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1.
Int J Mol Sci ; 23(9)2022 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-35563635

RESUMO

Cancer cell lines responded differentially to type I interferon treatment in models of oncolytic therapy using vesicular stomatitis virus (VSV). Two opposite cases were considered in this study, glioblastoma DBTRG-05MG and osteosarcoma HOS cell lines exhibiting resistance and sensitivity to VSV after the treatment, respectively. Type I interferon responses were compared for these cell lines by integrative analysis of the transcriptome, proteome, and RNA editome to identify molecular factors determining differential effects observed. Adenosine-to-inosine RNA editing was equally induced in both cell lines. However, transcriptome analysis showed that the number of differentially expressed genes was much higher in DBTRG-05MG with a specific enrichment in inflammatory proteins. Further, it was found that two genes, EGFR and HER2, were overexpressed in HOS cells compared with DBTRG-05MG, supporting recent reports that EGF receptor signaling attenuates interferon responses via HER2 co-receptor activity. Accordingly, combined treatment of cells with EGF receptor inhibitors such as gefitinib and type I interferon increases the resistance of sensitive cell lines to VSV. Moreover, sensitive cell lines had increased levels of HER2 protein compared with non-sensitive DBTRG-05MG. Presumably, the level of this protein expression in tumor cells might be a predictive biomarker of their resistance to oncolytic viral therapy.


Assuntos
Interferon Tipo I , Terapia Viral Oncolítica , Vírus Oncolíticos , Estomatite Vesicular , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Interferon Tipo I/metabolismo , Vírus Oncolíticos/fisiologia , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/fisiologia
2.
Mol Immunol ; 143: 1-6, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34990937

RESUMO

TANK-binding kinase 1 (TBK1) plays a pivotal role in antiviral innate immunity. TBK1 mediates the activation of interferon regulatory factor (IRF) 3, leading to the induction of type I IFNs (IFN-α/ß) and of NF-κB signal transduction following viral infections. TBK1 must be tightly regulated to effectively control viral infections and maintain immune homeostasis. Here, we found that E3 ubiquitin ligase RNF19a mediated K48-linked ubiquitination and proteasomal degradation of TBK1. Specifically, the silence of RNF19a enhanced the production of type I interferons and suppressed RNA viral replication. Our results uncover that RNF19a acts as a negative mediator in the RIG-I signaling pathway to attenuate antiviral immune responses and suggest RNF19a as a potential therapy target in clinical infectious and inflammatory diseases.


Assuntos
Antivirais/imunologia , Imunidade , Proteólise , Vírus de RNA/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Herpesvirus Humano 1/fisiologia , Interferon Tipo I/metabolismo , Lisina/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/virologia , Masculino , Camundongos Endogâmicos C57BL , Ubiquitinação , Vesiculovirus/fisiologia
3.
J Med Virol ; 94(2): 480-490, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-31017674

RESUMO

Chandipura virus (CHPV) is a neurotropic virus, known to cause encephalitis in humans. The microRNAs (miRNA/miR) play an important role in the pathogenesis of viral infection. The present study is focused on the role of miRNAs during CHPV (strain 1653514) infection in human microglial cells. The deep sequencing of CHPV-infected human microglial cells identified a total of 12 differentially expressed miRNA (DEMs). To elucidate the role of DEMs, the target gene prediction, Gene Ontology term (GO Term), pathway enrichment analysis, and miRNA-messenger RNA (mRNA) interaction network analysis was performed. The GO terms and pathway enrichment analysis provided 146 enriched genes; which were involved in interferon response, cytokine and chemokine signaling. Further, the WGCNA (weighted gene coexpression network analysis) of the enriched genes were discretely categorized into three modules (blue, brown, and turquoise). The hub genes in the blue module may correlate to CHPV induced neuroinflammation. Altogether, the miRNA-mRNA interaction network and WGCNA study revealed the following pairs, hsa-miR-542-3p and FAF1, hsa-miR-92a-1-5p and MYD88, and hsa-miR-3187-3p and TNFRSF21, which may contribute to neuroinflammation during CHPV infection in human microglial cells.


Assuntos
Redes Reguladoras de Genes/genética , MicroRNAs/genética , Microglia/metabolismo , Vesiculovirus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Humanos , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/genética , /virologia , Receptores do Fator de Necrose Tumoral/genética , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/virologia
4.
Bioengineered ; 12(2): 11847-11857, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34720036

RESUMO

Blockage of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signal pathway is effective to increase the cytotoxic effects of oncolytic virus on cancer cells, but the detailed mechanisms are still largely unknown. Based on this, the present study managed to investigate the anti-tumor effects of PI3K inhibitor ZSTK474 and oncolytic vesicular stomatitis virus VSVΔ51 combination treatments on osteosarcoma (OS) in vitro and in vivo. Specifically, ZSTK474 aggravated the inhibiting effects of VSVΔ51 on osteosarcoma development by triggering endoplasmic reticulum (ER)-stress mediated apoptotic cell death. Mechanistically, either ZSTK474 or VSVΔ51 alone had limited effects on cell viability in osteosarcoma cells, while ZSTK474 and VSVΔ51 combination treatments significantly induced osteosarcoma cell apoptosis. Interestingly, VSVΔ51 increased the expression levels of IRE1α and p-PERK to initiate ER stress in osteosarcoma cells, which were aggravated by co-treating cells with ZSTK474. Next, the promoting effects of ZSTK474-VSVΔ51 combined treatment on osteosarcoma cell death were abrogated by the ER-stress inhibitor 4-phenyl butyric acid (4-PBA), indicating that ZSTK474 enhanced the cytotoxic effects of VSVΔ51 on osteosarcoma cells in an ER-stress dependent manner. Finally, the xenograft tumor-bearing mice models were established, and the results showed that ZSTK474-VSVΔ51 combined treatment synergistically hindered tumorigenesis of osteosarcoma cells in vivo. Taken together, our data suggested that ZSTK474 was a novel agent to enhance the cytotoxic effects of VSVΔ51 on osteosarcoma by aggravating ER-stress, and the present study might provide alternative therapy treatments for osteosarcoma in clinic.


Assuntos
Estresse do Retículo Endoplasmático , Vírus Oncolíticos/fisiologia , Osteossarcoma/patologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Triazinas/farmacologia , Vesiculovirus/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Osteossarcoma/ultraestrutura , Ensaios Antitumorais Modelo de Xenoenxerto
5.
mBio ; 12(6): e0271221, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34781742

RESUMO

Poliomyelitis-like illness is a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis virus (JEV) and Chandipura virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we demonstrated that upon infection, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and in vivo morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic virus infections. IMPORTANCE Neurotropic viral infections are an increasingly common cause of immediate or delayed neuropsychiatric sequelae, cognitive impairment, and movement disorders or, in severe cases, death. Given the highest reported disability-adjusted life years and mortality rate worldwide, a better understanding of molecular mechanisms for underlying clinical manifestations like AFP will help in development of more effective tools for therapeutic solutions.


Assuntos
Viroses do Sistema Nervoso Central/metabolismo , Viroses do Sistema Nervoso Central/fisiopatologia , Proteína DEAD-box 58/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Neurônios Motores/citologia , Mielite/metabolismo , Mielite/fisiopatologia , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/fisiopatologia , Vesiculovirus/fisiologia , Animais , Morte Celular , Viroses do Sistema Nervoso Central/genética , Viroses do Sistema Nervoso Central/virologia , Proteína DEAD-box 58/genética , Vírus da Encefalite Japonesa (Espécie)/genética , Feminino , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Masculino , Camundongos , Atividade Motora , Neurônios Motores/metabolismo , Neurônios Motores/virologia , Mielite/genética , Mielite/virologia , Doenças Neuromusculares/genética , Doenças Neuromusculares/virologia , Vesiculovirus/genética
6.
Arch Virol ; 166(11): 3143-3150, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34533641

RESUMO

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies.


Assuntos
Ciclofilina A/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/patogenicidade , Ciclofilina A/antagonistas & inibidores , Ciclofilina A/química , Ciclosporina/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Replicação Viral/efeitos dos fármacos
7.
Cell Rep ; 36(12): 109731, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34551290

RESUMO

TBK1 is an essential kinase for the innate immune response against viral infection. However, the key molecular mechanisms regulating the TBK1 activation remain elusive. Here, we identify PRMT1, a type I protein arginine methyltransferase, as an essential regulator of TBK1 activation. PRMT1 directly interacts with TBK1 and catalyzes asymmetric methylation of R54, R134, and R228 on TBK1. This modification enhances TBK1 oligomerization after viral infection, which subsequently promotes TBK1 phosphorylation and downstream type I interferon production. More important, myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA and RNA viruses than Prmt1fl/fl mice. Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.


Assuntos
Arginina/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Linhagem Celular , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Metilação , Camundongos , Camundongos Knockout , Fosforilação , Agregados Proteicos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/deficiência , Proteína-Arginina N-Metiltransferases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Vesiculovirus/fisiologia
8.
Cell ; 184(17): 4447-4463.e20, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34363755

RESUMO

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.


Assuntos
Inflamação/enzimologia , Fator de Necrose Tumoral alfa/farmacologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Autoimunidade/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Feminino , Células HEK293 , Homozigoto , Humanos , Quinase I-kappa B/metabolismo , Imunofenotipagem , Inflamação/patologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Mutação com Perda de Função/genética , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , /metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptor 3 Toll-Like/metabolismo , Transcriptoma/genética , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia
9.
Cell Death Dis ; 12(8): 743, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315861

RESUMO

Transcription factor IRF3 is critical for the induction of antiviral type I interferon (IFN-I). The epigenetic regulation of IFN-I production in antiviral innate immunity needs to be further identified. Here, we reported that epigenetic remodeler ARID1A, a critical component of the mSWI/SNF complex, could bind IRF3 and then was recruited to the Ifn-I promoter by IRF3, thus selectively promoting IFN-I but not TNF-α, IL-6 production in macrophages upon viral infection. Myeloid cell-specific deficiency of Arid1a rendered mice more susceptible to viral infection, accompanied with less IFN-I production. Mechanistically, ARID1A facilitates chromatin accessibility of IRF3 at the Ifn-I promoters by interacting with histone methyltransferase NSD2, which methylates H3K4 and H3K36 of the promoter regions. Our findings demonstrated the new roles of ARID1A and NSD2 in innate immunity, providing insight into the crosstalks of chromatin remodeling, histone modification, and transcription factors in the epigenetic regulation of antiviral innate immunity.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferons/biossíntese , Macrófagos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antivirais/metabolismo , Cromatina/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Imunidade Inata , Interferons/genética , Lisina/metabolismo , Metilação , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Células RAW 264.7 , Vesiculovirus/fisiologia
10.
J Biomed Sci ; 28(1): 52, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233673

RESUMO

BACKGROUND: Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1ß, in human microglial cells during CHPV infection. METHODS: To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1ß in this study. RESULTS: The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. CONCLUSIONS: This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.


Assuntos
MicroRNAs/genética , Microglia/metabolismo , NF-kappa B/genética , Vesiculovirus/fisiologia , Humanos , MicroRNAs/metabolismo , NF-kappa B/metabolismo
11.
J Virol ; 95(16): e0059421, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037421

RESUMO

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction caused by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction, and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensures the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help us to understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. IMPORTANCE It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help us to understand the polymerase function of negative-stranded RNA viruses.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vesiculovirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Peixes , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Fosfoproteínas/metabolismo , Estabilidade Proteica , RNA Viral/biossíntese , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/virologia , Vesiculovirus/metabolismo
12.
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 , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Carga Viral
13.
J Biol Chem ; 296: 100701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895135

RESUMO

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.


Assuntos
Ambroxol/farmacologia , Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Vesiculovirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Administração por Inalação , Animais , Transporte Biológico , Ceramidas/metabolismo , Chlorocebus aethiops , Reposicionamento de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Expectorantes , Expressão Gênica , Humanos , Cultura Primária de Células , Vírus Reordenados/efeitos dos fármacos , Vírus Reordenados/fisiologia , SARS-CoV-2/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Vesiculovirus/fisiologia
14.
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
15.
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 , SARS-CoV-2/fisiologia , Vesiculovirus/fisiologia , Viroses/virologia , Internalização do Vírus
16.
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
17.
J Gen Virol ; 102(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33507144

RESUMO

The zebrafish (Danio rerio) possesses evolutionarily conserved innate and adaptive immunity as a mammal and has recently become a popular vertebrate model to exploit infection and immunity. Antiviral RNA interference (RNAi) has been illuminated in various model organisms, including Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and mice. However, to date, there is no report on the antiviral RNAi pathway of zebrafish. Here, we have evaluated the possible use of zebrafish to study antiviral RNAi with Sindbis virus (SINV), vesicular stomatitis virus (VSV) and Nodamura virus (NoV). We find that SINVs and NoVs induce the production of virus-derived small interfering RNAs (vsiRNAs), the hallmark of antiviral RNAi, with a preference for a length of 22 nucleotides, after infection of larval zebrafish. Meanwhile, the suppressor of RNAi (VSR) protein, NoV B2, may affect the accumulation of the NoV in zebrafish. Furthermore, taking advantage of the fact that zebrafish argonaute-2 (Ago2) protein is naturally deficient in cleavage compared with that of mammals, we provide evidence that the slicing activity of human Ago2 can virtually inhibit the accumulation of RNA virus after being ectopically expressed in larval zebrafish. Thus, zebrafish may be a unique model organism to study the antiviral RNAi pathway.


Assuntos
Interferência de RNA , Infecções por Vírus de RNA/virologia , Vírus de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Peixe-Zebra/virologia , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Imunidade Inata , Modelos Animais , Nodaviridae/imunologia , Nodaviridae/fisiologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Vírus Sindbis/imunologia , Vírus Sindbis/fisiologia , Vesiculovirus/imunologia , Vesiculovirus/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/metabolismo
18.
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 , /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
19.
RNA Biol ; 18(4): 537-546, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32940118

RESUMO

Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.


Assuntos
Regiões 5' não Traduzidas/fisiologia , Vesiculovirus/fisiologia , Replicação Viral/genética , Animais , Células Cultivadas , Mapeamento Cromossômico , Feminino , Peixes/virologia , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Novirhabdovirus/fisiologia , Proteínas do Nucleocapsídeo/genética , Pequeno RNA não Traduzido/fisiologia , RNA Viral/genética , RNA Viral/fisiologia , Análise de Sequência de RNA , Vesiculovirus/genética
20.
Int J Mol Sci ; 21(23)2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33261178

RESUMO

Mast cells (MCs) are critical for initiating inflammatory responses to pathogens including viruses. Type I interferons (IFNs) that exert their antiviral functions by interacting with the type I IFN receptor (IFNAR) play a central role in host cellular responses to viruses. Given that virus-induced excessive toxic inflammatory responses are associated with aberrant IFNAR signaling and considering MCs are an early source of inflammatory cytokines during viral infections, we sought to determine whether IFNAR signaling plays a role in antiviral cytokine responses of MCs. IFNAR-intact, IFNAR-blocked, and IFNAR-knockout (IFNAR-/-) bone-marrow-derived MCs (BMMCs) were treated in vitro with a recombinant vesicular stomatitis virus (rVSVΔm51) to assess cytokine production by these cells. All groups of MCs produced the cytokines interleukin-6 and tumor necrosis factor-α in response to rVSVΔm51. However, production of the cytokines was lowest in IFNAR-intact cells as compared with IFNAR-/- or IFNAR-blocked cells at 20 h post-stimulation. Surprisingly, rVSVΔm51 was capable of infecting BMMCs, but functional IFNAR signaling was able to protect these cells from virus-induced death. This study showed that BMMCs produced pro-inflammatory cytokines in response to rVSVΔm51 and that IFNAR signaling was required to down-modulate these responses and protect the cells from dying from viral infection.


Assuntos
Células da Medula Óssea/patologia , Citocinas/biossíntese , Citoproteção , Mastócitos/virologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Vesiculovirus/fisiologia , Animais , Morte Celular , Regulação para Baixo , Interleucina-6/metabolismo , Cinética , Camundongos Knockout , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
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