RESUMEN
Oncolytic virotherapy is a promising therapy for cancer. We previously established a recombinant measles virus (rMV-SLAMblind) that targets NECTIN4-expressing cancer cells and demonstrated its antitumor effects using a xenograft model in an immunodeficient mouse. In the current study, to investigate the immune response after rMV-SLAMblind therapy, we developed an immunocompetent cancer mouse model by introducing the NECTIN4 gene into mouse cancer cell lines. NECTIN4-expressing mouse cancer cells were successfully killed by rMV-SLAMblind in vitro. After transplantation of the NECTIN4-expressing tumor cells, rMV-SLAMblind significantly suppressed tumor growth in immunocompetent mice. Thus, this immunocompetent mouse cancer model could be a powerful tool in which to study the effect of rMV-SLAMblind therapy on the immune response. Using this model we found that rMV-SLAMblind elicited significant activation of natural killer cells, type 1 helper T cells and the tumor-specific CD8+ T-cell response in the tumor microenvironment. Immune cell depletion study revealed that CD8+ cells particularly played significant roles in the therapeutic efficacy of rMV-SLAMblind. Thus, rMV-SLAMblind exerts a therapeutic effect, not only directly by tumor cell killing, but also indirectly by efficient induction of antitumor immunity.
Asunto(s)
Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Animales , Ratones , Virus Oncolíticos/fisiología , Línea Celular Tumoral , Microambiente Tumoral , Moléculas de Adhesión Celular/metabolismo , Inmunidad , Neoplasias/terapiaRESUMEN
In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.
Asunto(s)
Inmunidad Innata/inmunología , Sarampión/metabolismo , Mitocondrias/metabolismo , Nucleotidiltransferasas/metabolismo , Animales , Regulación hacia Abajo , Humanos , Virus del Sarampión , Ratones , Ratones Endogámicos C57BL , Mitocondrias/virología , Biogénesis de Organelos , Infecciones por Virus ARN/metabolismo , Virus ARNRESUMEN
Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.
Asunto(s)
Inmunidad Innata/genética , Interleucina-8/genética , ARN Largo no Codificante/fisiología , Proteínas de Unión al ARN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Herpesvirus Humano 1/inmunología , Humanos , Virus del Sarampión/inmunología , Orthomyxoviridae/inmunología , Factor de Empalme Asociado a PTB , Regiones Promotoras Genéticas , Transporte de Proteínas , ARN Largo no Codificante/genética , Transcripción GenéticaRESUMEN
The tail domain of the measles virus (MeV) N protein is typically phosphorylated at S479 and S510. However, the protein kinase responsible for this phosphorylation has not been identified. To identify the protein kinase responsible, we conducted an in vitro kinase assay in the presence of various protein kinase inhibitors. Phosphorylation of S479 and S510 was suppressed in the presence of SP600125. We demonstrated that purified PIM 3 kinase, which is sensitive to SP600125, successfully phosphorylated both phosphorylation sites. Inhibitors of PIM kinase, CX6258 and LY294002, also suppressed phosphorylation of the N protein. These findings indicate that PIM 3 kinase is associated with the tail domain of the N protein and that PIM 3 kinase regulates N protein phosphorylation.
Asunto(s)
Virus del Sarampión/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Antracenos/farmacología , Línea Celular , Humanos , Proteínas de la Nucleocápside , Fosforilación/efectos de los fármacos , Dominios Proteicos , Proto-Oncogenes MasRESUMEN
Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impß1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses.IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism.
Asunto(s)
Núcleo Celular/metabolismo , Virus Hendra/fisiología , Infecciones por Henipavirus/metabolismo , Virus Nipah/fisiología , Nucleoproteínas/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Núcleo Celular/genética , Células HEK293 , Células HeLa , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Inmunidad Innata/inmunología , Nucleoproteínas/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Transducción de SeñalRESUMEN
In rare cases, measles virus (MV) in children leads to fatal neurological complications such as primary measles encephalitis, post-acute measles encephalitis, subacute sclerosing panencephalitis and measles inclusion-body encephalitis. To investigate the pathogenesis of MV-induced encephalitis, rodent brain-adapted MV strains CAM/RB and CAMR40 were generated. These strains acquired mutations to adapt to the rodent brain during 40 passages in rat brain. However, it is still unknown which genes confer the neurovirulence of MV. We previously established a rescue system for recombinant MVs possessing the backbone of wild-type strain HL, an avirulent strain in mice. In the present study, to identify the genes in CAMR40 that elicit neurovirulence, we generated chimeric recombinant MVs based on strain HL. As a result, recombinant wild-type MV in which the haemagglutinin (H) gene was substituted with that of CAMR40 caused a non-lethal mild disease in mice, while additional substitution of the HL phosphoprotein (P) gene with that of strain CAMR40 caused lethal severe neurological signs comparable to those of CAMR40. These results clearly indicated that, in addition to the H gene, the P gene is required for the neurovirulence of MV CAMR40.
Asunto(s)
Encéfalo/patología , Hemaglutininas/genética , Virus del Sarampión/genética , Virus del Sarampión/patogenicidad , Fosfoproteínas/genética , Panencefalitis Esclerosante Subaguda/patología , Proteínas Virales/genética , Animales , Encéfalo/virología , Callithrix , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Panencefalitis Esclerosante Subaguda/genética , Panencefalitis Esclerosante Subaguda/virología , Células VeroRESUMEN
UNLABELLED: In the current study, we generated recombinant chimeric canine distemper viruses (CDVs) by replacing the hemagglutinin (H) and/or phosphoprotein (P) gene in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain. An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity for neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. The recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only with a recombinant CDV that possessed both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that both genes are important to drive virulence of CDV in mice. Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation. IMPORTANCE: CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous system subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease.
Asunto(s)
Líquido Cefalorraquídeo/virología , Virus del Moquillo Canino/patogenicidad , Moquillo/virología , Hipocampo/virología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/virología , Línea Celular , Línea Celular Tumoral , Líquido Cefalorraquídeo/metabolismo , Chlorocebus aethiops , Moquillo/metabolismo , Perros , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Sarampión/metabolismo , Sarampión/virología , Virus del Sarampión/patogenicidad , Ratones , Neuronas/metabolismo , Neuronas/virología , Receptores Virales/metabolismo , Células VeroRESUMEN
Pancreatic cancer is one of the most intractable cancers and has a devastating prognosis; over the past three decades the 5-year survival rate has been <10%. Therefore, development of a novel anticancer treatment for pancreatic cancer is a matter of urgency. We previously developed an oncolytic recombinant measles virus (MV), rMV-SLAMblind, that had lost the ability to bind to its principal receptor, signaling lymphocyte activity molecule (SLAM), but which selectively infected and efficiently killed nectin-4-expressing breast and lung cancer cells. In this study, we analyzed the antitumor effect of this virus against pancreatic cancer. Nectin-4 was expressed on the surface of 4/16 tested pancreatic cancer cell lines, which were efficiently infected and killed by rMV-SLAMblind in vitro. The intratumoral inoculation of rMV-SLAMblind suppressed the growth of KLM1 and Capan-2 cells xenografted in SCID mice. The sequence analysis of MV isolated from the tumor revealed that the designed mutation in the H protein of rMV-SLAMblind had been stably maintained for 47 days after the last inoculation. These results suggest that rMV-SLAMblind is a promising candidate for the novel treatment of pancreatic cancer.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Linfocitos/metabolismo , Virus del Sarampión/fisiología , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Transducción de Señal , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/virología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Measles virus (MeV) causes several unique syndromes, including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase, which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions. IMPORTANCE: Measles virus (MeV) is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by cellular protein phosphatase 5 (PP5) and DNA-dependent protein kinase. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV.
Asunto(s)
Células Epiteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Virus del Sarampión , Sarampión/fisiopatología , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Células HEK293 , Humanos , Inmunoprecipitación , Luciferasas , Análisis por Micromatrices , Fosforilación , Plásmidos/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Nipah virus belongs to the genus Henipavirus in the family Paramyxoviridae, and its RNA genome is larger than those of other paramyxoviruses because it has long untranslated regions (UTRs) in each gene. However, the functions of these UTRs are not fully understood. In this study, we investigated the functions of the 5' UTRs and found that the 5' UTR of the M gene upregulated the translation of a reporter gene. Using an RNA pull-down assay, we showed that eukaryotic elongation factor 1-beta (EEF1B2) interacts with nucleotides 81-100 of the M 5' UTR and specifically enhances its translation efficiency. Our results suggest that the M 5' UTR promotes the production of M protein and viral budding by recruiting EEF1B2.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Virus Nipah/genética , Factor 1 de Elongación Peptídica/metabolismo , Proteínas Virales/metabolismo , Regiones no Traducidas 5' , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Factor 1 de Elongación Peptídica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral , Proteínas Virales/genéticaRESUMEN
Nipah and Hendra virus were first identified in mid 1990s in Australia and Malaysia, caus- ing epidemics with high mortality rate in affected animals and humans. Since their first emer- gence, they continued to re-emerge in Australia and South East Asia almost every year. Nipah and Hendra virus were classified in the new genus Henipavirus because of their un- common features amongst Paramyxoviridae. Henipaviruses are zoonotic paramyxoviruses with a broad tropism, and cause severe acute respiratory disease and encephalitis. Their high virulence and wide host range make them to be given Biosecurity Level 4 status. This review summarizes details of Henipavirus emergence, reservoir hosts and pathology, and introduce recent progress in vaccines and antivirals.
Asunto(s)
Infecciones por Henipavirus , Antivirales/uso terapéutico , Infecciones por Henipavirus/tratamiento farmacológico , Infecciones por Henipavirus/prevención & control , Vacunas Virales/uso terapéuticoRESUMEN
Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcription and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phosphorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine putative phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant. Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron microscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphorylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these results indicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation.
Asunto(s)
Virus del Sarampión/metabolismo , Sarampión/virología , Nucleocápside/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Treonina/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Humanos , Virus del Sarampión/química , Virus del Sarampión/genética , Datos de Secuencia Molecular , Nucleocápside/genética , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Fosforilación , Treonina/genética , Proteínas Virales/genéticaRESUMEN
The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus del Sarampión/fisiología , Nucleoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad del ARN , Proteínas Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Análisis Mutacional de ADN , Humanos , Virus del Sarampión/genética , Mutagénesis Sitio-Dirigida , Mutación Missense , Proteínas de la Nucleocápside , Fosforilación , Genética Inversa , Serina/genética , Carga ViralRESUMEN
Nipah virus (NiV) is a nonsegmented, single-stranded, negative-sense RNA virus belonging to the genus Henipavirus, family Paramyxoviridae. NiV causes acute encephalitis and respiratory disease in humans, is associated with high mortality, and poses a threat in southern Asia. The genomes of henipaviruses are about 18,246 nucleotides (nt) long, which is longer than those of other paramyxoviruses (around 15,384 nt). This difference is caused by the noncoding RNA region, particularly the 3' untranslated region (UTR), which occupies more than half of the noncoding RNA region. To determine the function(s) of the NiV noncoding RNA region, we investigated the effects of NiV 3' UTRs on reporter gene expression. The NiV N 3' UTR (nt 1 to 100) demonstrated strong repressor activity associated with hnRNP D protein binding to that region. Mutation of the hnRNP D binding site or knockdown of hnRNP D resulted in increased expression of the NiV N 3' UTR reporter. Our findings suggest that NiV N expression is repressed by hnRNP D through the NiV N 3' UTR and demonstrate the involvement of posttranscriptional regulation in the NiV life cycle. To the best of our knowledge, this provides the first report of the functions of the NiV noncoding RNA region.
Asunto(s)
Regiones no Traducidas 3'/genética , Regulación hacia Abajo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Virus Nipah/metabolismo , Regiones no Traducidas 3'/fisiología , Sitios de Unión/genética , Eliminación de Gen , Regulación Viral de la Expresión Génica , Genes Reporteros , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , Mutación , Virus Nipah/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans, and incurred a high fatality rate in humans. We established a system that enabled the rescue of replicating NiVs from a cloned DNA. Using the system, we analyzed the functions of accessory proteins in infected cells and the implications in in vivo pathogenicity. Further, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins, which appeared to be an appropriate to NiV vaccine candidate for use in humans.
Asunto(s)
Descubrimiento de Drogas , Infecciones por Henipavirus/virología , Virus Nipah/genética , Virus Nipah/patogenicidad , Vacunas Virales , Animales , ADN Viral , Proteínas de Unión al GTP/inmunología , Genes Virales/genética , Glicoproteínas , Infecciones por Henipavirus/prevención & control , Humanos , Vacuna Antisarampión/genética , Virus del Sarampión/genética , Virus Nipah/fisiología , Plásmidos/genética , Recombinación Genética , Genética Inversa , Vacunas Sintéticas/genética , Proteínas Reguladoras y Accesorias Virales/fisiología , Replicación ViralRESUMEN
PVRL4 (or nectin4) is a promising therapeutic target since its upregulated expression is found in a wide range of human cancer types. Enfortumab vedotin, an antibodydrug conjugate targeting PVRL4, is clinically used for the treatment of urothelial bladder cancer. In addition, rMVSLAMblind, a genetically engineered oncolytic measles virus, can infect cancer cells and induce apoptosis through interaction with PVRL4. Although PVRL4 transcript levels are elevated in breast, lung and ovarian cancer, the mechanisms of its upregulation have not yet been uncovered. To clarify the regulatory mechanisms of elevated PVRL4 expression in breast cancer cells, Assay for TransposaseAccessible Chromatinsequencing and chromatin immunoprecipitationsequencing (ChIPseq) data were used to search for its regulatory regions. Using breast cancer cells, an enhancer region was ultimately identified. Additional analyses, including ChIP and reporter assays, demonstrated that FOS interacted with the PVRL4 enhancer region, and that alterations of the FOSbinding motifs in the enhancer region decreased reporter activity. Consistent with these data, exogenous expression of FOS enhanced the reporter activity and PVRL4 expression in breast cancer cells. Furthermore, RNAseq analysis using breast cancer cells treated with PVRL4 small interfering RNA revealed its possible involvement in the cytokine response and immune system. These data suggested that FOS was involved, at least partly, in the regulation of PVRL4 expression in breast cancer cells, and that elevated PVRL4 expression may regulate the response of cancer cells to cytokines and the immune system.
Asunto(s)
Neoplasias de la Mama , Nectinas , Virus Oncolíticos , Femenino , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Virus del Sarampión/genética , Virus del Sarampión/metabolismo , Virus Oncolíticos/genética , ARN Interferente Pequeño , Nectinas/genética , Nectinas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
Canine primary lung cancer with metastasis has a poor prognosis with no effective treatment. We previously generated a recombinant measles virus (MV) that lost binding affinity to a principal receptor, SLAM, to eliminate its virulence as a new cancer treatment strategy. The virus, rMV-SLAMblind, targets nectin-4, recently listed as a tumor marker, and exerts antitumor activity against nectin-4-positive canine mammary cancer and urinary bladder transitional cell carcinoma cells. However, the effectivity of rMV-SLAMblind for other types of canine cancers is still unknown. Here we evaluated the antitumor effect of rMV-SLAMblind to canine lung cancer. Nectin-4 is expressed on three canine lung cancer cell lines (CLAC, AZACL1, AZACL2) and rMV-SLAMblind was able to infect these cell lines. CLAC cells showed reduced cell viability after virus infection. In the CLAC xenograft nude mouse model, intratumoral administration of rMV-SLAMblind significantly suppressed tumor growth. In rMV-SLAMblind-treated mice, natural killer cells were activated, and Cxcl10 and Il12a levels were significantly increased in comparison with levels in the control group. In addition, the depletion of NK cells reduced the anti-tumor effect. To understand difference in efficacy among canine lung cancer cell lines, we compared virus growth and gene expression pattern after virus treatment in the three canine lung cancer cell lines; virus growth was highest in CLAC cells compared with the other cell lines and the induction of interferon (IFN)-beta and IFN-stimulated genes was at lower levels in CLAC cells. These results suggested that rMV-SLAMblind exhibits oncolytic effect against some canine lung cancer cells and the cellular response after the virus infection may influence its efficacy.
Asunto(s)
Neoplasias Pulmonares , Viroterapia Oncolítica , Virus Oncolíticos , Virosis , Humanos , Animales , Perros , Ratones , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Virus del Sarampión/metabolismo , Viroterapia Oncolítica/métodos , Nectinas/metabolismo , Línea Celular Tumoral , Moléculas de Adhesión Celular/metabolismo , Virosis/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Virus Oncolíticos/genéticaRESUMEN
Measles is a highly contagious human disease caused by the measles virus (MeV). In this study, by proteomic analysis, we identified peroxiredoxin 1 (Prdx1) as a host factor that binds to the C-terminal region of the nucleoprotein (N; N(TAIL)) of MeV. Glutathione S-transferase (GST) pulldown experiments showed that the Prdx1-binding site overlapped with the MeV phosphoprotein (P)-binding site on N(TAIL) and that Prdx1 competed for the binding to N(TAIL) with the P protein, which is a component of RNA-dependent RNA polymerase (RdRp). Furthermore, RNA interference for Prdx1 resulted in a significant reduction in MeV growth in HEK293-SLAM cells. A minigenome assay indicated that Prdx1 suppression affected the viral RNA transcription and/or replication step. Relative quantification of viral RNA by real-time PCR (RT-PCR) showed that Prdx1 suppression not only reduced viral RNA transcription and replication but also enhanced polar attenuation in viral mRNA transcription. Surface plasmon resonance analysis showed that the binding affinity of Prdx1 to MeV-N was 40-fold lower than that of MeV-P to MeV-N, which suggested that Prdx1 might be involved in the early stage of MeV infection, when the expression level of Prdx1 was much higher than that of MeV-P. Since Prdx1 was expressed abundantly and constitutively in various cells, the results in this study indicate that Prdx1 is one of the inherent host factors implicated in MeV RNA synthesis.
Asunto(s)
Virus del Sarampión/fisiología , Sarampión/enzimología , Peroxirredoxinas/metabolismo , Transcripción Genética , Replicación Viral , Sitios de Unión , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Sarampión/virología , Virus del Sarampión/química , Virus del Sarampión/genética , Proteínas de la Nucleocápside , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/genética , Unión Proteica , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Many viruses use their host's cellular machinery to regulate the functions of viral proteins. The phosphorylation of viral proteins is known to play a role in genome transcription and replication in paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected cells, is a component of the N-RNA complex and supports the transcription and replication of virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in steady-state cells, but was detected after inhibition of cellular protein phosphatases. We identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by electrospray ionization-quadrupole time-of-flight mass spectrometry. In the NiV minigenome assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted in a reduction in luciferase activity of approximately 45â% compared with the wild-type protein. Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a more significant decrease in luciferase activity - approximately 81â%. Northern blot analysis showed that both virus transcription and replication were reduced by these mutations. These results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in virus transcription and replication.