62 research outputs found
Modulation of Tumor Necrosis Factor by Microbial Pathogens
In response to invasion by microbial pathogens, host defense mechanisms get activated by both the innate and adaptive arms of the immune responses. TNF (tumor necrosis factor) is a potent proinflammatory cytokine expressed by activated macrophages and lymphocytes that induces diverse cellular responses that can vary from apoptosis to the expression of genes involved in both early inflammatory and acquired immune responses. A wide spectrum of microbes has acquired elegant mechanisms to overcome or deflect the host responses mediated by TNF. For example, modulatory proteins encoded by multiple families of viruses can block TNF and TNF-mediated responses at multiple levels, such as the inhibition of the TNF ligand or its receptors, or by modulating key transduction molecules of the TNF signaling pathway. Bacteria, on the other hand, tend to modify TNF-mediated responses specifically by regulating components of the TNF signaling pathway. Investigation of these diverse strategies employed by viral and bacterial pathogens has significantly advanced our understanding of both host TNF responses and microbial pathogenesis. This review summarizes the diverse microbial strategies to regulate TNF and how such insights into TNF modulation could benefit the treatment of inflammatory or autoimmune diseases
Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.
Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells
Oncolytic Virotherapy for Hematological Malignancies
Hematological malignancies such as leukemias, lymphomas, multiple myeloma (MM), and the myelodysplastic syndromes (MDSs) primarily affect adults and are difficult to treat. For high-risk disease, hematopoietic stem cell transplant (HCT) can be used. However, in the setting of autologous HCT, relapse due to contamination of the autograft with cancer cells remains a major challenge. Ex vivo manipulations of the autograft to purge cancer cells using chemotherapies and toxins have been attempted. Because these past strategies lack specificity for malignant cells and often impair the normal hematopoietic stem and progenitor cells, prior efforts to ex vivo purge autografts have resulted in prolonged cytopenias and graft failure. The ideal ex vivo purging agent would selectively target the contaminating cancer cells while spare normal stem and progenitor cells and would be applied quickly without toxicities to the recipient. One agent which meets these criteria is oncolytic viruses. This paper details experimental progress with reovirus, myxoma virus, measles virus, vesicular stomatitis virus, coxsackievirus, and vaccinia virus as well as requirements for translation of these results to the clinic
Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma
Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects
Parallel adaptation of rabbit populations to myxoma virus.
In the 1950s the myxoma virus was released into European rabbit populations in Australia and Europe, decimating populations and resulting in the rapid evolution of resistance. We investigated the genetic basis of resistance by comparing the exomes of rabbits collected before and after the pandemic. We found a strong pattern of parallel evolution, with selection on standing genetic variation favoring the same alleles in Australia, France, and the United Kingdom. Many of these changes occurred in immunity-related genes, supporting a polygenic basis of resistance. We experimentally validated the role of several genes in viral replication and showed that selection acting on an interferon protein has increased the protein's antiviral effect.This work was supported by grants from the Programa Operacional Potencial HumanoâQuadro de ReferĂȘncia EstratĂ©gica Nacional funds from the European Social Fund and Portuguese MinistĂ©rio da CiĂȘncia, Tecnologia e Ensino Superior to M.C. (IF/00283/2014/CP1256/CT0012), to P.J.E. (IF/00376/2015) and to J.M.A. (SFRH/BD/72381/2010). AM was supported by the European Research Council (grant 647787-LocalAdaptation). FJ was supported by the European Research Council (grant 281668). LL was supported by the European Research Council grant (339941-ADAPT). McFadden Lab is supported by National Institute of Health (NIH) grant R01 AI080607. S.C.G. holds a Sir Henry Dale Fellowship, co-funded by the Wellcome Trust and the Royal Society (098406/Z/12/Z)
Co-Regulation of NF-ÎșB and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013
NF-ÎșB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1ÎČ and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-ÎșB. The induction of these NF-ÎșB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IÎșBα, which was followed by nuclear translocation of NF-ÎșB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-ÎșB-mediated reporter gene expression and nuclear translocation of NF-ÎșB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-ÎșB1, suggesting a direct physical and functional linkage between NF-ÎșB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-ÎșB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1ÎČ. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-ÎșB-mediated pro-inflammatory responses
Oncolytic Virotherapy with Myxoma Virus
Oncolytic viruses are one of the most promising novel therapeutics for malignant cancers. They selectively infect and kill cancer cells while sparing the normal counterparts, expose cancer- specific antigens and activate the host immune system against both viral and tumor determinants. Oncolytic viruses can be used as monotherapy or combined with existing cancer therapies to become more potent. Among the many types of oncolytic viruses that have been developed thus far, members of poxviruses are the most promising candidates against diverse cancer types. This review summarizes recent advances that are made with oncolytic myxoma virus (MYXV), a member of the Leporipoxvirus genus. Unlike other oncolytic viruses, MYXV infects only rabbits in nature and causes no harm to humans or any other non-leporid animals. However, MYXV can selectively infect and kill cancer cells originating from human, mouse and other host species. This selective cancer tropism and safety profile have led to the testing of MYXV in various types of preclinical cancer models. The next stage will be successful GMP manufacturing and clinical trials that will bring MYXV from bench to bedside for the treatment of currently intractable malignancies
Myxoma Virus dsRNA Binding Protein M029 Inhibits the Type I IFNâInduced Antiviral State in a Highly SpeciesâSpecific Fashion
Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbitârestricted host tropism but exhibits a much broader cellular host range in cultured cells. MYXV is able to efficiently block all aspects of the type I interferon (IFN)âinduced antiviral state in rabbit cells, partially in human cells and very poorly in mouse cells. The mechanism(s) of this speciesâspecific inhibition of type I IFNâinduced antiviral state is not well understood. Here we demonstrate that MYXV encoded protein M029, a truncated relative of the vaccinia virus (VACV) E3 doubleâstranded RNA (dsRNA) binding protein that inhibits protein kinase R (PKR), can also antagonize the type I IFNâinduced antiviral state in a highly speciesâspecific manner. In cells preâtreated with type I IFN prior to infection, MYXV exploits M029 to overcome the induced antiviral state completely in rabbit cells, partially in human cells, but not at all in mouse cells. However, in cells preâinfected with MYXV, IFNâinduced signaling is fully inhibited even in the absence of M029 in cells from all three species, suggesting that other MYXV protein(s) apart from M029 block IFN signaling in a speciesindependent manner. We also show that the antiviral state induced in rabbit, human or mouse cells by type I IFN can inhibit M029âknockout MYXV even when PKR is genetically knockedâout, suggesting that M029 targets other host proteins for this antiviral state inhibition. Thus, the MYXV dsRNA binding protein M029 not only antagonizes PKR from multiple species but also blocks the type I IFN antiviral state independently of PKR in a highly speciesâspecific fashion
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