RESUMEN
Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts. IMPORTANCE: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic.
Asunto(s)
Proteínas de la Cápside , Infecciones por Reoviridae , Replicación Viral , Animales , Ratones , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Infecciones por Reoviridae/virología , Infecciones por Reoviridae/metabolismo , Acoplamiento Viral , Polimorfismo Genético , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/fisiología , Orthoreovirus de los Mamíferos/metabolismo , Ensamble de Virus , Línea Celular , Cápside/metabolismo , HumanosRESUMEN
Mammalian orthoreovirus (reovirus) is a nonenveloped virus that establishes primary infection in the intestine and disseminates to sites of secondary infection, including the CNS. Reovirus entry involves multiple engagement factors, but how the virus disseminates systemically and targets neurons remains unclear. In this study, we identified murine neuropilin 1 (mNRP1) as a receptor for reovirus. mNRP1 binds reovirus with nanomolar affinity using a unique mechanism of virus-receptor interaction, which is coordinated by multiple interactions between distinct reovirus capsid subunits and multiple NRP1 extracellular domains. By exchanging essential capsid protein-encoding gene segments, we determined that the multivalent interaction is mediated by outer-capsid protein σ3 and capsid turret protein λ2. Using capsid mutants incapable of binding NRP1, we found that NRP1 contributes to reovirus dissemination and neurovirulence in mice. Collectively, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis.
Asunto(s)
Proteínas de la Cápside , Neuropilina-1 , Orthoreovirus de los Mamíferos , Receptores Virales , Infecciones por Reoviridae , Internalización del Virus , Animales , Ratones , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/fisiología , Orthoreovirus de los Mamíferos/metabolismo , Infecciones por Reoviridae/virología , Infecciones por Reoviridae/metabolismo , Receptores Virales/metabolismo , Humanos , Cápside/metabolismo , Línea Celular , Células HEK293 , Unión Proteica , Ratones Endogámicos C57BLRESUMEN
Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein µ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was nonlethal in wild-type mice, whereas more than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, as well as extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCE Reovirus is a nonenveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the nonlethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.
Asunto(s)
Proteínas de la Cápside/metabolismo , Orthoreovirus Mamífero 3/patogenicidad , Miocarditis/virología , Infecciones por Reoviridae/virología , Animales , Animales Recién Nacidos , Proteínas de la Cápside/genética , Inflamación , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocarditis/mortalidad , Miocarditis/patología , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/metabolismo , Orthoreovirus de los Mamíferos/patogenicidad , Infecciones por Reoviridae/mortalidad , Infecciones por Reoviridae/patología , Carga Viral , Virulencia , Replicación ViralRESUMEN
Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV µ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a µ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-µ2 and µNS. Thus, µ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs.
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Interacciones Huésped-Patógeno/genética , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Orthoreovirus de los Mamíferos/genética , Tubulina (Proteína)/genética , Proteínas Virales/genética , Animales , Línea Celular , Chlorocebus aethiops , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/ultraestructura , Centro Organizador de los Microtúbulos/virología , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Microtúbulos/virología , Nocodazol/farmacología , Orthoreovirus de los Mamíferos/efectos de los fármacos , Orthoreovirus de los Mamíferos/metabolismo , Transducción de Señal , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.
Asunto(s)
Orthoreovirus Mamífero 3/genética , Viroterapia Oncolítica/métodos , Replicación Viral/genética , Aminoácidos/genética , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Variación Genética/genética , Humanos , Orthoreovirus Mamífero 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/metabolismo , Filogenia , Reoviridae/genética , Proteínas Virales/metabolismoRESUMEN
Wild-type mammalian reoviruses (MRVs) have been evaluated as oncolytic agents against various cancers; however, genetic modification methods for improving MRV agents have not been exploited fully. In the present study, using MRV strain T1L, we generated a reporter MRV that expresses a NanoLuc luciferase (NLuc) gene and used it for noninvasive imaging of MRV infection in tumor xenograft mice. NLuc and a P2A self-cleaving peptide gene cassette were placed upstream of the L1 gene open reading frame to enable bicistronic expression of NLuc and the L1 gene product. BALB/c nude mice intranasally infected with MRV expressing NLuc (rsT1L-NLuc) displayed bioluminescent signals in the chest area at 4 days postinfection (dpi), which is consistent with natural MRV infection in the lung. Furthermore, to monitor tumor-selective infection by MRV, nude mice bearing human cancer xenografts were infected intravenously with rsT1L-NLuc. Bioluminescent signals were detected in tumors as early as 3 dpi and persisted for 2 months. The results demonstrate the utility of an autonomous replicating reporter MRV for noninvasive live imaging of replicating oncolytic MRV agents.IMPORTANCE Engineering of recombinant MRV for improved oncolytic activity has not yet been achieved due to difficulty in generating autonomous replicating MRV harboring transgenes. Here, we constructed a reporter MRV that can be used to monitor cancer-selective infection by oncolytic MRV in a mouse model. Among the numerous oncolytic viruses, MRV has an advantage in that the wild-type virus shows marked oncolytic activity in patients without any notable adverse effects. The reporter MRV developed herein will open avenues to the development of recombinant MRV vectors armed with anticancer transgenes.
Asunto(s)
Regulación Viral de la Expresión Génica , Luciferasas/biosíntesis , Mediciones Luminiscentes , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Animales , Línea Celular Tumoral , Humanos , Luciferasas/genética , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Neoplasias/virología , Virus Oncolíticos/genética , Orthoreovirus de los Mamíferos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The reovirus outer capsid protein µ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized µ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized µ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of µ1 also affects aspects of viral replication. We found that, while µ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the µ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when µ1 is knocked down. Following µ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of µ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to µ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCE We used mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for the reovirus outer capsid protein µ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection; µ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.
Asunto(s)
Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Animales , Apoptosis , Cápside/metabolismo , Muerte Celular , Línea Celular , Células HeLa , Humanos , Ratones , Necroptosis/fisiología , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismo , Reoviridae/metabolismo , Reoviridae/fisiología , Proteínas Virales/metabolismo , Virión/genética , Internalización del Virus , Replicación ViralRESUMEN
The mammalian orthoreovirus (reovirus) outer capsid, which is composed of 200 µ1/σ3 heterohexamers and a maximum of 12 σ1 trimers, contains all of the proteins that are necessary for attaching to and entering host cells. Following attachment, reovirus is internalized by receptor-mediated endocytosis and acid-dependent cathepsin proteases degrade the σ3 protein. This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the µ1 membrane penetration protein is exposed. ISVPs undergo a second structural rearrangement to deposit the genome-containing core into the host cytoplasm. The conformationally altered particle is called ISVP*. ISVP-to-ISVP* conversion culminates in the release of µ1 N- and C-terminal fragments, µ1N and Φ, respectively. Released µ1N is thought to facilitate core delivery by generating size-selective pores within the endosomal membrane, whereas the precise role of Φ, particularly in the context of viral entry, is undefined. In this report, we characterize a recombinant reovirus that fails to cleave Φ from µ1 in vitro Φ cleavage, which is not required for ISVP-to-ISVP* conversion, enhances the disruption of liposomal membranes and facilitates the recruitment of ISVP*s to the site of pore formation. Moreover, the Φ cleavage-deficient strain initiates infection of host cells less efficiently than the parental strain. These results indicate that µ1N and Φ contribute to reovirus pore forming activity.IMPORTANCE Host membranes represent a physical barrier that prevents infection. To overcome this barrier, viruses utilize diverse strategies, such as membrane fusion or membrane disruption, to access internal components of the cell. These strategies are characterized by discrete protein-protein and protein-lipid interactions. The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of well-defined conformational changes, which conclude with pore formation and delivery of the viral genetic material. In this report, we characterize the role of the small, reovirus-derived Φ peptide in pore formation. Φ cleavage from the outer capsid enhances membrane disruption and facilitates the recruitment of virions to membrane-associated pores. Moreover, Φ cleavage promotes the initiation of infection. Together, these results reveal an additional component of the reovirus pore forming apparatus and highlight a strategy for penetrating host membranes.
Asunto(s)
Proteínas de la Cápside/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Proteolisis , Infecciones por Reoviridae/metabolismo , Virión/metabolismo , Animales , Proteínas de la Cápside/genética , Línea Celular , Ratones , Orthoreovirus de los Mamíferos/genética , Dominios Proteicos , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/patología , Virión/genéticaRESUMEN
Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).
Asunto(s)
Antivirales/farmacología , Quelantes/farmacología , Hidrazonas/farmacología , Simplexvirus/efectos de los fármacos , Virus Vaccinia/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Línea Celular , Línea Celular Tumoral , Quelantes/síntesis química , Quelantes/metabolismo , Chlorocebus aethiops , Cobre/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Humanos , Hidrazonas/síntesis química , Hidrazonas/metabolismo , Concentración 50 Inhibidora , Magnesio/metabolismo , Manganeso/metabolismo , Orthoreovirus de los Mamíferos/efectos de los fármacos , Orthoreovirus de los Mamíferos/crecimiento & desarrollo , Orthoreovirus de los Mamíferos/metabolismo , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Virus de la Parainfluenza 3 Humana/crecimiento & desarrollo , Virus de la Parainfluenza 3 Humana/metabolismo , Phlebovirus/efectos de los fármacos , Phlebovirus/crecimiento & desarrollo , Phlebovirus/metabolismo , Virus Sincitiales Respiratorios/efectos de los fármacos , Virus Sincitiales Respiratorios/crecimiento & desarrollo , Virus Sincitiales Respiratorios/metabolismo , Simplexvirus/crecimiento & desarrollo , Simplexvirus/metabolismo , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/metabolismo , Células Vero , Vesiculovirus/efectos de los fármacos , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/metabolismoRESUMEN
Mammalian orthoreovirus protein µ2 is a component of the viral core particle. Its activities include RNA binding and hydrolysis of the γ-phosphate from NTPs and RNA 5´-termini, suggesting roles as a cofactor for the viral RNA-dependent RNA polymerase, λ3, first enzyme in 5´-capping of viral plus-strand RNAs, and/or prohibitory of RNA-5´-triphosphate-activated antiviral signaling. Within infected cells, µ2 also contributes to viral factories, cytoplasmic structures in which genome replication and particle assembly occur. By associating with both microtubules (MTs) and viral factory matrix protein µNS, µ2 can anchor the factories to MTs, the full effects of which remain unknown. In this study, a protease-hypersensitive region allowed µ2 to be dissected into two large fragments corresponding to residues 1-282 and 283-736. Fusions with enhanced green fluorescent protein revealed that these amino- and carboxyl-terminal regions of µ2 associate in cells with either MTs or µNS, respectively. More exhaustive deletion analysis defined µ2 residues 1-325 as the minimal contiguous region that associates with MTs in the absence of the self-associating tag. A region involved in µ2 self-association was mapped to residues 283-325, and self-association involving this region was essential for MT-association as well. Likewise, we mapped that µNS-binding site in µ2 relates to residues 290-453 which is independent of µ2 self-association. These findings suggest that µ2 monomers or oligomers can bind to MTs and µNS, but that self-association involving µ2 residues 283-325 is specifically relevant for MT-association during viral factories formation.
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Microtúbulos/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Microscopía Fluorescente , Unión Proteica , ARN Viral/metabolismo , Proteínas del Núcleo Viral/metabolismo , Replicación ViralRESUMEN
Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.
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Metiltransferasas/genética , Complejos Multienzimáticos/genética , Nucleotidiltransferasas/genética , Orthoreovirus de los Mamíferos/genética , Orthoreovirus/genética , Monoéster Fosfórico Hidrolasas/genética , Plásmidos/metabolismo , Genética Inversa/métodos , Proteínas no Estructurales Virales/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Cricetulus , ADN Complementario/genética , ADN Complementario/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Epiteliales/virología , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Metiltransferasas/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleotidiltransferasas/metabolismo , Orthoreovirus/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Plásmidos/química , Transducción Genética , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismoRESUMEN
Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.
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Adenocarcinoma/terapia , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Ensayos Clínicos como Asunto , Desoxicitidina Quinasa/genética , Desoxicitidina Quinasa/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , TransgenesRESUMEN
UNLABELLED: Viruses utilize host cell machinery for propagation and manage to evade cellular host defense mechanisms in the process. Much remains unknown regarding how the host responds to viral infection. We recently performed global proteomic screens of mammalian reovirus TIL- and T3D-infected and herpesvirus (herpes simplex virus 1 [HSV-1])-infected HEK293 cells. The nonenveloped RNA reoviruses caused an upregulation, whereas the enveloped DNA HSV-1 caused a downregulation, of cellular secretogranin II (SCG2). SCG2, a member of the granin family that functions in hormonal peptide sorting into secretory vesicles, has not been linked to virus infections previously. We confirmed SCG2 upregulation and found SCG2 phosphorylation by 18 h postinfection (hpi) in reovirus-infected cells. We also found a decrease in the amount of reovirus secretion from SCG2 knockdown cells. Similar analyses of cells infected with HSV-1 showed an increase in the amount of secreted virus. Analysis of the stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) pathway indicated that each virus activates different pathways leading to activator protein 1 (AP-1) activation, which is the known SCG2 transcription activator. We conclude from these experiments that the negative correlation between SCG2 quantity and virus secretion for both viruses indicates a virus-specific role for SCG2 during infection. IMPORTANCE: Mammalian reoviruses affect the gastrointestinal system or cause respiratory infections in humans. Recent work has shown that all mammalian reovirus strains (most specifically T3D) may be useful oncolytic agents. The ubiquitous herpes simplex viruses cause common sores in mucosal areas of their host and have coevolved with hosts over many years. Both of these virus species are prototypical representatives of their viral families, and investigation of these viruses can lead to further knowledge of how they and the other more pathogenic members of their respective families interact with the host. Here we show that secretogranin II (SCG2), a protein not previously studied in the context of virus infections, alters virus output in a virus-specific manner and that the quantity of SCG2 is inversely related to amounts of infectious-virus secretion. Herpesviruses may target this protein to facilitate enhanced virus release from the host.
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Regulación de la Expresión Génica/fisiología , Herpesvirus Humano 1/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Secretogranina II/metabolismo , Factor de Transcripción AP-1/metabolismo , Liberación del Virus/fisiología , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Immunoblotting , Ratones , Microscopía Fluorescente , Fosforilación , Células VeroRESUMEN
As prostate tumor cell growth depends on hormones, androgen ablation is an effective therapy for prostate cancer (PCa). However, progression of PCa cells to androgen independent growth (castrate resistant prostate cancer, CRPC) results in relapse and mortality. Hypoxia, a microenvironment of low oxygen that modifies the activity of PCa regulatory proteins including the androgen receptor (AR), plays a critical role in progression to CRPC. Therapies targeting hypoxia and the AR may lengthen the time to CRPC progression thereby increasing survival time of PCa patients. Mammalian Orthoreovirus (MRV) has shown promise for the treatment of prostate tumors in vitro and in vivo. In this study, we found that MRV infection induces downregulation of proteins implicated in CRPC progression, interferes with hypoxia-induced AR activity, and induces apoptosis in androgen dependent cells. This suggests MRV possesses traits that could be exploited to create novel therapies for the inhibition of progression to CRPC.
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Regulación Neoplásica de la Expresión Génica , Virus Oncolíticos/genética , Orthoreovirus de los Mamíferos/genética , Próstata/virología , Receptores Androgénicos/genética , Andrógenos/metabolismo , Apoptosis/genética , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Viroterapia Oncolítica/métodos , Virus Oncolíticos/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Próstata/metabolismo , Próstata/patología , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/metabolismo , Transducción de SeñalRESUMEN
Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.
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Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/metabolismo , Animales , Ratones , Mutación/genética , Virión/metabolismo , Ensamble de Virus/genéticaRESUMEN
Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.
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Gangliosidosis GM2/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Receptores Virales/metabolismo , Infecciones por Reoviridae/metabolismo , Proteínas Virales/metabolismo , Animales , Cricetinae , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión de Mamíferos/virología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Gangliosidosis GM2/genética , Células L , Ratones , Mutación , Orthoreovirus de los Mamíferos/genética , Estructura Terciaria de Proteína , Receptores Virales/genética , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/patología , Proteínas Virales/genéticaRESUMEN
Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.
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Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Proteolisis , Sustitución de Aminoácidos , Animales , Proteínas de la Cápside/genética , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular , Ratones , Mutación Missense , Orthoreovirus de los Mamíferos/genética , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/metabolismoRESUMEN
Mammalian orthoreoviruses replicate and assemble in the cytosol of infected cells. A viral nonstructural protein, µNS, forms large inclusion-like structures called viral factories (VFs) in which assembling viral particles can be identified. Here we examined the localization of the cellular chaperone Hsc70 and found that it colocalizes with VFs in infected cells and also with viral factory-like structures (VFLs) formed by ectopically expressed µNS. Small interfering RNA (siRNA)-mediated knockdown of Hsc70 did not affect the formation or maintenance of VFLs. We further showed that dominant negative mutants of Hsc70 were also recruited to VFLs, indicating that Hsc70 recruitment to VFLs is independent of the chaperone function. In support of this finding, µNS was immunoprecipitated with wild-type Hsc70, with a dominant negative mutant of Hsc70, and with the minimal substrate-binding site of Hsc70 (amino acids 395 to 540). We identified a minimal region of µNS between amino acids 222 and 271 that was sufficient for the interaction with Hsc70. This region of µNS has not been assigned any function previously. However, neither point mutants with alterations in this region nor the complete deletion of this domain abrogated the µNS-Hsc70 interaction, indicating that a second portion of µNS also interacts with Hsc70. Taken together, these findings suggest a specific chaperone function for Hsc70 within viral factories, the sites of reovirus replication and assembly in cells.
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Proteínas del Choque Térmico HSC70/metabolismo , Cuerpos de Inclusión Viral/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Infecciones por Reoviridae/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Proteínas del Choque Térmico HSC70/genética , Humanos , Cuerpos de Inclusión Viral/genética , Cuerpos de Inclusión Viral/virología , Orthoreovirus de los Mamíferos/química , Orthoreovirus de los Mamíferos/genética , Unión Proteica , Transporte de Proteínas , Infecciones por Reoviridae/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/ß) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/ß response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-ß stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/ß through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/ß are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded µ2 protein is a strain-specific repressor of IFN-ß signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that µ2 amino acid 208 determines repression of IFN-ß signaling and modulates reovirus induction of IFN-ß in cardiac myocytes. Moreover, µ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-ß response. Amino acid 208 of µ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, µ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-ß signaling mediated by reovirus µ2 amino acid 208 is a determinant of the IFN-ß response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-ß response and, consequently, damage to the heart.
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Regulación hacia Abajo , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Miocarditis/metabolismo , Orthoreovirus de los Mamíferos/genética , Polimorfismo de Nucleótido Simple , Infecciones por Reoviridae/metabolismo , Transducción de Señal , Proteínas Virales/genética , Animales , Línea Celular , Células Cultivadas , Humanos , Interferón-alfa/genética , Interferón beta/genética , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/metabolismo , Ratones , Miocarditis/genética , Miocarditis/virología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Orthoreovirus de los Mamíferos/metabolismo , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/virologíaRESUMEN
Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.