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1.
PLoS Pathog ; 13(9): e1006610, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28957419

RESUMO

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.


Assuntos
Antígenos de Diferenciação/metabolismo , Vírion , Replicação Viral , Linhagem Celular , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Internalização do Vírus
2.
J Virol ; 89(20): 10467-81, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26246581

RESUMO

UNLABELLED: Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE: BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.


Assuntos
Vírus Bluetongue/genética , Células Endoteliais/virologia , Regulação Viral da Expressão Gênica , Genoma Viral , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/virologia , Vírus Bluetongue/química , Vírus Bluetongue/metabolismo , Linhagem Celular Transformada , Ceratopogonidae , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Plexo Corióideo/virologia , Cricetulus , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Especificidade de Hospedeiro , Camundongos , Dados de Sequência Molecular , Cultura Primária de Células , Estabilidade Proteica , Proteólise , Genética Reversa , Ovinos , Transdução de Sinais , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Liberação de Vírus/genética
3.
J Virol ; 89(1): 535-44, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25339764

RESUMO

UNLABELLED: Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A. IMPORTANCE: BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.


Assuntos
Retrovirus Jaagsiekte de Ovinos/imunologia , Retrovirus Jaagsiekte de Ovinos/fisiologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/antagonistas & inibidores , Vírion/metabolismo , Montagem de Vírus , Animais , Complexo de Golgi/metabolismo , Transporte Proteico , Ovinos
4.
J Virol ; 86(17): 9015-24, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22674991

RESUMO

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT), a hemorrhagic disease of ruminants that can cause high levels of morbidity and mortality. BTV is an arbovirus transmitted between its ruminant hosts by Culicoides biting midges (Diptera: Ceratopogonidae). Recently, Europe has experienced some of the largest BT outbreaks ever recorded, including areas with no known history of the disease, leading to unprecedented economic and animal welfare issues. The current lack of genomic resources and genetic tools for Culicoides restricts any detailed study of the mechanisms involved in the virus-insect interactions. In contrast, the genome of the fruit fly (Drosophila melanogaster) has been successfully sequenced, and it is used extensively as a model of molecular pathways due to the existence of powerful genetic technology. In this study, D. melanogaster is investigated as a model for the replication and tropism of BTV. Using reverse genetics, a modified BTV-1 that expresses the fluorescent mCherry protein fused to the viral nonstructural protein NS3 (BTV-1/NS3mCherry) was generated. We demonstrate that BTV-1/NS3mCherry is not only replication competent as it retains many characteristics of the wild-type virus but also replicates efficiently in D. melanogaster after removal of the bacterial endosymbiont Wolbachia pipientis by antibiotic treatment. Furthermore, confocal microscopy shows that the tissue tropism of BTV-1/NS3mCherry in D. melanogaster resembles that described previously for BTV in Culicoides. Overall, the data presented in this study demonstrate the feasibility of using D. melanogaster as a genetic model to investigate BTV-insect interactions that cannot be otherwise addressed in vector species.


Assuntos
Vírus Bluetongue/fisiologia , Bluetongue/virologia , Doenças dos Bovinos/virologia , Modelos Animais de Doenças , Drosophila melanogaster/virologia , Tropismo Viral , Replicação Viral , Animais , Vírus Bluetongue/genética , Bovinos , Linhagem Celular , Ceratopogonidae/virologia , Drosophila melanogaster/genética , Insetos Vetores/virologia
5.
Hum Gene Ther ; 27(2): 166-83, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26886833

RESUMO

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA(GFP-CFTR) to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA(GFP-CFTR) was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.


Assuntos
Micropartículas Derivadas de Células/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Vesículas Extracelulares/química , Terapia Genética/métodos , RNA Mensageiro/genética , Transdução Genética/métodos , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/patologia , Exossomos/química , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
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