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1.
mBio ; 12(2)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849978

RESUMO

Mammalian cells detect microbial molecules known as pathogen-associated molecular patterns (PAMPs) as indicators of potential infection. Upon PAMP detection, diverse defensive responses are induced by the host, including those that promote inflammation and cell-intrinsic antimicrobial activities. Host-encoded molecules released from dying or damaged cells, known as damage-associated molecular patterns (DAMPs), also induce defensive responses. Both DAMPs and PAMPs are recognized for their inflammatory potential, but only the latter are well established to stimulate cell-intrinsic host defense. Here, we report a class of DAMPs that engender an antiviral state in human epithelial cells. These DAMPs include oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine), PGPC (1-palmitoyl-2-glutaryl phosphatidylcholine), and POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine], oxidized lipids that are naturally released from dead or dying cells. Exposing cells to these DAMPs prior to vesicular stomatitis virus (VSV) infection limits viral replication. Mechanistically, these DAMPs prevent viral entry, thereby limiting the percentage of cells that are productively infected and consequently restricting viral load. We found that the antiviral actions of oxidized lipids are distinct from those mediated by the PAMP Poly I:C, in that the former induces a more rapid antiviral response without the induction of the interferon response. These data support a model whereby interferon-independent defensive activities can be induced by DAMPs, which may limit viral replication before PAMP-mediated interferon responses are induced. This antiviral activity may impact viruses that disrupt interferon responses in the oxygenated environment of the lung, such as influenza virus and SARS-CoV-2.IMPORTANCE In this work, we explored how a class of oxidized lipids, spontaneously created during tissue damage and unprogrammed cell lysis, block the earliest events in RNA virus infection in the human epithelium. This gives us novel insight into the ways that we view infection models, unveiling a built-in mechanism to slow viral growth that neither engages the interferon response nor is subject to known viral antagonism. These oxidized phospholipids act prior to infection, allowing time for other, better-known innate immune mechanisms to take effect. This discovery broadens our understanding of host defenses, introducing a soluble factor that alters the cellular environment to protect from RNA virus infection.


Assuntos
Alarminas/farmacologia , Antivirais/farmacologia , Vírus de RNA/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Morte Celular/efeitos dos fármacos , Humanos , Imunidade Inata , Interferons/genética , Interferons/metabolismo , Cinética , Padrões Moleculares Associados a Patógenos/farmacologia , Fosfatidilcolinas/farmacologia , Vírus de RNA/fisiologia , /fisiologia , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Carga Viral
2.
Fish Shellfish Immunol ; 113: 24-34, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33757800

RESUMO

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.


Assuntos
Apoptose , Doenças dos Peixes/imunologia , Peixes , Infecções por Rhabdoviridae/veterinária , Transdução de Sinais/imunologia , Vesiculovirus/fisiologia , Proteínas da Matriz Viral/fisiologia , Animais , Linhagem Celular , Doenças dos Peixes/virologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia
3.
Viruses ; 13(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669141

RESUMO

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus-host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral-host interplay at differing levels of complexity-in individual hosts, organs, tissues and cells-and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid-liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Fenômenos Fisiológicos Virais , Animais , Fenômenos Biofísicos , HIV/fisiologia , Humanos , Vírus da Influenza A/fisiologia , Morbillivirus/fisiologia , Organelas/virologia , Vesiculovirus/fisiologia , Viroses/virologia , Internalização do Vírus
4.
Nat Commun ; 12(1): 1362, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649317

RESUMO

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.


Assuntos
Vírus de RNA/genética , Vírus de RNA/fisiologia , Replicação Viral/genética , Animais , Linhagem Celular Tumoral , Genoma Viral , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Humanos , Camundongos Endogâmicos NOD , Fosfoproteínas/metabolismo , Multimerização Proteica , Vírus de RNA/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/genética , Vesiculovirus/fisiologia , Replicação Viral/efeitos dos fármacos
5.
PLoS Pathog ; 17(1): e1009111, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33411856

RESUMO

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.


Assuntos
Antivirais/metabolismo , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Proteínas/metabolismo , Infecções por Rhabdoviridae/imunologia , Via Secretória , Vesiculovirus/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Vesiculovirus/fisiologia
6.
Virology ; 543: 54-62, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32056847

RESUMO

Hantaviruses are rodent-borne hemorrhagic fever viruses leading to serious diseases. Viral attachment and entry represent the first steps in virus transmission and are promising targets for antiviral therapeutic intervention. Here we investigated receptor use in human airway epithelium of the Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV). Using a biocontained recombinant vesicular stomatitis virus pseudotype platform, we provide first evidence for a role of the cellular phosphatidylserine (PS) receptors of the T-cell immunoglobulin and mucin (TIM) protein family in HTNV and ANDV infection. In line with previous studies, HTNV, but not ANDV, was able to use glycosaminoglycan heparan sulfate and αvß3 integrin as co-receptors. In sum, our studies demonstrate for the first time that hantaviruses make use of apoptotic mimicry for infection of human airway epithelium, which may explain why these viruses can easily break the species barrier.


Assuntos
Vírus Hantaan/metabolismo , Hantavirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Bacteriocinas/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vírus Hantaan/efeitos dos fármacos , Vírus Hantaan/patogenicidade , Vírus Hantaan/fisiologia , Hantavirus/fisiologia , Haplorrinos , Heparitina Sulfato/farmacologia , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Mimetismo Molecular , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Vesiculovirus/metabolismo , Vesiculovirus/fisiologia
7.
Microb Pathog ; 140: 103957, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31891795

RESUMO

BACKGROUND: Activation of the immune system to fight cancer is a major goal in immunology and oncology. Although cancer treatment using oncolytic viruses shows promising results, virus mediated oncolysis induces a weak anti-tumor immune response. Upon application of viruses, immune responses against the virus play a significant role in limiting tumor virotherapy. Although suppression of host immunity increases the efficacy of virotherapy against the tumor, but inhibits anti-tumor immune responses. Induction of viral specific tolerance before viral replication may cause the virus to efficiently replicate in tumor cells without affecting the immune responses against tumor antigens. Investigation of the combined strategy of virotherapy and immunotherapy using irradiated tumor cells along with IL-2 and interferon-alpha in virus specific tolerant mice was the goal of this study. MATERIALS AND METHODS: For tolerance induction, the newborn mice were injected with vesicular stomatitis virus (VSV) subcutaneously. After injection of TC-1 tumor cells to adult tolerant mice and formation of a tumor, irradiated TC-1 cells along with IL-2 and Interferon-alpha expression plasmid were injected twice in mice and followed by virotherapy. Size of tumors and CTL activity against the virus and tumor cells were measured. RESULT: The results showed increased efficacy of virotherapy in combination with immune-stimulators and tumor cells injection in tolerant mice compared to normal mice. CONCLUSION: Specific tolerance against the oncolytic virus enhances the efficacy of virotherapy both in monotherapy and in combination with immunotherapy.


Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Animais , Feminino , Humanos , Tolerância Imunológica , Interleucina-2/genética , Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/virologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Vírus Oncolíticos/fisiologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Vesiculovirus/fisiologia , Replicação Viral
8.
Viruses ; 13(1)2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33396704

RESUMO

We previously demonstrated, using the Piry virus model, that environmental enrichment promotes higher T-cell infiltration, fewer microglial changes, and faster central nervous system (CNS) virus clearance in adult mice. However, little is known about disease progression, behavioral changes, CNS cytokine concentration, and neuropathology in limbic encephalitis in experimental models. Using Cocal virus, we infected C57Bl6 adult mice and studied the neuroanatomical distribution of viral antigens in correlation with the microglial morphological response, measured the CNS cytokine concentration, and assessed behavioral changes. C57Bl6 adult mice were maintained in an impoverished environment (IE) or enriched environment (EE) for four months and then subjected to the open field test. Afterwards, an equal volume of normal or virus-infected brain homogenate was nasally instilled. The brains were processed to detect viral antigens and microglial morphological changes using selective immunolabeling. We demonstrated earlier significant weight loss and higher mortality in IE mice. Additionally, behavioral analysis revealed a significant influence of the environment on locomotor and exploratory activity that was associated with less neuroinvasion and a reduced microglial response. Thus, environmental enrichment was associated with a more effective immune response in a mouse model of limbic encephalitis, allowing faster viral clearance/decreased viral dissemination, reduced disease progression, and less CNS damage.


Assuntos
Encéfalo/patologia , Encéfalo/virologia , Encefalite Límbica/patologia , Encefalite Límbica/virologia , Vesiculovirus/fisiologia , Animais , Antígenos Virais/imunologia , Comportamento Animal , Biomarcadores , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Microglia/patologia , Microglia/virologia , Mortalidade , Neuropatologia , Avaliação de Sintomas , Carga Viral
9.
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
10.
Science ; 365(6458): 1171-1176, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31439758

RESUMO

Host cell metabolism can be modulated by viral infection, affecting viral survival or clearance. Yet the cellular metabolism rewiring mediated by the N 6-methyladenosine (m6A) modification in interactions between virus and host remains largely unknown. Here we report that in response to viral infection, host cells impair the enzymatic activity of the RNA m6A demethylase ALKBH5. This behavior increases the m6A methylation on α-ketoglutarate dehydrogenase (OGDH) messenger RNA (mRNA) to reduce its mRNA stability and protein expression. Reduced OGDH decreases the production of the metabolite itaconate that is required for viral replication. With reduced OGDH and itaconate production in vivo, Alkbh5-deficient mice display innate immune response-independent resistance to viral exposure. Our findings reveal that m6A RNA modification-mediated down-regulation of the OGDH-itaconate pathway reprograms cellular metabolism to inhibit viral replication, proposing potential targets for controlling viral infection.


Assuntos
Adenosina/análogos & derivados , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Imunidade Inata , Complexo Cetoglutarato Desidrogenase/metabolismo , Vesiculovirus/patogenicidade , Replicação Viral , Adenosina/química , Animais , Células Cultivadas , Reprogramação Celular , Humanos , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/química , Succinatos , Células THP-1 , Vesiculovirus/fisiologia
11.
J Immunother Cancer ; 7(1): 189, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315674

RESUMO

BACKGROUND: Cancer immunotherapies are emerging as promising treatment strategies for ovarian cancer patients that experience disease relapse following first line therapy. As such, identifying strategies to bolster anti-tumor immunity and limit immune suppression, while recognizing diverse patterns of tumor response to immunotherapy is critical to selecting treatment combinations that lead to durable therapeutic benefit. METHODS: Using a pre-clinical mouse model, we evaluated a heterologous prime/boost vaccine in combination with checkpoint blockade to treat metastatic intraperitoneal ovarian cancer. Vaccine-elicited CD8+ T cell responses and changes in the tumor microenvironment following treatment were analyzed and compared to treatment outcome. Kinetics of intraperitoneal tumor growth were assessed using non-invasive magnetic resonance imaging (MRI). RESULTS: Vaccine priming followed by antigen-armed oncolytic Maraba virus boosting elicited robust tumor-specific CD8+ T cell responses that improved tumor control and led to unique immunological changes in the tumor, including a signature that correlated with improved clinical outcome of ovarian cancer patients. However, this treatment was not curative and T cells in the tumor microenvironment (TME) were functionally suppressed. Combination PD-1 blockade partially overcame the adaptive resistance in the tumor observed in response to prime/boost vaccination, restoring CD8+ T cell function in the TME and enhancing the therapeutic response. Non-invasive MRI of tumors during the course of combination treatment revealed heterogeneous radiologic response patterns following treatment, including pseudo-progression, which was associated with improved tumor control prior to relapse. CONCLUSIONS: Our findings point to a key hierarchical role for PD-1 signaling and adaptive immune resistance in the ovarian TME in determining the functional fate of tumor-specific CD8+ T cells, even in the context of robust therapy mediated anti-tumor immunity, as well as the ability of multiple unique patterns of therapeutic response to result in durable tumor control.


Assuntos
Antígenos de Neoplasias/genética , Vacinas Anticâncer/administração & dosagem , Oxirredutases Intramoleculares/genética , Ovalbumina/genética , Neoplasias Ovarianas/terapia , Vesiculovirus/fisiologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Humanos , Oxirredutases Intramoleculares/imunologia , Camundongos , Metástase Neoplásica , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Ovalbumina/imunologia , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/imunologia , Resultado do Tratamento , Microambiente Tumoral , Vesiculovirus/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
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
13.
J Virol ; 93(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31043529

RESUMO

Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-ß) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-ß in response to virus infections. Unexpectedly, infection of Rela -/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.IMPORTANCE RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-ß, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.


Assuntos
Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno , Infecções por Rhabdoviridae/metabolismo , Fator de Transcrição RelA/metabolismo , Vesiculovirus/fisiologia , Células 3T3 , Animais , Linhagem Celular , Chlorocebus aethiops , Embrião de Mamíferos/patologia , Embrião de Mamíferos/virologia , Fibroblastos/patologia , Fibroblastos/virologia , Camundongos , Infecções por Rhabdoviridae/patologia , Células Vero
14.
Front Immunol ; 10: 466, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30930901

RESUMO

Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7-/- mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7-/- mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7-/- mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.


Assuntos
Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Infecções por Rhabdoviridae/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Receptor 7 Toll-Like/imunologia , Vesiculovirus/fisiologia , Replicação Viral/imunologia , Animais , Encéfalo/imunologia , Encéfalo/virologia , Macrófagos/virologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/patologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Medula Espinal/imunologia , Medula Espinal/virologia , Receptor 7 Toll-Like/genética , Replicação Viral/genética
15.
Cell Death Dis ; 10(5): 346, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31024004

RESUMO

Early detection of viruses by the innate immune system is crucial for host defense. The NLRP3 inflammasome, through activation of caspase-1, promotes the maturation of IL-1ß and IL-18, which are critical for antiviral immunity and inflammatory response. However, the mechanism by which viruses activate this inflammasome is still debated. Here, we report that the replication of cytopathogenic RNA viruses such as vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) induced a lytic cell death leading to potassium efflux, the common trigger of NLRP3 inflammasome activation. This lytic cell death was not prevented by a chemical or genetic inhibition of apoptosis, pyroptosis, or necroptosis but required the viral replication. Hence, the viruses that stimulated type I IFNs production after their sensing did not activate NLRP3 inflammasome due to an inhibition of their replication. In contrast, NLRP3 inflammasome activation induced by RNA virus infection was stimulated in IFNAR-deficient or MAVS-deficient cells consequently to an increased viral replication and ensuing lytic cell death. Therefore, in a context of inefficient IFN response, viral replication-induced lytic cell death activates of the NLRP3 inflammasome to fight against infection.


Assuntos
Vírus da Encefalomiocardite/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , Vesiculovirus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células da Medula Óssea/citologia , Dinaminas/antagonistas & inibidores , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Necroptose , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Replicação Viral
16.
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
17.
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
18.
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 , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Proteínas de Fluorescência Verde , Humanos , Replicação Viral
19.
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
20.
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
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