting in the induction of members of the type I interferon (IFN) family, which are regarded among the most important effectors of the innate immune system. Although RIG-I is expressed ubiquitously in the cytoplasm, its levels are subject to transcriptional and post-transcriptional regulation. RIG-I belongs to the IFN-stimulated gene (ISG) family, but certain cells regulate its expression through IFN-independent mechanisms. Several lines of evidence indicate that deregulated RIG-I signaling is associated with autoimmune disorders. Further studies suggest that RIG-I has functions in addition to those directly related to its role in RNA sensing and host defense. We have much to learn and discover regarding this interesting cytoplasmic sensor so that we can capitalize on its properties for the treatment of viral infections, immune disorders, cancer, and perhaps other conditions.
retinoic acid-inducible gene-I; innate immunity; RNA virus
The ribonucleic acid (RNA) helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) recognize distinct viral and synthetic RNAs, leading to the production of interferons. Although 5′-triphosphate single-stranded RNA is a RIG-I ligand, the role of RIG-I and MDA5 in double-stranded (ds) RNA recognition remains to be characterized. In this study, we show that the length of dsRNA is important for differential recognition by RIG-I and MDA5. The MDA5 ligand, polyinosinic-polycytidylic acid, was converted to a RIG-I ligand after shortening of the dsRNA length. In addition, viral dsRNAs differentially activated RIG-I and MDA5, depending on their length. Vesicular stomatitis virus infection generated dsRNA, which is responsible for RIG-I–mediated recognition. Collectively, RIG-I detects dsRNAs without a 5′-triphosphate end, and RIG-I and MDA5 selectively recognize short and long dsRNAs, respectively.
Innate immune defenses are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs) 1. Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people 2. Infection is governed by hepatic immune defenses triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals IRF-3 activation to induce the expression of α/β interferon (IFN) and antiviral/interferon-stimulated genes (ISGs) that limit infection 3–10. Here we identified the poly-uridine motif of the HCV genome 3’ nontranslated region (NTR) as the PAMP substrate of RIG-I, and show that this and similar homopoly-uridine motifs present in the genome of RNA viruses is the chief feature of RIG-I recognition and immune triggering 8. 5’ terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent upon homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signaling to induce a hepatic innate immune response in vivo, and triggered IFN and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by identifying homopoly-uridine motfis present in the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and define immunogenic features of the PAMP/RIG-I interaction that could be utilized as an immune adjuvant for vaccine and immunotherapy approaches.
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.
The nonstructural proteins 1 (NS1) from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN) response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I) signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-β promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-β promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism.
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.
The innate immune system is responsible for recognizing invading pathogens and initiating a protective response. In particular, the retinoic acid-inducible gene 1 protein (RIG-I) participates in the recognition of single- and double-stranded RNA viruses. RIG-I activation leads to the production of an appropriate cytokine and chemokine cocktail that stimulates an antiviral state and drives the adaptive immune system toward an efficient and specific response against the ongoing infection. One of the best-characterized natural RIG-I agonists is the defective interfering (DI) RNA produced by Sendai virus strain Cantell. This 546-nucleotide RNA is a well-known activator of the innate immune system and an extremely potent inducer of type I interferon. We designed an in vitro-transcribed RNA that retains the type I interferon stimulatory properties, and the RIG-I affinity of the Sendai virus produced DI RNA both in vitro and in vivo. This in vitro-synthesized RNA is capable of enhancing the production of anti-influenza virus hemagglutinin (HA)-specific IgG after intramuscular or intranasal coadministration with inactivated H1N1 2009 pandemic vaccine. Furthermore, our adjuvant is equally effective at increasing the efficiency of an influenza A/Puerto Rico/8/34 virus inactivated vaccine as a poly(I·C)- or a squalene-based adjuvant. Our in vitro-transcribed DI RNA represents an excellent tool for the study of RIG-I agonists as vaccine adjuvants and a starting point in the development of such a vaccine.
Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.
Dengue fever, dengue haemmorhagic fever and dengue shock syndrome, which are caused by dengue virus infection, are a major public health problem in many parts of the world, especially South East Asia. The investigation of host cell transcriptional changes in response to virus infection using DNA microarray technology has been an area of great interest. In our previous study, we used microarray technology to study expression of individual human genes in relation to dengue virus infection. Most of the genes that were upregulated were type 1 interferon related genes. To gain a better understanding of the innate immune response to dengue virus, we knocked down RIG-I, MDA5 and TLR3 genes in HUH-7 cells. Silencing these genes using siRNA technology resulted in significant increase in viral replication. This increase in viral load induced ER stress leading to apoptosis. This study demonstrates a synergistic role for RIG-I, MDA5 and TLR3 in restricting dengue virus infection.
Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells are potent antigen presenting cells and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived dendritic cells (moDCs), as downregulation of its expression by siRNA significantly blocked hMPV-induced chemokine and type I interferon expression. Similar results were found in bone marrow derived-dendritic cells (BM-DCs) from TLR4 deficient mice. MoDCs infected with a virus lacking G protein expression (rhMPV-ΔG) produced higher levels of cytokines and chemokines, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as rhMPV-ΔG infection of moDCs inhibited LPS-induced production of cytokine and chemokines significantly less than rhMPV-WT, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.
Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-ΔG) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-ΔG enhanced activation of transcription factors belonging to the nuclear factor (NF)-κB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-ΔG also increased IRF- and NF-κB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor.
Human metapneumovirus (hMPV), a member of the Paramyxoviridae family, is an important cause of respiratory morbidity throughout life. The contribution of viral-specific proteins to the pathogenesis of hMPV infection and immune evasion is largely unknown. Previous work has suggested that the glycoprotein G of hMPV is not necessary for the process of viral fusion and attachment to host cells, and a recombinant hMPV lacking the G protein (rhMPV-ΔG) shows an attenuated phenotype in the respiratory tract of animal models of infection. Airway epithelial cells, a major component of the innate immune system, are a primary target of hMPV infection. In this study, we show that hMPV G protein functions as a major inhibitory factor of the host antiviral response by blocking production of inducible chemokines and IFN-α/β. A major finding of this work is the demonstration that hMPV G protein interacts with RIG-I, a cytoplasmic viral sensor. As result, hMPV G protein inhibits RIG-I-dependent signaling pathways, including activation of NF-κB and IRF-3, two transcription factors necessary for the synthesis of inflammatory and antiviral cytokines. Understanding the function of hMPV proteins is critical for the future design of effective antiviral therapies and rationale design of vaccine candidates.
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family, which includes several major human and animal pathogens. Epidemiological studies indicate that hMPV is a significant human respiratory pathogen with worldwide distribution. It is associated with respiratory illnesses in children, adults, and immunocompromised patients, ranging from upper respiratory tract infections to severe bronchiolitis and pneumonia. Interferon (IFN) represents a major line of defense against virus infection, and in response, viruses have evolved countermeasures to inhibit IFN production as well as IFN signaling. Although the strategies of IFN evasion are similar, the specific mechanisms by which paramyxoviruses inhibit IFN responses are quite diverse. In this review, we will present an overview of the strategies that hMPV uses to subvert cellular signaling in airway epithelial cells, the major target of infection, as well as in primary immune cells.
metapneumovirus; viral proteins; innate immune system; interferon antagonism
IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.
Vitamin A can significantly decrease measles-associated morbidity and mortality. Vitamin A can inhibit the replication of measles virus (MeV) in vitro through an RARα- and type I interferon (IFN)-dependent mechanism. Retinoid-induced gene I (RIG-I) expression is induced by retinoids, activated by MeV RNA and is important for IFN signaling. We hypothesized that RIG-I is central to retinoid-mediated inhibition of MeV in vitro. We demonstrate that RIG-I expression is increased in cells treated with retinoids and infected with MeV. The central role of RIG-I in the retinoid-anti-MeV effect was demonstrated in the Huh-7/7.5 model; the latter cells having non-functional RIG-I. RAR-dependent retinoid signaling was required for the induction of RIG-I by retinoids and MeV. Retinoid signaling was also found to act in combination with IFN to induce high levels of RIG-I expression. RIG-I promoter activation required both retinoids and MeV, as indicated by markers of active chromatin. IRF-1 is known to be regulated by retinoids and MeV, but we found recruitment of IRF-1 to the RIG-I promoter by retinoids alone. Using luciferase expression constructs, we further demonstrated that the IRF-1 response element of RIG-I was required for RIG-I activation by retinoids or IFN. These results reveal that retinoid treatment and MeV infection induces significant RIG-I. RIG-I is required for the retinoid-MeV antiviral response. The induction is dependent on IFN, retinoids and IRF-1.