Interferon regulatory factor-1 protects from fatal neurotropic infection with vesicular stomatitis virus by specific inhibition of viral replication in neurons.
ABSTRACT: The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1-/- mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1-/- mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.
Project description:Virus infection of a cell generally evokes an immune response by the host to defeat the intruder in its effort. Many viruses have developed an array of strategies to evade or antagonize host antiviral responses. Kaposi's sarcoma-associated herpesvirus (KSHV) is demonstrated in this report to be able to prevent activation of host antiviral defense mechanisms upon infection. Cells infected with wild-type KSHV were permissive for superinfection with vesicular stomatitis virus (VSV), suggesting that KSHV virions fail to induce host antiviral responses. We previously showed that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu et al., Proc. Natl. Acad. Sci. USA. 99:5573-5578, 2002). Here, using an ORF45-null recombinant virus, we demonstrate a profound role of ORF45 in inhibiting host antiviral responses. Infection of cells with an ORF45-null mutant recombinant KSHV (BAC-stop45) triggered an immune response that resisted VSV super-infection, concomitantly associated with appreciable increases in transcription of type I IFN and downstream anti-viral effector genes. Gain-of-function analysis showed that ectopic expression of ORF45 in human fibroblast cells by a lentivirus vector decreased the antiviral responses of the cells. shRNA-mediated silencing of IRF-7, that predominantly regulates both the early and late phase induction of type I IFNs, clearly indicated its critical contribution to the innate antiviral responses generated against incoming KSHV particles. Thus ORF45 through its targeting of the crucial IRF-7 regulated type I IFN antiviral responses significantly contributes to the KSHV survival immediately following a primary infection allowing for progression onto subsequent stages in its life-cycle.
Project description:Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.
Project description:Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKepsilon kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKepsilon activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKepsilon following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKepsilon kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKepsilon in epithelial cells. Induction of IKKepsilon potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.
Project description:The negative regulation of antiviral immune responses is essential for the host to maintain homeostasis. Jumonji domain-containing protein 6 (JMJD6) was previously identified with a number of functions during RNA virus infection. Upon viral RNA recognition, retinoic acid-inducible gene-I-like receptors (RLRs) physically interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type-I interferon (IFN-I) production. Here, JMJD6 was demonstrated to reduce type-I interferon (IFN-I) production in response to cytosolic poly (I:C) and RNA virus infections, including Sendai virus (SeV) and Vesicular stomatitis virus (VSV). Genetic inactivation of JMJD6 enhanced IFN-I production and impaired viral replication. Our unbiased proteomic screen demonstrated JMJD6 contributes to IRF3 K48 ubiquitination degradation in an RNF5-dependent manner. Mice with gene deletion of JMJD6 through piggyBac transposon-mediated gene transfer showed increased VSV-triggered IFN-I production and reduced susceptibility to the virus. These findings classify JMJD6 as a negative regulator of the host's innate immune responses to cytosolic viral RNA.
Project description:Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity but some cancer cells are resistant to VSV killing, either constitutively or due to type I interferon (IFN) inducing an antiviral state in the cells. Here, we evaluated VSV oncolysis of a panel of human head and neck cancer cells and showed that VSV resistance in SCC25 and SCC15 cells could be reversed with Janus kinase (JAK) 1/2 inhibitors (JAK inhibitor I and ruxolitinib). Pre-treatment of cells with JAK1/2 inhibitors before or in conjunction with VSV enhanced viral infection, spread and progeny yield (100- to 1000-fold increase). In contrast, inhibitors of histone deacetylase (LBH589), phosphatidylinositol 3-kinase (GDC-0941, LY294002), mammalian target of rapamycin (rapamycin) or signal transducer and activator of transcription 3 (STAT3 inhibitor VII) were ineffective. Compared with VSV-sensitive SW579 cells, IFN?/? responsive antiviral genes (IRF-9, IRF-7, OAS1 but not MxA) are constitutively expressed in SCC25 cells. Pretreatment with JAK inhibitors reduced mRNA levels of these genes, increasing VSV expression in the cells. Interestingly, 1?h of drug exposure was sufficient to reverse SCC25 resistance to VSV and was still effective if virus was added 24?h later. Overall, we show here that JAK inhibitor I and ruxolitinib (Jakafi) can reverse resistance to VSV, supporting the rationale to incorporate JAK1/2 inhibitors in future VSV virotherapy trials.
Project description:Interferon regulatory factor (IRF)-3 is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-3 promotes antiviral control by inducing interferon (IFN)-beta, near normal levels of IFN-alpha and IFN-beta were observed in IRF-3(-/-) mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-3 modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-3-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-3 coordinates an antiviral response, we infected IRF-3(-/-) mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-3(-/-) mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-3(-/-) macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-alpha and IFN-beta production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-3(-/-) neurons lacked induction of host defense genes and had blunted IFN-alpha and IFN-beta production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-3 responses protect against WNV infection through both IFN-dependent and -independent programs.
Project description:Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-?? expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1(-/-) mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1(-/-) cells and mice. IRF-1(-/-) mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1(-/-) macrophages supported enhanced WNV replication but infection was unaltered in IRF-1(-/-) fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8(+) T cell expansion. Although markedly fewer CD8(+) T cells were observed in naïve animals as described previously, remarkably, IRF-1(-/-) mice rapidly expanded their pool of WNV-specific cytolytic CD8(+) T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8(+) T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8(+) T cell response.
Project description:Interferon-regulatory factors (IRFs) are a family of transcription factors (TFs) that translate viral recognition into antiviral responses, including type I interferon (IFN) production. Dengue virus (DENV) and other clinically important flaviviruses are suppressed by type I IFN. While mice lacking the type I IFN receptor (Ifnar1-/-) succumb to DENV infection, we found that mice deficient in three transcription factors controlling type I IFN production (Irf3-/-Irf5-/-Irf7-/- triple knockout [TKO]) survive DENV challenge. DENV infection of TKO mice resulted in minimal type I IFN production but a robust type II IFN (IFN-?) response. Using loss-of-function approaches for various molecules, we demonstrate that the IRF-3-, IRF-5-, IRF-7-independent pathway predominantly utilizes IFN-? and, to a lesser degree, type I IFNs. This pathway signals via IRF-1 to stimulate interleukin-12 (IL-12) production and IFN-? response. These results reveal a key antiviral role for IRF-1 by activating both type I and II IFN responses during DENV infection.
Project description: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.
Project description:Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, limiting the systemic spread of pathogens. Recent work has shown that CD11b(+)CD169(+) macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for the clearance of viruses from the lymph and for initiating antiviral humoral immune responses. Here we show, using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the central nervous system (CNS). On local depletion of LN macrophages, about 60% of mice developed ascending paralysis and died 7-10 days after subcutaneous infection with a small dose of VSV, whereas macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system through peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially in SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but decreased type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I-producing plasmacytoid dendritic cells to the SCS and in addition were a major source of IFN-I themselves. Experiments in bone marrow chimaeric mice revealed that IFN-I must act on both haematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal infection with VSV. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral invasion of the nervous system on peripheral infection.