The A20 protein has emerged as an important negative regulator of Toll like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-mediated anti-viral signaling. A20 functions both as a RING-type E3 ubiquitin ligase and as a de-ubiquitinating enzyme. Nuclear factor kappa B (NF-kappaB) and interferon regulatory factor (IRF) pathways are targeted by A20 through mechanisms that appear to be both overlapping and distinct, resulting in the downregulation of interferon alpha/beta (IFNalpha/beta) production. This review specifically details the impact of A20 on the cytosolic RIG-I/MDA5 pathway, a process that is less understood than that of NF-kappaB but is essential for the regulation of the innate immune response to viral infection.
Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-?B to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.
Retroviruses can selectively trigger an array of innate immune responses through various PRR. The identification and the characterization of the molecular basis of retroviral DNA sensing by the DNA sensors IFI16 and cGAS has been one of the most exciting developments in viral immunology in recent years. DNA sensing by these cytosolic sensors not only leads to the initiation of the type I interferon (IFN) antiviral response and the induction of the inflammatory response, but also triggers cell death mechanisms including pyroptosis and apoptosis in retrovirus-infected cells, thereby providing important insights into the pathophysiology of chronic retroviral infection. Host restriction factors such as SAMHD1 and Trex1 play important roles in regulating innate immune sensing, and have led to the idea that innate immune defense and host restriction actually converge at different levels to determine the outcome of retroviral infection. In this review, we discuss the sensing of retroviruses by cytosolic DNA sensors, the relevance of host factors during retroviral infection, and the interplay between host factors and the innate antiviral response in different cell types, within the context of two human pathogenic retroviruses - human immunodeficiency virus (HIV-1) and human T cell-leukemia virus type I (HTLV-1).
The 16th International Conference on Human Retrovirology: HTLV and Related Retroviruses was held in Montreal, Québec from June 26th to June 30th, 2013 and was therefore hosted by a Canadian city for the first time. The major topic of the meeting was human T-lymphotropic viruses (HTLVs) and was covered through distinct oral and poster presentation sessions: clinical research, animal models, immunology, molecular and cellular biology, human endogenous and emerging exogenous retroviruses and virology. In this review, highlights of the meeting are provided by different experts for each of these research areas.
RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5'triphosphorylated RNA (5'pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5'pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5'pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.
I?B kinase ? (IKK-?) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-?B and interferon (IFN) signaling. In the present study, we investigated IKK-? activation following T cell receptor (TCR)/CD28 stimulation of primary CD4(+) T cells and its role in the stimulation of a type I IFN response. IKK-? was activated following TCR/CD28 stimulation of primary CD4(+) T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-? transcription. We demonstrated that IKK-? phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4(+) T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-?B RelA subunit and interfered with PCAF-mediated acetylation of NF-?B RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-? activation, which in turn contributes to suppression of IFN-? production.
Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.
Vesicular stomatitis virus (VSV) is an oncolytic virus that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to VSV. In resistant tumor models, VSV oncolytic potential can be reversibly stimulated by combination with epigenetic modulators such as the histone deacetylase inhibitor Vorinostat. Based on this reversible effect of Vorinostat, we reasoned that critical host genes involved in oncolysis may likewise be reversibly regulated by Vorinostat. A transcriptome analysis in prostate cancer PC3 cells identified a subset of NF-?B target genes reversibly regulated by Vorinostat, as well as a group of interferon (IFN)-stimulated genes (ISGs). Consistent with the induction of NF-?B target genes, Vorinostat-mediated enhancement of VSV oncolysis increased hyper-acetylation of NF-?B RELA/p65. Additional bioinformatics analysis revealed that NF-?B signaling also increased expression of several autophagy-related genes. Kinetically, autophagy preceded apoptosis and apoptosis was only observed when cells were treated with both VSV and Vorinostat. VSV replication and cell killing were suppressed when NF-?B signaling was inhibited using pharmacological or genetic approaches. Inhibition of autophagy by 3-methyladenine (3-MA) enhanced expression of ISGs, and either 3-MA treatment or genetic ablation of the autophagic marker Atg5 decreased VSV replication and oncolysis. Together, these data demonstrate that Vorinostat stimulates NF-?B activity in a reversible manner via modulation of RELA/p65 signaling, leading to induction of autophagy, suppression of the IFN-mediated response, and subsequent enhancement of VSV replication and apoptosis.
SAMHD1 [sterile alpha motif and histidine-aspartic domain (HD) containing protein 1] is the most recent addition to a unique group of host restriction factors that limit retroviral replication at distinct stages of the viral life cycle. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase that degrades the intracellular pool of deoxynucleoside triphosphates available during early reverse transcription. SAMHD1 activity is blocked by the Vpx accessory function present in human immunodeficiency virus type 2 and SIVsm. Mutations in SAMHD1 are associated with the autoimmune disorder Aicardi-Goutières syndrome, thus emphasizing its role in regulation of the immune response. SAMHD1 antiretroviral activity is modulated by post-translational modifications, cell-cycle-dependent functions and cytokine-mediated changes. Innate receptors that sense retroviral DNA intermediates are the focus of intense study, and recent studies have established a link among SAMHD1 restriction, innate sensing of DNA and protective immune responses. Cell-cycle-dependent regulation of SAMHD1 by phosphorylation and the increasingly broad range of viruses inhibited by SAMHD1 further emphasize the importance of these mechanisms of host restriction. This review highlights current knowledge regarding SAMHD1 regulation and its impact on innate immune signaling and retroviral restriction.
In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-?) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-? responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-? responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-? promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-? and IFN-? mRNA, and levels of IFN-? production by pDCs similar to those of RMs or humans. These data establish that IFN-? and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.
Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia and HTLV-1-associated myelopathies. In addition to T cells, HTLV-1 infects cells of the myeloid lineage, which play critical roles in the host innate response to viral infection. Investigating the monocyte depletion observed during HTLV-1 infection, we discovered that primary human monocytes infected with HTLV-1 undergo abortive infection accompanied by apoptosis dependent on SAMHD1, a host restriction factor that hydrolyzes endogenous dNTPs to below the levels required for productive reverse transcription. Reverse transcription intermediates (RTI) produced in the presence of SAMHD1 induced IRF3-mediated antiviral and apoptotic responses. Viral RTIs complexed with the DNA sensor STING to trigger formation of an IRF3-Bax complex leading to apoptosis. This study provides a mechanistic explanation for abortive HTLV-1 infection of monocytes and reports a link between SAMHD1 restriction, HTLV-1 RTI sensing by STING, and initiation of IRF3-Bax driven apoptosis.
Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.
The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5 triphosphate (5ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5pppRNA, and not by IFN?-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
Many primary cancers including chronic lymphocytic leukemia (CLL) are resistant to vesicular stomatitis virus (VSV)-induced oncolysis due to overexpression of the antiapoptotic and antiautophagic members of the B-cell lymphoma-2 (BCL-2) family. In the present study, we investigated the mechanisms of CLL cell death induced as a consequence of VSV infection in the presence of BCL-2 inhibitors, obatoclax, and ABT-737 in primary ex vivo CLL patient samples. Microarray analysis of primary CD19? CD5? CLL cells treated with obatoclax and VSV revealed changes in expression of genes regulating apoptosis, the mechanistic target of rapamycin (mTOR) pathway, and cellular metabolism. A combined therapeutic effect was observed for VSV and BCL-2 inhibitors in cells from untreated patients and from patients unresponsive to standard of care therapy. In addition, combination treatment induced several markers of autophagy--LC3-II accumulation, p62 degradation, and staining of autophagic vacuoles. Inhibition of early stage autophagy using 3-methyladenine (3-MA) led to increased apoptosis in CLL samples. Mechanistically, a combination of BCL-2 inhibitors and VSV disrupted inhibitory interactions of Beclin-1 with BCL-2 and myeloid cell leukemia-1 (MCL-1), thus biasing cells toward autophagy. We propose a mechanism in which changes in cellular metabolism, coupled with pharmacologic disruption of the BCL-2-Beclin-1 interactions, facilitate induction of apoptosis and autophagy to mediate the cytolytic effect of VSV.
X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase 3-, 7-, and 9-dependent apoptotic activities that functions as an E3 ubiquitin ligase, and it targets caspases for degradation. In this study, we demonstrate that Sendai virus (SeV) infection results in the IKK?- or TBK1-mediated phosphorylation of XIAP in vivo at Ser430, resulting in Lys(48)-linked autoubiquitination at Lys322/328 residues, followed by the subsequent proteasomal degradation of XIAP. Interestingly, IKK? expression and XIAP turnover increases SeV-triggered mitochondrion-dependent apoptosis via the release of caspase 3, whereas TBK1 expression does not increase apoptosis. Interestingly, phosphorylation also regulates XIAP interaction with the transcription factor IRF3, suggesting a role in IRF3-Bax-mediated apoptosis. Our findings reveal a novel function of IKK? as a regulator of the virus-induced triggering of apoptosis via the phosphorylation-dependent turnover of XIAP.
Infection with the Human T-cell Leukemia virus type I (HTLV-1) retrovirus results in a number of diverse pathologies, including the aggressive, fatal T-cell malignancy adult T-cell leukemia (ATL) and the chronic, progressive neurologic disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Worldwide, it is estimated there are 15-20 million HTLV-1-infected individuals; although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% of AC develops either ATL or HAM/TSP, but never both. Regardless of asymptomatic status or clinical outcome, HTLV-1 carriers are at high risk of opportunistic infection. The progression to pathological HTLV-1 disease is in part attributed to the failure of the innate and adaptive immune system to control virus spread. The innate immune response against retroviral infection requires recognition of viral pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRR) dependent pathways, leading to the induction of host antiviral and inflammatory responses. Recent studies have begun to characterize the interplay between HTLV-1 infection and the innate immune response and have identified distinct gene expression profiles in patients with ATL or HAM/TSP--upregulation of growth regulatory pathways in ATL and constitutive activation of antiviral and inflammatory pathways in HAM/STP. In this review, we provide an overview of the replicative lifecycle of HTLV-1 and the distinct pathologies associated with HTLV-1 infection. We also explore the innate immune mechanisms that respond to HTLV-1 infection, the strategies used by HTLV-1 to subvert these defenses and their contribution to HTLV-1-associated diseases.
Oncolytic virotherapy is a promising biological approach to cancer treatment that contributes to tumor eradication via immune- and non-immune-mediated mechanisms. One of the remaining challenges for these experimental therapies is the necessity to develop a durable adaptive immune response against the tumor. Vesicular stomatitis virus (VSV) is a prototypical oncolytic virus (OV) that exemplifies the multiple mechanisms of oncolysis, including direct cell lysis, cellular hypoxia resulting from the shutdown of tumor vasculature, and inflammatory cytokine release. Despite these properties, the generation of sustained antitumor immunity is observed only when VSV is engineered to express a tumor antigen directly. In the present study, we sought to increase the number of tumor-associated dendritic cells (DC) in vivo and tumor antigen presentation by combining VSV treatment with recombinant Fms-like tyrosine kinase 3 ligand (rFlt3L), a growth factor promoting the differentiation and proliferation of DC. The combination of VSV oncolysis and rFLt3L improved animal survival in two different tumor models, i.e., VSV-resistant B16 melanoma and VSV-sensitive E.G7 T lymphoma; however, increased survival was independent of the adaptive CD8 T cell response. Tumor-associated DC were actively infected by VSV in vivo, which reduced their viability and prevented their migration to the draining lymph nodes to prime a tumor-specific CD8 T cell response. These results demonstrate that VSV interferes with tumor DC functions and blocks tumor antigen presentation.
IFN-? plays a critical role in tumor immunosurveillance by affecting either immune cells or tumor cells; however, IFN-mediated effects on tumor elimination are largely unknown. In this study, we showed that IFN regulatory factors (IRF) modulated by IFNs up- and downregulated Noxa expression, a prodeath BH3 protein, in various cancer cells. Inhibition of Noxa expression using short hairpin RNA in tumor cells leads to resistance against lipopolysaccharide (LPS)-induced tumor elimination, in which IFN-? is known as a critical effecter in mice. Chromatin immunoprecipitation analysis in both CT26 cells and SP2/0 cells, sensitive and resistant to LPS-induced tumor elimination, respectively, revealed that the responsiveness of IRF1, 3, 4, and 7 in the Noxa promoter region in response to IFN-? might be crucial in LPS-induced tumor elimination. IRF1, 3, and 7 were upregulated by IFN-? and activated Noxa expression, leading to the death of Noxa wild-type baby mouse kidney (BMK) cells but not of Noxa-deficient BMK cells. In contrast, IRF4 acts as a repressor for Noxa expression and inhibits cell death induced by IRF1, 3, or 7. Therefore, although IFN-? alone are not able to induce cell death in tumor cells in vitro, Noxa induction by IFN-?, which is regulated by the balance between its activators (IRF1, 3, and 7) and its repressor (IRF4), is crucial to increasing the susceptibility of tumor cells to immune cell-mediated cytotoxicity.
Sensing of RNA virus infection by the RIG-I-like receptors (RLRs) engages a complex signaling cascade that utilizes the mitochondrial antiviral signaling (MAVS) adapter protein to orchestrate the innate host response to pathogen, ultimately leading to the induction of antiviral and inflammatory responses mediated by type I interferon (IFN) and NF-?B pathways. MAVS is localized to the outer mitochondrial membrane, and has been associated with peroxisomes, the endoplasmic reticulum and autophagosomes, where it coordinates signaling events downstream of RLRs. MAVS not only plays a pivotal role in the induction of antiviral and inflammatory pathways but is also involved in the coordination of apoptotic and metabolic functions. This review summarizes recent findings related to the MAVS adapter and its essential role in the innate immune response to RNA viruses.
Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKK? is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS(-/-) mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKK? to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKK?-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.
The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the de novo infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR(-). Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the de novo HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.
The genus ? human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus ? HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5 phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.
In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)-a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials-and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.
Influenza viruses have to overcome the type I interferon induced antiviral response to successfully propagate in target cells. A major antiviral factor induced by interferons is the protein kinase R (PKR) that is further activated by dsRNA and phosphorylates the eukaryotic initiation factor 2 (eIF2?). This results in inhibition of protein translation thereby limiting viral replication. Here we describe a novel mechanism by which influenza A viruses escape the antiviral action of PKR. We demonstrate that the mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) MK2 and MK3 are activated on virus infection and, in their active form, directly interact with the repressor of the inhibitor of PKR p88(rIPK). This leads to recruitment of a tetrameric protein complex consisting of p88(rIPK), the inhibitor of PKR p58(IPK) and PKR itself, and finally results in inhibition of the kinase. The importance of MKs for influenza virus propagation was further underscored by demonstrating reduced viral progeny in cells genetically deficient in MK2 or MK3 genes as well as in highly proliferating tumor cells, in which expression of MKs was diminished by specific small interfering RNA. Accordingly, knockdown of MKs resulted in enhanced phosphorylation of PKR and its substrate eIF2?.
Oncolytic viruses (OVs) have shown promise as cancer therapeutics in pre-clinical and clinical testing; however, it is unlikely that OVs will constitute a stand-alone treatment. Histone deacetylase inhibitors (HDIs) represent a class of anticancer agents known to influence epigenetic modifications of chromatin, alter gene expression and manipulate a variety of signaling pathways, in some cases blunting the cellular antiviral response. Recent studies have shown that combining OV therapy with HDI treatment enhances viral replication and synergistically induces the killing of cancer cells in vitro and in vivo, an effect that has now been demonstrated in variety of virus/HDI combinations. This review discusses the results obtained with the different OV/HDI combinations, the rationale supporting these combinations and the advantages for oncolytic virus therapy.
Oncolytic viruses (OVs) are promising anticancer agents but like other cancer monotherapies, the genetic heterogeneity of human malignancies can lead to treatment resistance. We used a virus/cell-based assay to screen diverse chemical libraries to identify small molecules that could act in synergy with OVs to destroy tumor cells that resist viral infection. Several molecules were identified that aid in viral oncolysis, enhancing virus replication and spread as much as 1,000-fold in tumor cells. One of these molecules we named virus-sensitizers 1 (VSe1), was found to target tumor innate immune response and could enhance OV efficacy in animal tumor models and within primary human tumor explants while remaining benign to normal tissues. We believe this is the first example of a virus/cell-based "pharmacoviral" screen aimed to identify small molecules that modulate cellular response to virus infection and enhance oncolytic virotherapy.
Inhibition of the expression and replication of human retroviruses by different families of host restriction factors has emerged as an important component of antiviral innate immunity. The term "intrinsic immunity" is used to define this specific arm of innate immunity and suggests that host restriction factors are constitutively present within infected cells. The essential role of the interferon (IFN) signaling pathways in eliciting host restriction factor gene transcription - triggered a consequence of pattern recognition receptor signaling - may be an under-recognized aspect of intrinsic immunity. This review discusses the relevance of innate IFN signaling in the induction of retroviral restriction factors, the mechanisms of action of these factors, as well as the counter-regulation of IFN response that results from the plethora of retrovirus-restriction factor interactions.
Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP, but never both. To better understand the gene expression changes in HTLV-1-associated diseases, we examined the mRNA profiles of CD4+ T cells isolated from 7 ATL, 12 HAM/TSP, 11 AC and 8 non-infected controls. Using genomic approaches followed by bioinformatic analysis, we identified gene expression pattern characteristic of HTLV-1 infected individuals and particular disease states. Of particular interest, the suppressor of cytokine signaling 1--SOCS1--was upregulated in HAM/TSP and AC patients but not in ATL. Moreover, SOCS1 was positively correlated with the expression of HTLV-1 mRNA in HAM/TSP patient samples. In primary PBMCs transfected with a HTLV-1 proviral clone and in HTLV-1-transformed MT-2 cells, HTLV-1 replication correlated with induction of SOCS1 and inhibition of IFN-?/? and IFN-stimulated gene expression. Targeting SOCS1 with siRNA restored type I IFN production and reduced HTLV-1 replication in MT-2 cells. Conversely, exogenous expression of SOCS1 resulted in enhanced HTLV-1 mRNA synthesis. In addition to inhibiting signaling downstream of the IFN receptor, SOCS1 inhibited IFN-? production by targeting IRF3 for ubiquitination and proteasomal degradation. These observations identify a novel SOCS1 driven mechanism of evasion of the type I IFN antiviral response against HTLV-1.
The cytosolic DNA sensing pathway has remained poorly defined thus far. A recent study by Ishikawa et al. demonstrates that STING is essential for DNA-mediated type I IFN production and host defence against DNA pathogens.
The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-kappaB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-kappaB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity.
Oncolytic viruses (OVs) represent an exciting new biological approach to cancer therapy. In particular, RNA viruses have emerged as potent agents for oncolytic virotherapy because of their capacity to specifically target and destroy tumour cells while sparing normal cells and tissues. Several barriers remain in the development of OV therapy, including poor penetration into the tumour mass, inefficient virus replication in primary cancers, and tumour-specific resistance to OV-mediated killing. The combination of OVs with cytotoxic agents, such as small molecule inhibitors of signalling or immunomodulators, as well as stealth delivery of therapeutic viruses have shown promise as novel experimental strategies to overcome resistance to viral oncolysis. These agents complement OV therapy by unblocking host pathways, delivering viruses with greater efficiency and/or increasing virus proliferation at the tumour site. In this review, we summarize recent development of these concepts, the potential obstacles, and future prospects for the clinical utilization of RNA OVs in cancer therapy.
Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-kappaB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IkappaB kinase epsilon (IKKepsilon) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKepsilon to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKepsilon to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-kappaB activation. Thus, recruitment of IKKepsilon to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-kappaB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKepsilon and TBK-1 in the RIG-I/Mda5 pathway.
Differential expression of the human interferon A (IFN-A) gene cluster is modulated following paramyxovirus infection by the relative amounts of active interferon regulatory factor 3 (IRF-3) and IRF-7. IRF-3 expression activates predominantly IFN-A1 and IFN-B, while IRF-7 expression induces multiple IFN-A genes. IFN-A1 gene expression is dependent on three promoter proximal IRF elements (B, C, and D modules, located at positions -98 to -45 relative to the mRNA start site). IRF-3 binds the C module of IFN-A1, while other IFN-A gene promoters are responsive to the binding of IRF-7 to the B and D modules. Maximal expression of IFN-A1 is observed with complete occupancy of the three modules in the presence of IRF-7. Nucleotide substitutions in the C modules of other IFN-A genes disrupt IRF-3-mediated transcription, whereas a G/A substitution in the D modules enhances IRF7-mediated expression. IRF-3 exerts dual effects on IFN-A gene expression, as follows: a synergistic effect with IRF-7 on IFN-A1 expression and an inhibitory effect on other IFN-A gene promoters. Chromatin immunoprecipitation experiments reveal that transient binding of both IRF-3 and IRF-7, accompanied by CBP/p300 recruitment to the endogenous IFN-A gene promoters, is associated with transcriptional activation, whereas a biphasic recruitment of IRF-3 and CBP/p300 represses IFN-A gene expression. This regulatory mechanism contributes to differential expression of IFN-A genes and may be critical for alpha interferon production in different cell types by RIG-I-dependent signals, leading to innate antiviral immune responses.
Interferon regulatory factor (IRF) family members have been implicated as critical transcription factors that function in immune response, hematopoietic differentiation and cell growth regulation. Activation of IRF-5 results in the production of pro-inflammatory cytokines such as TNFalpha, IL6 and IL12p40, as well as type I interferons.
The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys48-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442--previously shown to be important for association with the MAVS adapter--were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin "immuno-editing" following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.
Viral and microbial pathogens contain specific motifs or pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface- and endosome-associated Toll-like receptors (TLRs). RNA virus infection is also detected through TLR-independent mechanisms. Early viral replicative intermediates are detected by two recently characterized cystolic viral RNA receptors-RIG-I and MDA-5. Both are DExDH/box RNA helicases, and RIG-I specifically recognizes 5-triphosphate containing viral RNA and transmits signals that induce type I interferon-mediated host immunity against virus infection. In this review, we will focus on RIG-I-like receptor (RLR) signal transduction and the regulatory mechanisms - ubiquitination, deubiquitination, ISGylation - underlying this important host response.
The immunoregulatory transcriptional modulators - IFN-regulatory factor (IRF)-3 and IRF-7 - possess similar structural features but distinct gene-regulatory potentials. For example, adenovirus-mediated transduction of the constitutively active form of IRF-3 triggered cell death in primary human MPhi, whereas expression of active IRF-7 induced a strong anti-tumoral activity in vitro. To further characterize target genes involved in these distinct cellular responses, transcriptional profiles of active IRF-3- or IRF-7-transduced primary human MPhi were compared and used to direct further mechanistic studies. The pro-apoptotic BH3-only protein Noxa was identified as a primary IRF-3 target gene and an essential regulator of IRF-3, dsRNA and vesicular stomatitis virus-induced cell death. The critical role of IRF-7 and type I IFN production in increasing the immunostimulatory capacity of MPhi was also evaluated; IRF-7 increased the expression of a broad range of IFN-stimulated genes including immunomodulatory cytokines and genes involved in antigen processing and presentation. Furthermore, active IRF-7 augmented the cross-presentation capacity and tumoricidal activity of MPhi and led to an anti-tumor response against the B16 melanoma model in vivo. Altogether, these data further highlight the respective functions of IRF-3 and IRF-7 to program apoptotic, immune and anti-tumor responses.
Intrinsic and acquired drug resistance remains a fundamental obstacle to successful applications of anticancer therapies for lung cancer. Combining conventional therapies with immunotherapeutic approaches is a promising strategy to circumvent lung cancer drug resistance. Genetically modified oncolytic viruses (OVs) kill tumor cells via completely unique mechanisms compared to small molecule chemotherapeutics typically used in lung cancer treatment and can also be used to deliver specific toxic, therapeutic or immunomodulatory genes to tumor cells. Recent pre-clinical and clinical studies with oncolytic vaccine approaches have revealed promising combination strategies that enhance oncolysis of tumor cells and circumvent tumor resistance mechanisms. As clinical trials with oncolytic vaccines progress, and as the knowledge acquired from these studies builds a foundation demonstrating OVs safety and efficacy, novel combination approaches could soon have a major impact on the clinical management of patients diagnosed with lung cancer.
Cyclophilin A (CypA) is vital for HCV replication. Cyp inhibitors successfully decrease viral loads in HCV-infected patients. However, their mechanisms of action remain unknown. Since interferon (IFN) can also suppress HCV replication, we asked whether a link between CypA and the IFN response exists.
The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-?B, respectively. Both the IRF and NF-?B pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-?B-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.
The sensing of viral infection by the innate immune system is dominated by the recognition of nucleic acids. New data now demonstrate that the fusion of viral and target-cell membranes leads to the activation of an immune response dependent on the adaptor STING.
The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.
Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.
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