RESUMEN
The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.
Asunto(s)
Virus de la Viruela Vacuna/fisiología , Viruela Vacuna/inmunología , ARN Interferente Pequeño/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales/metabolismo , Animales , Evolución Molecular , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Inflamación , Ratones , Ratones Noqueados , Necroptosis/genética , Orthopoxvirus , Filogenia , Proteínas Quinasas/genética , Proteolisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Análisis de Secuencia de ARN , Proteínas Virales/genética , Replicación ViralRESUMEN
The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.
Asunto(s)
Exocitosis , Herpes Simple , Herpesvirus Humano 1 , Liberación del Virus , Animales , Humanos , Transporte Biológico , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , NeuronasRESUMEN
RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , ARN Helicasas DEAD-box/fisiología , Myxoma virus/fisiología , Proteínas de Neoplasias/fisiología , Animales , Antivirales , Línea Celular Tumoral , Gránulos Citoplasmáticos/química , ARN Helicasas DEAD-box/genética , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Biosíntesis de Proteínas , Conejos , Estrés Fisiológico , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Schistosomiasis is a devastating disease caused by parasitic flatworms of the genus Schistosoma. Praziquantel (PZQ), the current treatment of choice, is ineffective against immature worms and cannot prevent reinfection. The continued reliance on a single drug for treatment increases the risk of the development of PZQ-resistant parasites. Reports of PZQ insusceptibility lends urgency to the need for new therapeutics. Here, we report that Myxoma virus (MYXV), an oncolytic pox virus which is non-pathogenic in all mammals except leporids, infects and replicates in S. mansoni schistosomula, juveniles, and adult male and female worms. MYXV infection results in the shredding of the tegument and reduced egg production in vitro, identifying MYXV as the first viral pathogen of schistosomes. MYXV is currently in preclinical studies to manage multiple human cancers, supporting its use in human therapeutics. Our findings raise the exciting possibility that MYXV virus represents a novel and safe class of potential anthelmintic therapeutics.
Asunto(s)
Antihelmínticos , Myxoma virus , Virus Oncolíticos , Esquistosomiasis mansoni , Animales , Antihelmínticos/farmacología , Femenino , Humanos , Masculino , Mamíferos , Praziquantel/farmacología , Schistosoma mansoni , Esquistosomiasis mansoni/tratamiento farmacológicoRESUMEN
Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.
Asunto(s)
Myxoma virus , FN-kappa B/inmunología , Transducción de Señal/inmunología , Proteínas Virales , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Caspasa 1/genética , Caspasa 1/inmunología , Células HeLa , Humanos , Inflamasomas/genética , Mutagénesis Sitio-Dirigida , Mutación Missense , Myxoma virus/química , Myxoma virus/genética , Myxoma virus/inmunología , FN-kappa B/genética , Dominios Proteicos , Transducción de Señal/genética , Células THP-1 , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Myxoma virus (MYXV) is a rabbit-specific poxvirus, which is highly virulent in European rabbits. The attenuation of MYXV and the increased resistance of rabbits following the release of MYXV in Australia is one of the best-documented examples of host-pathogen coevolution. To elucidate the molecular mechanisms that contribute to the restriction of MYXV infection to rabbits and MYXV attenuation in the field, we have studied the interaction of the MYXV protein M156 with the host antiviral protein kinase R (PKR). In yeast and cell-culture transfection assays, M156 only inhibited rabbit PKR but not PKR from other tested mammalian species. Infection assays with human HeLa PKR knock-down cells, which were stably transfected with human or rabbit PKR, revealed that only human but not rabbit PKR was able to restrict MYXV infection, whereas both PKRs were able to restrict replication of a vaccinia virus (VACV) strain that lacks the PKR inhibitors E3 and K3. Inactivation of M156R led to MYXV virus attenuation in rabbit cells, which was rescued by the ectopic expression of VACV E3 and K3. We further show that a mutation in the M156 encoding gene that was identified in more than 50% of MYXV field isolates from Australia resulted in an M156 variant that lost its ability to inhibit rabbit PKR and led to virus attenuation. The species-specific inhibition of rabbit PKR by M156 and the M156 loss-of-function in Australian MYXV field isolates might thus contribute to the species specificity of MYXV and to the attenuation in the field, respectively.
Asunto(s)
Myxoma virus/genética , Proteínas Virales/genética , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genética , Animales , Australia , Línea Celular Tumoral , Células HeLa , Humanos , Mutación/genética , Myxoma virus/patogenicidad , Conejos , Proteínas Virales/metabolismo , Virulencia/genética , Replicación Viral/genéticaRESUMEN
BACKGROUND: Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy. METHODS: Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay. RESULTS: MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation. CONCLUSIONS: Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Neoplasias Hematológicas/patología , Células Madre Hematopoyéticas/citología , Myxoma virus/fisiología , Viroterapia Oncolítica/métodos , Adulto , Antígenos CD34/metabolismo , Autoinjertos/normas , Médula Ósea/patología , Células de la Médula Ósea/patología , Células Cultivadas , Femenino , Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante de Células Madre Hematopoyéticas/normas , Células Madre Hematopoyéticas/fisiología , Humanos , Masculino , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/prevención & control , Acondicionamiento Pretrasplante/métodosRESUMEN
Epstein-Barr virus (EBV) has been associated with a variety of epithelial and hematologic malignancies, including B-, T- and NK cell-lymphomas, Hodgkin's disease (HD), post-transplant lymphoproliferative diseases (LPDs), nasopharyngeal and gastric carcinomas, smooth muscle tumors, and HIV-associated lymphomas. Currently, treatment options for EBV-associated malignancies are limited. We have previously shown that myxoma virus specifically targets various human solid tumors and leukemia cells in a variety of animal models, while sparing normal human or murine tissues. Since transplant recipients of bone marrow or solid organs often develop EBV-associated post-transplant LPDs and lymphoma, myxoma virus may be of utility to prevent EBV-associated malignancies in immunocompromised transplant patients where treatment options are frequently limited. In this report, we demonstrate the safety and efficacy of myxoma virus purging as a prophylactic strategy for preventing post-transplant EBV-transformed human lymphomas, using a highly immunosuppressed mouse xenotransplantation model. This provides support for developing myxoma virus as a potential oncolytic therapy for preventing EBV-associated LPDs following transplantation of bone marrow or solid organ allografts.
Asunto(s)
Herpesvirus Humano 4/patogenicidad , Linfoma de Células B/prevención & control , Trastornos Linfoproliferativos/complicaciones , Viroterapia Oncolítica , Trasplante/efectos adversos , Animales , Modelos Animales de Enfermedad , Xenoinjertos , Linfoma de Células B/virología , Trastornos Linfoproliferativos/etiología , RatonesRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Monocitos/inmunología , Myxoma virus/fisiología , Proteínas de Neoplasias/metabolismo , Proteínas Virales/metabolismo , Tropismo Viral , Replicación Viral , eIF-2 Quinasa/metabolismo , Animales , Antivirales/metabolismo , Antivirales/uso terapéutico , Línea Celular , Células Cultivadas , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Susceptibilidad a Enfermedades , Femenino , Técnicas de Inactivación de Genes , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/uso terapéutico , Monocitos/metabolismo , Monocitos/virología , Mutación , Mixomatosis Infecciosa/prevención & control , Mixomatosis Infecciosa/virología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in pancreatic cancer cells, but its efficacy in animal models of pancreatic cancer has not been determined. The efficacy of MYXV as monotherapy or in combination with gemcitabine was evaluated in intraperitoneal dissemination (IPD) models of pancreatic cancer. The effects of an intact immune system on the efficacy of MYXV therapy was tested by comparing immunodeficient versus immunocompetent murine models and combination therapy with gemcitabine was also evaluated. In cell culture, MYXV replication was robust in a broad range of pancreatic cancer cells and also showed increased oncolysis in combination with gemcitabine. In animal models, MYXV treatment conferred survival benefits over control or gemcitabine-treated cohorts regardless of the cell line or animal model used. MYXV monotherapy was most effective in an immunocompetent IPD model, and resulted in 60% long-term survivors. In Pan02 engrafted immunocompetent IPD models, sequential treatment in which MYXV was administered first, followed by gemcitabine, was the most effective and resulted in 100% long-term survivors. MYXV is an effective oncolytic virus for pancreatic cancer and can be combined with gemcitabine to enhance survival, particularly in the presence of an intact host immune system.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Myxoma virus/fisiología , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/terapia , Animales , Línea Celular Tumoral , Supervivencia Celular , Desoxicitidina/uso terapéutico , Femenino , Citometría de Flujo , Humanos , Mediciones Luminiscentes , Ratones , Ratones Desnudos , Microscopía Fluorescente , Myxoma virus/genética , Virus Oncolíticos/genética , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses.
Asunto(s)
Herpesvirus Humano 1 , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Herpesvirus Humano 1/genética , Virus Oncolíticos/genética , Tropismo , Virus ADNRESUMEN
Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting XPO1-mediated nuclear export with selective inhibitors activates multiple antiviral and anti-inflammatory pathways. The XPO1 inhibitor, Selinexor, is an FDA-approved anticancer drug predicted to have antiviral function against many viruses, including SARS-CoV-2. Unexpectedly, we observed that pretreatment of cultured human cells with Selinexor actually enhanced protein expression and replication of coronaviruses, including SARS-CoV-2. Knockdown of cellular XPO1 protein expression significantly enhanced the replication of coronaviruses in human cells. We further demonstrate that Selinexor treatment reduced the formation of unique cytoplasmic antiviral granules that include RNA helicase DHX9 in the virus-infected cells. These results, for the first time, show that the anti-cancer drug Selinexor enhances the replication of coronaviruses in human cells in vitro and thus should be further explored in vivo for the potential impact on the dual use for anticancer and antiviral therapy.
RESUMEN
The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.
RESUMEN
Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. In addition, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy. Significance: We demonstrated that a combination of nuclear export inhibitor selinexor and oncolytic MYXV significantly enhanced viral replication, reduced cancer cell proliferation, reduced tumor burden, and enhanced the overall survival of animals. Thus, selinexor and oncolytic MYXV can be used as potential new anticancer therapy.
Asunto(s)
Myxoma virus , Neoplasias , Virus Oncolíticos , Humanos , Animales , Ratones , Myxoma virus/genética , Transporte Activo de Núcleo Celular , Proteómica , Virus Oncolíticos/genéticaRESUMEN
The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1ß are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1ß in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1ß was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1ß in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends on sensing by the NLRP3 receptor in response to vMyxM013-KO infection of human myeloid cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Células Mieloides/metabolismo , Myxoma virus/genética , FN-kappa B/metabolismo , Infecciones por Poxviridae/inmunología , Receptores Toll-Like/metabolismo , Infecciones Tumorales por Virus/inmunología , Proteínas Virales/fisiología , Western Blotting , Proteínas Portadoras/genética , Caspasa 1/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunidad Innata , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/metabolismo , Myxoma virus/inmunología , FN-kappa B/genética , Proteína con Dominio Pirina 3 de la Familia NLR , Infecciones por Poxviridae/genética , Infecciones por Poxviridae/virología , Estructura Terciaria de Proteína , Pirina , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virologíaRESUMEN
Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-ß, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
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Células Dendríticas/inmunología , Factor 7 Regulador del Interferón/inmunología , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Myxoma virus/inmunología , Receptor Toll-Like 9/inmunología , Animales , Células Cultivadas , Femenino , Factor 7 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/metabolismo , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Receptor Toll-Like 9/metabolismo , Virus Vaccinia/inmunologíaRESUMEN
OBJECTIVE: We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV). METHODS: Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus protein expression, and cell viability by alamarBlue assay. Akti-1/2 (5 µM) and rapamycin (20 nM) were used to assay the role of PI3K-AKT signaling in mediating MYXV infection. RESULTS: Ascites-derived EOC cells grown in adherent culture are effectively killed by MYXV infection. EOC cells grown in suspension to form three-dimensional EOC spheroids readily permit MYXV entry into cells, yet are protected from the cytopathic effects of late MYXV infection. Upon reattachment (to model secondary metastasis), EOC spheroids are re-sensitized to MYXV-mediated oncolysis. The critical determinant that facilitates efficient MYXV infection is the presence of an activated PI3K-AKT signaling pathway. Treatment with the specific AKT inhibitor Akti-1/2 reduces infection of monolayer EOC cells and spheroids. Direct infection of freshly-collected ascites demonstrated that 54.5% of patient samples were sensitive to MYXV-mediated oncolytic cell killing. We also demonstrate that factor(s) present in ascites may negatively impact MYXV infection and oncolysis of EOC cells, which may be due to a down-regulation in endogenous AKT activity. CONCLUSIONS: Differential activity of AKT serves as the mechanistic basis for regulating MYXV-mediated oncolysis of EOC spheroids during key steps of the metastatic program. In addition, we provide the first evidence that MYXV oncolytic therapy may be efficacious for a significant proportion of ovarian cancer patients with metastatic disease.
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Myxoma virus/fisiología , Neoplasias Glandulares y Epiteliales/terapia , Proteína Oncogénica v-akt/metabolismo , Viroterapia Oncolítica/métodos , Neoplasias Ováricas/terapia , Ascitis/patología , Carcinoma Epitelial de Ovario , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Fluorescente/métodos , Myxoma virus/genética , Myxoma virus/metabolismo , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/virología , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/virología , Fosfatidilinositol 3-Quinasas/metabolismo , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-kappaB1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-kappaB1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-kappaB signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.
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Interacciones Huésped-Patógeno , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Proteómica , Virus de la Viruela/metabolismo , Proteínas Virales/metabolismo , Línea Celular , Biblioteca de Genes , Humanos , Subunidad p50 de NF-kappa B/metabolismo , Orthopoxvirus/metabolismo , Orthopoxvirus/patogenicidad , Técnicas del Sistema de Dos HíbridosRESUMEN
Poxviruses are a diverse family of double-stranded DNA viruses that cause mild-to-severe disease in selective hosts, including humans. Although most poxviruses are restricted to their hosts, some members can leap host species and cause zoonotic diseases and, therefore, are genuine threats to human and animal health. The recent global spread of monkeypox in humans suggests that zoonotic poxviruses can adapt to a new host, spread rapidly in the new host, and evolve to better evade host innate barriers. Unlike many other viruses, poxviruses express an extensive repertoire of self-defense proteins that play a vital role in the evasion of host innate and adaptive immune responses in their newest host species. The function of these viral immune modulators and host-specific cytokine responses can result in different host tropism and poxvirus disease progression. Here, we review the role of different cytokines that control poxvirus host tropism and adaptation.
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Infecciones por Poxviridae , Poxviridae , Animales , Humanos , Tropismo Viral , Citocinas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Poxviridae/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-ß ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.