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1.
Adv Virus Res ; 104: 147-183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31439148

RESUMO

Rhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus.


Assuntos
Interações Hospedeiro-Patógeno , Vírus da Raiva/fisiologia , Vesiculovirus/fisiologia , Ligação Viral , Internalização do Vírus , Endocitose , Glicoproteínas/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo
2.
PLoS Pathog ; 15(6): e1007875, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226162

RESUMO

Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5' cap-structure and 3'-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)-encoded by 2 such mRNAs-support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function.


Assuntos
Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Replicação Viral/fisiologia , Células HeLa , Humanos
3.
Neuroimage ; 197: 133-142, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022567

RESUMO

The elucidation of neural networks is essential to understanding the mechanisms of brain functions and brain disorders. Neurotropic virus-based trans-synaptic tracing tools have become an effective method for dissecting the structure and analyzing the function of neural-circuitry. However, these tracing systems rely on fluorescent signals, making it hard to visualize the panorama of the labeled networks in mammalian brain in vivo. One MRI method, Diffusion Tensor Imaging (DTI), is capable of imaging the networks of the whole brain in live animals but without information of anatomical connections through synapses. In this report, a chimeric gene coding for ferritin and enhanced green fluorescent protein (EGFP) was integrated into Vesicular stomatitis virus (VSV), a neurotropic virus that is able to spread anterogradely in synaptically connected networks. After the animal was injected with the recombinant VSV (rVSV), rVSV-Ferritin-EGFP, into the somatosensory cortex (SC) for four days, the labeled neural-network was visualized in the postmortem whole brain with a T2-weighted MRI sequence. The modified virus transmitted from SC to synaptically connected downstream regions. The results demonstrate that rVSV-Ferritin-EGFP could be used as a bimodal imaging vector for detecting synaptically connected neural-network with both ex vivo MRI and fluorescent imaging. The strategy in the current study has the potential to longitudinally monitor the global structure of a given neural-network in living animals.


Assuntos
Mapeamento Encefálico/métodos , Imagem por Ressonância Magnética , Neurônios/citologia , Córtex Somatossensorial/citologia , Vesiculovirus/fisiologia , Animais , Ferritinas/genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/virologia , Neurônios/virologia , Córtex Somatossensorial/virologia , Vesiculovirus/genética
4.
mBio ; 10(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622188

RESUMO

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (biosafety level 3 [BSL-3]) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn and Gc (Gn/Gc) entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantaviruses, Hantaan virus (HTNV) or Dobrava-Belgrade virus (DOBV), were refractory to rescue. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations: I532K in the cytoplasmic tail of Gn and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Finally, I532K/S1094L mutations in DOBV Gn/Gc permitted the rescue of rVSV-DOBV Gn/Gc, demonstrating that incorporation of cognate mutations into other hantaviral Gn/Gc proteins could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSVs, bearing HTNV and DOBV Gn/Gc, reported herein may also have utility as vaccines.IMPORTANCE Human hantavirus infections cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSVs encoding Hantaan virus and Dobrava-Belgrade virus glycoproteins described in this work provide a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as hantavirus vaccines.


Assuntos
Vetores Genéticos , Hantavirus/genética , Proteínas Mutantes/genética , Mutação Puntual , Proteínas Recombinantes/genética , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Linhagem Celular , Glicoproteínas/genética , Humanos , Genética Reversa , Inoculações Seriadas , Vesiculovirus/fisiologia , Liberação de Vírus , Replicação Viral
5.
J Biotechnol ; 289: 144-149, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30508556

RESUMO

During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium.


Assuntos
Vesiculovirus/fisiologia , Animais , Contagem de Células , Linhagem Celular , Meios de Cultura Livres de Soro , Proteínas de Fluorescência Verde , Humanos , Replicação Viral
6.
Fish Shellfish Immunol ; 86: 46-52, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30447429

RESUMO

Snakehead vesiculovirus (SHVV) has caused great economic loss in snakehead fish culture in China. However, there is no effective strategy to prevent the epidemic of the virus. Understanding the host factors in response to virus infection is the basis for the prevention of viral disease. In this study, the transcriptomic profiles of SHVV-infected and mock-infected SSN-1 cells (derived from striped snakehead, Channa striatus) at 3 and 24 h (h) post of infection (poi) were obtained using high-throughput sequencing technique. A total of 93,372 unigenes were obtained. The differently expressed genes (DEGs) of SSN-1 cells upon SHVV infection were thereby identified, including 3668 and 3536 DEGs at 3 and 24 h poi, respectively. These DEGs were involved in many pathways of viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Therefore, several immune-related DEGs were randomly selected and confirmed by quantitative real-time PCR (qRT-PCR). In addition, the effects of the interferon inducible protein 35 (IFI35) on SHVV replication were further investigated. Over-expression or inhibition of IFI35 significantly promoted or reduced SHVV replication at the level of viral gene expression, which indicated that IFI35 might be a positive factor for SHVV replication in SSN-1 cells. Our findings presented some valuable information, which will benefit for future study on SHVV-host interactions.


Assuntos
Quimiocinas/metabolismo , Peixes , Transcriptoma/fisiologia , Vesiculovirus/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/imunologia
7.
Clin Exp Immunol ; 195(1): 64-73, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30444956

RESUMO

Viral infections can be fatal because of the direct cytopathic effects of the virus or the induction of a strong, uncontrolled inflammatory response. Virus and host intrinsic characteristics strongly modulate the outcome of viral infections. Recently we determined the circumstances under which enhanced replication of virus within the lymphoid tissue is beneficial for the outcome of a disease. This enforced viral replication promotes anti-viral immune activation and, counterintuitively, accelerates virus control. In this review we summarize the mechanisms that contribute to enforced viral replication. Antigen-presenting cells and CD169+ macrophages exhibit enforced viral replication after infection with the model viruses lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). Ubiquitin-specific peptidase 18 (Usp18), an endogenous type I interferon blocker in CD169+ macrophages, has been identified as a proviral gene, as are B cell activating factor (BAFF) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Lymphotoxins (LT) strongly enhance viral replication in the spleen and lymph nodes. All these factors modulate splenic architecture and thereby promote the development of CD169+ macrophages. Tumor necrosis factor alpha (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cell signaling (NF-κB) have been found to promote the survival of infected CD169+ macrophages, thereby similarly promoting enforced viral replication. Association of autoimmune disease with infections is evident from (1) autoimmune phenomena described during a chronic virus infection; (2) onset of autoimmune disease simultaneous to viral infections; and (3) experimental evidence. Involvement of virus infection during onset of type I diabetes is strongly evident. Epstein-Bar virus (EBV) infection was discussed to be involved in the pathogenesis of systemic lupus erythematosus. In conclusion, several mechanisms promote viral replication in secondary lymphatic organs. Identifying such factors in humans is a challenge for future studies.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Sistema Linfático/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Vesiculovirus/fisiologia , Viroses/imunologia , Replicação Viral , Animais , Diabetes Mellitus Tipo 1/virologia , Herpesvirus Humano 4 , Interações Hospedeiro-Patógeno , Humanos , Lúpus Eritematoso Sistêmico/virologia , Sistema Linfático/virologia , Especificidade de Órgãos , Viroses/virologia
8.
Eur J Immunol ; 49(1): 42-53, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30466171

RESUMO

Retinoic acid-inducible gene I (RIG-I) is a critical RNA virus sensor that initiates antiviral immune response through K63-linked ubiquitination. In this study, we demonstrated USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by directly deubiquitinating K63-linked RIG-I. USP14 knockdown significantly enhanced RIG-I-triggered type I IFN signaling and inhibited vesicular stomatitis virus (VSV) replication both in mouse peritoneal macrophages and THP1 cells. USP14 overexpression in HeLa cells attenuated RIG-I-triggered IFN-ß expression and promoted VSV replication. Besides, USP14-specific inhibitor, IU1, increased RIG-I-mediated type I IFN production and antiviral responses in vitro and in vivo. In addition, USP14 could interact with RIG-I and remove RIG-I K63-linked polyubiquitination chains. This article is the first to report that USP14 acts as a negative regulator in antiviral response through deubiquitinating K63-linked RIG-I. These findings provide insights into a potential new therapy targeting USP14 for RNA virus-related diseases.


Assuntos
Macrófagos/imunologia , Infecções por Rhabdoviridae/imunologia , Ubiquitina Tiolesterase/metabolismo , Vesiculovirus/fisiologia , Animais , Proteína DEAD-box 58/metabolismo , Feminino , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Transdução de Sinais , Células THP-1 , Ubiquitina Tiolesterase/genética , Ubiquitinação , Replicação Viral
9.
Fish Shellfish Immunol ; 84: 299-303, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30308292

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.


Assuntos
Doenças dos Peixes/fisiopatologia , Proteínas de Peixes/genética , Glicogênio Sintase/genética , MicroRNAs/genética , Perciformes , RNA Viral/genética , Infecções por Rhabdoviridae/veterinária , Animais , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Glicogênio Sintase/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Infecções por Rhabdoviridae/fisiopatologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/genética , Vesiculovirus/fisiologia
10.
Fish Shellfish Immunol ; 86: 1044-1052, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30590160

RESUMO

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.


Assuntos
Autofagia , Glutamina/metabolismo , Dissulfeto de Glutationa/metabolismo , Perciformes/virologia , Vesiculovirus/fisiologia , Animais , Butionina Sulfoximina , Linhagem Celular , Glutationa , Perciformes/fisiologia , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/veterinária , Replicação Viral
11.
Curr Biol ; 28(20): 3212-3219.e4, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30318351

RESUMO

In addition to the conventional release of free, individual virions, virus dispersal can involve multi-virion assemblies that collectively infect cells. However, the implications of collective infection for viral fitness remain largely unexplored. Using vesicular stomatitis virus, here, we compare the fitness of free versus saliva-aggregated viral particles. We find that aggregation has a positive effect on early progeny production, conferring a fitness advantage relative to equal numbers of free particles in most cell types. The advantage of aggregation resides, at least partially, in increasing the cellular multiplicity of infection. In mouse embryonic fibroblasts, the per capita, short-term viral progeny production peaked for a dose of ca. three infectious particles per cell. This reveals an Allee effect restricting early viral proliferation at the cellular level, which should select for dispersal in groups. We find that genetic complementation between deleterious mutants is probably not the mechanism underlying the fitness advantage of collective infection. Instead, this advantage is cell type dependent and correlates with cellular permissivity to the virus, as well as with the ability of host cells to mount an antiviral innate immune response.


Assuntos
Saliva/virologia , Seleção Genética , Vesiculovirus/fisiologia , Replicação Viral/fisiologia , Células A549 , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Vero , Vesiculovirus/genética
12.
mBio ; 9(5)2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30181255

RESUMO

RNA viruses that replicate in the cell cytoplasm typically concentrate their replication machinery within specialized compartments. This concentration favors enzymatic reactions and shields viral RNA from detection by cytosolic pattern recognition receptors. Nonsegmented negative-strand (NNS) RNA viruses, which include some of the most significant human, animal, and plant pathogens extant, form inclusions that are sites of RNA synthesis and are not circumscribed by a membrane. These inclusions share similarities with cellular protein/RNA structures such as P granules and nucleoli, which are phase-separated liquid compartments. Here we show that replication compartments of vesicular stomatitis virus (VSV) have the properties of liquid-like compartments that form by phase separation. Expression of the individual viral components of the replication machinery in cells demonstrates that the 3 viral proteins required for replication are sufficient to drive cytoplasmic phase separation. Therefore, liquid-liquid phase separation, previously linked to organization of P granules, nucleolus homeostasis, and cell signaling, plays a key role in host-pathogen interactions. This work suggests novel therapeutic approaches to the problem of combating NNS RNA viral infections.IMPORTANCE RNA viruses compartmentalize their replication machinery to evade detection by host pattern recognition receptors and concentrate the machinery of RNA synthesis. For positive-strand RNA viruses, RNA replication occurs in a virus-induced membrane-associated replication organelle. For NNS RNA viruses, the replication compartment is a cytoplasmic inclusion that is not circumscribed by a cellular membrane. Such structures were first observed in the cell bodies of neurons from humans infected with rabies virus and were termed Negri bodies. How the replication machinery that forms this inclusion remains associated in the absence of a membrane has been an enduring mystery. In this article, we present evidence that the VSV replication compartments form through phase separation. Phase separation is increasingly recognized as responsible for cellular structures as diverse as processing bodies (P-bodies) and nucleoli and was recently demonstrated for rabies virus. This article further links the fields of host-pathogen interaction with that of phase separation.


Assuntos
Grânulos Citoplasmáticos/virologia , Interações entre Hospedeiro e Microrganismos , Corpos de Inclusão Viral/fisiologia , Vesiculovirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Compartimento Celular , Linhagem Celular , RNA Viral , Células Vero , Proteínas Virais/genética
13.
PLoS Negl Trop Dis ; 12(7): e0006648, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30001342

RESUMO

Chandipura Virus (CHPV), a negative-stranded RNA virus belonging to the Rhabdoviridae family, has been previously reported to bring neuronal apoptosis by activating several factors leading to neurodegeneration. Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues gets activated and starts secreting chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the effect of CHPV on microglia and the activation of cellular signalling pathways leading to chemokine expression upon CHPV infection. Protein and mRNA expression profiles of chemokine genes revealed that CHPV infection strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. CHPV infection triggered the activation of signalling pathways mediated by mitogen-activated protein kinases, including p38, JNK 1 and 2, and nuclear factor κB (NF-kappaB). CHPV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-kappaB pathways. Considering the important role of inflammation in neurodegeneration, we have targeted NF-kappaB using a newly synthesised natural product nitrosporeusine analogue and showed incapability of microglial supernatant of inducing apoptosis in neurons after treatment.


Assuntos
Alcaloides/administração & dosagem , Antivirais/administração & dosagem , Doenças do Sistema Nervoso Central/tratamento farmacológico , Microglia/imunologia , NF-kappa B/imunologia , Infecções por Rhabdoviridae/imunologia , Vesiculovirus/fisiologia , Animais , Linhagem Celular , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/imunologia , Doenças do Sistema Nervoso Central/virologia , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Humanos , Camundongos , Microglia/efeitos dos fármacos , Microglia/virologia , NF-kappa B/genética , Infecções por Rhabdoviridae/tratamento farmacológico , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/virologia , Transdução de Sinais/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/genética
14.
Viruses ; 10(6)2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29874821

RESUMO

Cellular kinases are crucial for the transcription/replication of many negative-strand RNA viruses and might serve as targets for antiviral therapy. In this study, a library comprising 80 kinase inhibitors was screened for antiviral activity against vesicular stomatitis virus (VSV), a prototype member of the family Rhabdoviridae. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125), an inhibitor of eukaryotic elongation factor 2 (eEF2) kinase, significantly inhibited entry of single-cycle VSV encoding a luciferase reporter. Treatment of virus particles had only minimal effect on virus entry, indicating that the compound primarily acts on the host cell rather than on the virus. Accordingly, resistant mutant viruses were not detected when the virus was passaged in the presence of the drug. Unexpectedly, NH125 led to enhanced, rather than reduced, phosphorylation of eEF2, however, it did not significantly affect cellular protein synthesis. In contrast, NH125 revealed lysosomotropic features and showed structural similarity with N-dodecylimidazole, a known lysosomotropic agent. Related alkylated imidazolium compounds also exhibited antiviral activity, which was critically dependent on the length of the alkyl group. Apart from VSV, NH125 inhibited infection by VSV pseudotypes containing the envelope glycoproteins of viruses that are known to enter cells in a pH-dependent manner, i.e. avian influenza virus (H5N1), Ebola virus, and Lassa virus. In conclusion, we identified an alkylated imidazolium compound which inhibited entry of several viruses not because of the previously postulated inhibition of eEF2 kinase but most likely because of its lysosomotropic properties.


Assuntos
Antivirais/farmacologia , Imidazóis/farmacologia , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Ebolavirus/efeitos dos fármacos , Células HeLa , Humanos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Vírus Lassa/efeitos dos fármacos , Células Vero
15.
Jpn J Infect Dis ; 71(3): 205-208, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29709967

RESUMO

Crimean-Congo hemorrhagic fever virus is a risk group 4 pathogen, which mandates the use of maximum containment facilities, often termed biosafety level 4 or containment level 4 when working with infectious materials. Diagnostic and research work involving live viruses in such laboratories is time-consuming and inconvenient, resulting in delays. Herein, we show that serum neutralizing activity against the virus can be measured in low-containment laboratories using a pseudotyped virus.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia/diagnóstico , Testes de Neutralização/métodos , Vesiculovirus , Virologia/métodos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Vírus da Febre Hemorrágica da Crimeia-Congo/química , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Humanos , Células Vero , Vesiculovirus/genética , Vesiculovirus/fisiologia
16.
J Vis Exp ; (134)2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29683442

RESUMO

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Neoplasias/virologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Interferência de RNA , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Neoplasias/terapia , Vírus Oncolíticos/genética , RNA Interferente Pequeno/genética , Transfecção , Vírus Vaccinia/genética , Vírus Vaccinia/fisiologia , Vesiculovirus/genética , Vesiculovirus/fisiologia , Replicação Viral
18.
Biochem Biophys Res Commun ; 498(3): 537-543, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29518389

RESUMO

Protein ubiquitination and deubiquitination enzymes are widely involved in innate immune responses. The ubiquitin specific protease 25 (USP25), a deubiquitinating enzyme, has been demonstrated to play an important role in virus infection and immunity. However, how USP25 is degraded and regulated by E3 ubiquitin ligases remains poorly understood. Here, we identified Smad ubiquitin regulatory factor 1(Smurf1) as a first novel E3 ubiquitin ligase of USP25. Smurf1 overexpression decreases USP25 protein turnover, and the E3 ligase enzymatic activity of Smurf1 is required for USP25 degradation. Additionally, Smurf1-mediated degradation of USP25 is via promoting the K48-linkage polyubiquitination of USP25 in an ubiquitin proteasome dependent pathway. Importantly, USP25 overexpression restricts vesicular stomatitis virus (VSV) replication and the restriction of VSV replication by USP25 is enhanced in Smurf1 stable knock down cells. Therefore, our study firstly identified that Smurf1 negatively regulated the antiviral function mediated by USP25. Our findings revealed a previously unrecognized role of Smurf1 acting on USP25 and also their roles in the regulation of VSV replications.


Assuntos
Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Replicação Viral , Células HEK293 , Células HeLa , Humanos , Proteólise , Ubiquitina/metabolismo
19.
J Virol ; 92(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29142134

RESUMO

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.


Assuntos
Interferon Tipo I/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Estomatite Vesicular/imunologia , Imunidade Adaptativa , Animais , Imunidade Inata , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Fator de Transcrição RelA/metabolismo , Vesiculovirus/fisiologia , Replicação Viral
20.
Mol Cancer Ther ; 17(1): 316-326, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29158470

RESUMO

Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single-shot systemic therapy with a vesicular stomatitis virus (VSV)-IFNß-NIS, a novel recombinant oncolytic VSV, can induce curative remission in tumor-bearing mice. Clinical translation of VSV-IFNß-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression, and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNß-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNß-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNß-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNß-NIS therapy, and provided preliminary evidence of clinical efficacy and potential biomarkers that correlate with therapeutic response. Mol Cancer Ther; 17(1); 316-26. ©2017 AACR.


Assuntos
Doenças do Cão/terapia , Doenças do Cão/virologia , Neoplasias/veterinária , Terapia Viral Oncolítica/métodos , Vesiculovirus/fisiologia , Administração Intravenosa , Animais , Cães , Feminino , Neoplasias/terapia , Neoplasias/virologia , Animais de Estimação
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