Cutting Edge: Influenza A virus activates TLR3-dependent inflammatory and RIG-I-dependent antiviral responses in human lung epithelial cells

Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.     

MDA-5 is cleaved in poliovirus-infected cells

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is approximately 90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.     

Regulation of protective autophagy in anoikis-resistant glioma stem cells by SDCBP/MDA-9/Syntenin

Glioblastoma multiforme (GBM) is a frequent and aggressive glial tumor, containing a small population of therapy-resistant cells, glioma stem cells (GSCs). Current dogma suggests that tumors regrow from GSCs, and these cells contribute to therapy resistance, poor prognosis, and recurrence; highlighting the importance of GSCs in glioma pathophysiology and therapeutic targeting. Macroautophagy/autophagy-based cellular homeostasis can be changed from pro-survival to pro-cell death by modulating SDCBP/MDA-9/Syntenin (syndecan binding protein)-mediated signaling. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of SDCBP/MDA-9/Syntenin. Conversely, SDCBP/MDA-9/Syntenin silencing induces autophagic death in GSCs, indicating that SDCBP/MDA-9/Syntenin regulates protective autophagy in GSCs under anoikis conditions. This process is mediated through phosphorylation of the anti-apoptotic protein BCL2 accompanied with suppression of high levels of autophagic proteins (ATG5, LAMP1, LC3B) through EGFR signaling. SDCBP/MDA-9/Syntenin-mediated regulation of BCL2 and EGFR phosphorylation is achieved through PTK2/FAK and PRKC/PKC signaling. When SDCBP/MDA-9/Syntenin is absent, this protective mechanism is deregulated, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. Our recent paper reveals a novel functional link between SDCBP/MDA-9/Syntenin expression and protective autophagy in GSCs. These new insights into SDCBP/MDA-9/Syntenin-mediated regulation and maintenance of GSCs present leads for developing innovative combinatorial cancer therapies.     

Intranasal Administration of dsRNA Analog Poly(I:C) Induces Interferon-α Receptor-Dependent Accumulation of Antigen Experienced T Cells in the Airways

Polyriboinosinic-polyribocytoidylic acid (pIC), a synthetic dsRNA, acts as an adjuvant that boosts immune responses and protection. Intranasal (IN) administration of pIC has recently been used to adjuvant influenza virus vaccines; however, the effects of IN pIC administration on pulmonary T cell responses remain unclear. Here we show that a single IN administered dose of dsRNA into mice induced local Th1 chemokine production in the lungs and airways, and generated a biphasic and sustained migration of T lymphocytes to the airways. Furthermore, IN pIC-induced chemokine production and T cell recruitment to the airways were interferon-α receptor (IFNAR) signaling dependent. The effect of dsRNA on T cell recruitment to the airways was also dependent on the presence of high molecular weight (HMW) pIC, as a low molecular weight (LMW) pIC preparation known to only interact with TLR3 did not elicit the same effect on T cell migration to the airways, suggesting that the observed effects were dependent upon dsRNA recognition by multiple pattern recognition receptors (PPRs). IN pIC was additionally capable of stimulating low levels of T cell proliferation in the draining lymph nodes approximately 4–6 days after treatment that preceded a small population of de-novo T cells found in the airways by day 10. Taken together, these results demonstrate that the adjuvant effect of IN pIC that results in enhanced T cell proliferation and sustained T cell recruitment to the airways requires multiple PRRs and IFNAR signaling.     

Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)

MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy.     

Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication

The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.     

Herpes simplex virus 1 tegument protein US11 downmodulates the RLR signaling pathway via direct interaction with RIG-I and MDA-5

The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.     

Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)

MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy.     

NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase

The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-kappaB). The mechanism by which PKR mediates activation of NF-kappaB is unknown. Here we show that in response to poly(rI). poly(rC) (pIC), PKR activates IkappaB kinase (IKK), leading to the degradation of the inhibitors IkappaBalpha and IkappaBbeta and the concomitant release of NF-kappaB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-kappaB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-alpha) and to potentiate activation of NF-kappaB by cotreatment with TNF-alpha and IFN-gamma. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKbeta or the NF-kappaB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-kappaB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-kappaB is mediated by NIK and IKK activation.     

Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C)

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.     

Suppressing ROS-TFE3-dependent autophagy enhances ivermectin-induced apoptosis in human melanoma cells

Melanoma is an aggressive skin malignancy with a high mortality rate; however, successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to its observed antitumor effects. However, the molecular mechanisms of ivermectin remain poorly understood. In the current study, we tested the involvement of autophagy in the ivermectin mechanism of action in human melanoma cells. We exposed SK-MEL-28 cells to different concentrations of ivermectin (2.5, 5, and 10 μM) for 24 hours. Here, ivermectin-induced apoptosis, as evidenced by the upregulation of cleaved poly (ADP-ribose) polymerase, BAX expression, and caspase-3 activity and downregulation of BCL-2 expression. In line with the apoptosis response, ivermectin triggered autophagy. Pharmacological or genetic inhibition of autophagy further sensitized SK-MEL-28 cells to ivermectin-induced apoptosis. Mechanistically, ivermectin-induced TFE3^(Ser321) dephosphorylation, activated TFE3 nuclear translocation and increased TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes, and subsequently, initiated autophagy in SK-MEL-28 cells. Moreover, N-acetyl-cysteine, an reactive oxygen species (ROS) scavenger, abrogated the effects of ivermectin on TFE3-dependent autophagy. Taken together, we demonstrated that ivermectin increases TFE3-dependent autophagy through ROS signaling pathways in human melanoma cells and that inhibiting autophagy enhances ivermectin-induced apoptosis in human melanoma cells.     

An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA,Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II^-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.     

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.     

Autophagy is involved in the early step of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry-LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.     

The extrinsic RNA-sensing pathway for adjuvant immunotherapy of cancer

Infection with RNA viruses presents a typical pattern of virus products, double-stranded RNA (dsRNA), and induces the maturation of antigen-presenting dendritic cell (mDC). There are several dsRNA sensors that are differentially distributed on the cell membrane and in the cytoplasm and are variably expressed depending on the cell type. Among these sensors, TLR3 links to the adaptor TICAM-1 (TRIF), which is characterized by its unique multipronged signaling cascades for cytokine/chemokine production, apoptosis and autophagy in both immune and tumor cells. In the context of mDC maturation, various cellular events are further induced in response to dsRNA; these include cross-priming followed by CD8+ CTL induction, NK activation and proliferation of CD4+ T cells including Th1, Th2, Treg and Th17 cells. In this review, we focus on the potential role of dsRNA in modulating the inflammatory milieu around mDCs and tumor-associated antigens to drive specific cellular effectors against the tumor.