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1.
Signal Transduct Target Ther ; 6(1): 300, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381015

RESUMO

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.


Assuntos
COVID-19/genética , Diabetes Mellitus/genética , MicroRNAs/genética , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Fatores Etários , Idoso , COVID-19/sangue , COVID-19/patologia , COVID-19/virologia , China , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Estudos de Coortes , Diabetes Mellitus/sangue , Diabetes Mellitus/patologia , Diabetes Mellitus/virologia , Exercício Físico , Exossomos/genética , Exossomos/metabolismo , Exossomos/virologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , SARS-CoV-2/genética , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/sangue , Replicação Viral
2.
Signal Transduct Target Ther ; 6(1): 189, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980808

RESUMO

Since the outbreak of coronavirus disease 2019 (COVID-19), it has become a global pandemic. The spike (S) protein of etiologic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specifically recognizes human angiotensin-converting enzyme 2 (hACE2) as its receptor, which is recently identified as an interferon (IFN)-stimulated gene. Here, we find that hACE2 exists on the surface of exosomes released by different cell types, and the expression of exosomal hACE2 is increased by IFNα/ß treatment. In particular, exosomal hACE2 can specifically block the cell entry of SARS-CoV-2, subsequently inhibit the replication of SARS-CoV-2 in vitro and ex vivo. Our findings have indicated that IFN is able to upregulate a viral receptor on the exosomes which competitively block the virus entry, exhibiting a potential antiviral strategy.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Exossomos/metabolismo , Interferon-alfa/farmacologia , Interferon beta/farmacologia , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Animais , Chlorocebus aethiops , Exossomos/genética , Exossomos/virologia , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Células Vero
3.
FASEB J ; 35(4): e21505, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33723887

RESUMO

Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLß2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLß2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.


Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Vesículas Extracelulares/metabolismo , Linfoma/virologia , Microambiente Tumoral/fisiologia , Proliferação de Células/fisiologia , Infecções por Vírus Epstein-Barr/virologia , Exossomos/metabolismo , Exossomos/virologia , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 4/fisiologia , Humanos , Linfoma/metabolismo
4.
Fish Physiol Biochem ; 47(4): 857-867, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33745109

RESUMO

Exosomes are small membrane-enclosed vesicles secreted by various types of cells. Exosomes not only participate in different physiological processes in cells, but also involve in the cellular responses to viral infection. Grass carp reovirus (GCRV) is a non-enveloped virus with segmented, double-stranded RNA genome. Nowadays, the exact role of exosomes in regulating the life cycle of GCRV infection is still unclear. In this study, the exosomes secreted from Ctenopharyngodon idellus kidney (CIK) cells infected or uninfected with GCRV were isolated, and the differential protein expression profiles were analyzed by proteomic technologies. A total of 1297 proteins were identified in the isolated exosomes. The differentially abundant proteins were further analyzed with functional categories, and numerous important pathways were regulated by exosomes in GCRV-infected CIK cells. These exosomal proteins were estimated to interact with the genes (proteins) of the top 10 most enriched signaling pathways. Furthermore, GW4869 exosome inhibitor suppressed the expression level of VP7 in GCRV-infected cells, suggesting that exosomes play a crucial role in the life cycle of GCRV infection. These findings could shed new lights on understanding the functional roles of exosomes in the cellular responses to GCRV infection.


Assuntos
Exossomos/metabolismo , Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Rim/citologia , Infecções por Reoviridae/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Carpas , Células Cultivadas , Exossomos/efeitos dos fármacos , Exossomos/virologia , Doenças dos Peixes/virologia , Rim/virologia , Proteômica , Reoviridae , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/virologia
5.
Tohoku J Exp Med ; 252(4): 309-320, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33268600

RESUMO

The chronicity of Hepatitis B virus (HBV) infection relates to both viral factors and host factors. HBV could result in persistent infection and even serious liver disease, including chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma (HCC). Although the HBV vaccine can effectively prevent HBV infection, chronic HBV infection still endangers human health and results in a large social burden. Moreover, the mechanisms underlying the HBV-mediated imbalance of the immune response and persistent infection are not fully understood. Exosomes are extracellular vesicles (EVs) 40-160 nm in size that are released from many cells and transfer specific functional RNAs, proteins, lipids and viral components from donor to recipient cells. These exosome nanovesicles are associated with various biological processes, such as cellular homeostasis, immune response and cancer progression. Besides, previous studies on exosomes have shown that they take part in viral pathogenicity due to the similarity in structure and function between exosomes and enveloped viruses. Moreover, exosome as a novel immunomodulatory carrier plays a significant role in viral immunology. In this review, we focus on the latest progress in understanding the role of exosomes in HBV transmission as well as their vital roles in immune regulation during HBV infection. Furthermore, we discuss the potential clinical applications of exosomes in hepatitis B infection, including the use of exosomes in the auxiliary diagnosis and treatment of hepatitis B.


Assuntos
Exossomos/virologia , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Hepatite B/transmissão , Imunidade , Animais , Hepatite B/virologia , Humanos , Modelos Biológicos , Replicação Viral
6.
Cells ; 9(12)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33353065

RESUMO

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR-RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33+ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33+ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR-RUNX1-STAT3-miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Exossomos/virologia , Hepacivirus , MicroRNAs/metabolismo , Células Supressoras Mieloides/citologia , RNA Longo não Codificante , Fator de Transcrição STAT3/metabolismo , Adulto , Idoso , Arginase/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Feminino , Regulação Viral da Expressão Gênica , Genótipo , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Humanos , Imunossupressores , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Células Supressoras Mieloides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Regulação para Cima , Carga Viral
7.
PLoS Comput Biol ; 16(11): e1008421, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33151933

RESUMO

Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.


Assuntos
Hepacivirus/genética , Hepacivirus/fisiologia , Modelos Biológicos , Antivirais/farmacologia , Biologia Computacional , Simulação por Computador , Exossomos/virologia , Hepacivirus/patogenicidade , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Conceitos Matemáticos , RNA Viral/biossíntese , RNA Viral/genética , Compartimentos de Replicação Viral/fisiologia , Vírion/genética , Vírion/fisiologia , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Liberação de Vírus/genética , Liberação de Vírus/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/fisiologia
8.
Life Sci ; 263: 118564, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33075374

RESUMO

AIMS: Zika virus (ZIKV) infection causes a public health concern because of its potential association with the development of microcephaly. During viral infections, the host innate immune response is mounted quickly to produce some endogenous functional molecules to limit virus replication and spread. Exosomes contain molecules from their cell of origin following virus infection and can enter recipient cells for intercellular communication. Here, we aim to clarify whether ZIKV-induced exosomes can regulate viral pathogenicity by transferring specific RNAs. MAIN METHODS: In this study, exosomes were isolated from the supernatants of A549 cells with or without ZIKV infection. Human transcriptome array (HTA) was performed to analyze the profiling of RNAs wrapped in exosomes. Then qPCR, western blotting and ELISA were used to determine ZIKV replication. CCK-8 and flow cytometry were used to test the cell proliferation and cell cycles. Co-culture assay was used to analyze the effect of exosomes on the cell cycles of recipient cells. KEY FINDINGS: Through human transcriptome array (HTA) we found the defensin alpha 1B (DEFA1B) expression was significantly increased within exosomes isolated from ZIKV infected A549 cells. Additionally, we found that the extracellular DEFA1B exerts significant anti-ZIKV activity, mainly before ZIKV entering host cells. Interestingly, up-regulated DEFA1B retards the cell cycle of host cells. Further studies demonstrated that DEFA1B interacted with the origin recognition complex 1 (ORC1) which is required to initiate DNA replication during the cell cycle and increased DEFA1B expression decreased the ORC1 level in the cell nuclei. Accordingly, DEFA1B-containing exosomes can be internalized by the recipient cells to retard their cell cycles. SIGNIFICANCE: Together, our results demonstrated that the anti-ZIKV activity of DEFA1B can be mediated by exosomes, and DEFA1B interacts with ORC1 to retard cell cycles. Our study provides a novel concept that DEFA1B not only acts as an antiviral molecule during ZIKV infection but also may correlate with cell proliferation by retarding the progression of cell cycles.


Assuntos
Ciclo Celular , Exossomos/virologia , Complexo de Reconhecimento de Origem/metabolismo , Replicação Viral , Infecção por Zika virus/prevenção & controle , Zika virus/fisiologia , alfa-Defensinas/metabolismo , Células A549 , Antivirais/farmacologia , Efeito Citopatogênico Viral , Exossomos/genética , Exossomos/metabolismo , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/imunologia , Complexo de Reconhecimento de Origem/genética , Zika virus/isolamento & purificação , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia , alfa-Defensinas/genética
9.
PLoS One ; 15(9): e0239153, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941510

RESUMO

BACKGROUND: Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. AIM: To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. METHODS: We used Huh7 cells-JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. RESULTS: HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes. CONCLUSION: RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.


Assuntos
Proteínas de Transporte/metabolismo , Exossomos/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linhagem Celular , Exossomos/virologia , Feminino , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade
10.
Viruses ; 12(7)2020 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-32605316

RESUMO

Almost all types of cells release extracellular vesicles (EVs) into the extracellular space. EVs such as exosomes and microvesicles are membrane-bound vesicles ranging in size from 30 to 1000 nm in diameter. Under normal conditions, EVs mediate cell to cell as well as inter-organ communication via the shuttling of their cargoes which include RNA, DNA and proteins. Under pathological conditions, however, the number, size and content of EVs are found to be altered and have been shown to play crucial roles in disease progression. Emerging studies have demonstrated that EVs are involved in many aspects of viral infection-mediated neurodegenerative diseases. In the current review, we will describe the interactions between EV biogenesis and the release of virus particles while also reviewing the role of EVs in various viral infections, such as HIV-1, HTLV, Zika, CMV, EBV, Hepatitis B and C, JCV, and HSV-1. We will also discuss the potential uses of EVs and their cargoes as biomarkers and therapeutic vehicles for viral infections.


Assuntos
Vesículas Extracelulares/virologia , Doenças Neurodegenerativas/virologia , Viroses/virologia , Fenômenos Fisiológicos Virais , Animais , Exossomos/virologia , Humanos , Vírus/genética
11.
Vet Res ; 51(1): 91, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678013

RESUMO

Seneca Valley virus (SVV) is a non-encapsulated single-stranded positive-strand RNA virus whose transmission routes have not yet been fully elucidated. Exosomes have been implicated in the intercellular transport of a variety of materials, such as proteins, RNA, and liposomes. However, whether exosomes can mediate SVV intercellular transmission remains unknown. In this study, we extracted exosomes from SVV-infected IBRS-2 cells to investigate intercellular transmission. Our results suggest that the intercellular transmission of SVV is mediated by exosomes. The results of co-localization and RT-qPCR studies showed that exosomes harbor SVV and enable the virus to proliferate in both susceptible and non-susceptible cells. Furthermore, the replication of SVV was inhibited when IBRS-2 cells were treated with interfering RNA Rab27a and exosome inhibitor GW4869. Finally, neutralization experiments were performed to further verify whether the virus was encapsulated by the exosomes that mediated transmission between cells. It was found that exosome-mediated intercellular transmission was not blocked by SVV-specific neutralizing antibodies. This study reveals a new transmission route of SVV and provides clear evidence regarding the pathogenesis of SVV, information which can also be useful for identifying therapeutic interventions.


Assuntos
Exossomos/virologia , Infecções por Picornaviridae/veterinária , Picornaviridae/fisiologia , Doenças dos Suínos/transmissão , Animais , Infecções por Picornaviridae/transmissão , Infecções por Picornaviridae/virologia , Sus scrofa , Suínos , Doenças dos Suínos/virologia
12.
Front Immunol ; 11: 887, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32477358

RESUMO

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated in vivo, particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.


Assuntos
Exossomos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Infecções por Orthomyxoviridae/imunologia , Sistema Respiratório/imunologia , Animais , Transporte Biológico , Exossomos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Infecções por Orthomyxoviridae/virologia , Proteômica , Sistema Respiratório/virologia , Organismos Livres de Patógenos Específicos , Ligação Viral
13.
Nat Microbiol ; 5(9): 1096-1106, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32541946

RESUMO

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.


Assuntos
Endossomos/metabolismo , Exossomos/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Vírus da Hepatite A/metabolismo , Hepatite A/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Linhagem Celular , Ebolavirus , Endocitose , Endossomos/virologia , Exossomos/virologia , Técnicas de Inativação de Genes , Células HEK293 , Hepatite A/imunologia , Hepatite A/virologia , Receptor Celular 1 do Vírus da Hepatite A/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana , Transcriptoma , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Internalização do Vírus
14.
Curr Opin Insect Sci ; 40: 39-47, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32590312

RESUMO

Ticks and mosquitoes are medically important vectors that transmit several pathogens, including arboviruses, to humans. Understanding how these blood-feeding arthropods transmit pathogens to humans requires knowledge on the molecular and cellular interplay at vector-host interface. Recent studies have highlighted the role of tick and mosquito small extracellular vesicles (EVs), including exosomes, facilitating arbovirus transmission within arthropod cells and from arthropod to mammalian cells. In this review, we summarize this emerging line of investigation in understanding the role of tick and mosquito exosomes in vector-pathogen-host tripartite interactions. Understanding the role of arthropod exosomes in pathogen interactions could lead to the discovery of novel therapeutic targets to interfere with the life cycle of several pathogens transmitted by vectors.


Assuntos
Arbovírus/fisiologia , Artrópodes/fisiologia , Exossomos/fisiologia , Doenças Transmitidas por Vetores/transmissão , Animais , Exossomos/microbiologia , Exossomos/parasitologia , Exossomos/virologia , Doenças Transmitidas por Vetores/microbiologia , Doenças Transmitidas por Vetores/parasitologia , Doenças Transmitidas por Vetores/virologia
15.
Emerg Microbes Infect ; 9(1): 962-975, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32267217

RESUMO

The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus's pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient's plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment.


Assuntos
Exossomos/virologia , Subtipo H7N9 do Vírus da Influenza A , Influenza Aviária/virologia , Influenza Humana/virologia , Animais , Aves , Sangue/virologia , Encéfalo/virologia , Linhagem Celular , Citocinas/sangue , Genoma Viral , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Camundongos , Viremia
16.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 38(4): 159-165, abr. 2020. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-200681

RESUMO

INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSIÓN: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell


INTRODUCCIÓN: La principal causa del cáncer de cérvix es la infección de los queratinocitos de la capa basal del epitelio estratificado del cuello uterino por el virus del papiloma humano (VPH). El ADN del VPH se ha encontrado en muestras cervicales, pero también en suero y otros fluidos, aunque su origen en estos últimos no está claro. Las vesículas extracelulares (VE) podrían ser el medio de transporte del ADN viral considerando su papel emergente en la comunicación celular. El contenido de las VE derivadas de células cervicales ha sido poco explorado y la presencia en ellas de ADN de VPH sigue siendo desconocida. MÉTODOS: Evaluamos el ADN de exosomas purificados a partir de cultivos de células HeLa mediante secuenciación de nueva generación (NGS) y confirmamos su presencia a través de PCR. La presencia de ADN de VPH también se evaluó mediante PCR y NGS en VE de muestras cervicales positivas a VPH, sin lesión aparente o con LSIL. RESULTADOS: Detectamos la forma integrada del ADN viral en exosomas de células HeLa mediante NGS, y confirmamos su presencia a través de PCR. La búsqueda de secuencias de VPH en VE obtenidas a partir de muestras de exudado cervical sin lesión aparente o con LSIL, donde esperamos encontrar el genoma viral en forma episomal, indicó que el DNA de VPH incluyendo los oncogenes E6 y E7, está presente en estas VE. CONCLUSIÓN: El ADN del VPH incluyendo el correspondiente con los oncogenes virales E6/E7 se encuentra en exosomas independientemente del estado de integración del virus en la célula infectada


Assuntos
Humanos , Feminino , Papillomaviridae/genética , Exossomos/virologia , Genoma Viral , DNA Viral/isolamento & purificação , Células HeLa , Reação em Cadeia da Polimerase , Sequenciamento de Nucleotídeos em Larga Escala
17.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188736

RESUMO

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, and 600,000 deaths are caused by HBV-related hepatic failure, liver cirrhosis, and hepatocellular carcinoma annually. It is important to reveal the mechanism underlying the regulation of HBV replication. This study demonstrated that osteopetrosis-associated transmembrane protein 1 (Ostm1) plays an inhibitory role in HBV replication. Ostm1 represses the levels of HBeAg and HBsAg proteins, HBV 3.5-kb and 2.4/2.1-kb RNAs, and core-associated DNA in HepG2, Huh7, and NTCP-HepG2 cells. Notably, Ostm1 has no direct effect on the activity of HBV promoters or the transcription of HBV RNAs; instead, Ostm1 binds to HBV RNA to facilitate RNA decay. Detailed studies further demonstrated that Ostm1 binds to and recruits the RNA exosome complex to promote the degradation of HBV RNAs, and knockdown of the RNA exosome component exonuclease 3 (Exosc3) leads to the elimination of Ostm1-mediated repression of HBV replication. Mutant analyses revealed that the N-terminal domain, the transmembrane domain, and the C-terminal domain are responsible for the repression of HBV replication, and the C-terminal domain is required for interaction with the RNA exosome complex. Moreover, Ostm1 production is not regulated by interferon-α (IFN-α) or IFN-γ, and the expression of IFN signaling components is not affected by Ostm1, suggesting that Ostm1 anti-HBV activity is independent of the IFN signaling pathway. In conclusion, this study revealed a distinct mechanism underlying the repression of HBV replication, in which Ostm1 binds to HBV RNA and recruits RNA exosomes to degrade viral RNA, thereby restricting HBV replication.IMPORTANCE Hepatitis B virus (HBV) is a human pathogen infecting the liver to cause a variety of diseases ranging from acute hepatitis to advanced liver diseases, fulminate hepatitis, liver cirrhosis, and hepatocellular carcinoma, thereby causing a major health problem worldwide. In this study, we demonstrated that Ostm1 plays an inhibitory role in HBV protein production, RNA expression, and DNA replication. However, Ostm1 has no effect on the activities of the four HBV promoters; instead, it binds to HBV RNA and recruits RNA exosomes to promote HBV RNA degradation. We further demonstrated that the anti-HBV activity of Ostm1 is independent of the interferon signaling pathway. In conclusion, this study reveals a distinct mechanism underlying the repression of HBV replication and suggests that Ostm1 is a potential therapeutic agent for HBV infection.


Assuntos
Exossomos/metabolismo , Vírus da Hepatite B/fisiologia , Proteínas de Membrana/metabolismo , RNA Viral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Proteínas de Membrana/genética , Domínios Proteicos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/genética
18.
Emerg Microbes Infect ; 8(1): 1626-1635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711408

RESUMO

Dengue fever is one of those unique diseases where host immune responses largely determine the pathogenesis and its severity. Earlier studies have established the fact that dengue virus (DENV) infection causes haemorrhagic fever and shock syndrome, but it is not directly responsible for exhibiting these clinical symptoms. It is noteworthy that clinically, vascular leakage syndrome does not develop for several days after infection despite a robust innate immune response that elicits the production of proinflammatory and proangiogenic cytokines. The onset of hyperpermeability in severe cases of dengue disease takes place around the time of defervescence and after clearance of viraemia. Extracellular vesicles are known to carry biological information (mRNA, miRNA, transcription factors) from their cells of origin and have emerged as a significant vehicle for horizontal transfer of stress signals. In dengue virus infection, the relevance of exosomes can be instrumental since the majority of the immune responses in severe dengue involve heavy secretion and circulation of pro-inflammatory cytokines and chemokines. Here, we present an updated review which will address the unique and puzzling features of hyperpermeability associated with DENV infection with a special focus on the role of secreted extracellular vesicles.


Assuntos
Vírus da Dengue/fisiologia , Exossomos/virologia , Dengue Grave/virologia , Animais , Citocinas/genética , Citocinas/metabolismo , Vírus da Dengue/genética , Exossomos/genética , Exossomos/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Humanos , Dengue Grave/genética , Dengue Grave/metabolismo
19.
Viral Immunol ; 32(10): 453-462, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31755827

RESUMO

CD81 serves as an immune modulator, playing its role in tumor growth and metastasis of hepatitis C virus (HCV)-mediated hepatocellular carcinoma (HCC). CD81 serves as a coreceptor of viral entry and is found to be enriched in exosomes. HCV E2 protein when associated with CD81 may be responsible for B cell lymphoproliferative disorders, as extrahepatic manifestation. Studies predict that HCV association with exosomes, leads to the establishment of persistent infection, through immune evasion. Herein, we confirm the association of HCV particles with CD81+ exosomes. Breifly, exosomes were enriched from peripheral blood of chronic HCV patients who have developed HCC. Sideways, exosomes were also enriched from peripheral blood of healthy individuals, who exhibited normal liver function test profile and had no known infection. Isolation of subpopulation of CD81+ exosomes was performed through immunocapture, followed by detection using FACS. Scanning electron microscopy confirmed the physical association of a fraction of exosome with HCV. CD81+ exosomes from chronic HCV patients with HCC were more granulated and larger when compared with those enriched from a healthy individual and HCV RNA was also detected in enriched fractions of CD81+ exosomes from HCV-positive HCC patients only, through real-time quantitative polymerase chain reaction. We concluded that CD81+ exosomes carry HCV particles and the association plays a pivotal role in establishing persistent infection, through immune evasion, thus leading to HCC progression. Exosomal CD81 and its interacting proteins might, therefore, serve as a potential prognostic marker and therapeutic target in HCV progression mediated by active HCV infection.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Exossomos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Neoplasias Hepáticas/diagnóstico , Tetraspanina 28/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Estudos de Casos e Controles , Progressão da Doença , Exossomos/metabolismo , Exossomos/virologia , Feminino , Voluntários Saudáveis , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Paquistão , Prognóstico , Tetraspanina 28/metabolismo , Adulto Jovem
20.
PLoS Pathog ; 15(7): e1007907, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31344124

RESUMO

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Efeito Espectador , Colesterol/metabolismo , Exossomos/metabolismo , Exossomos/virologia , Células HEK293 , HIV-1 , Humanos , Inflamação/metabolismo , Inflamação/virologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
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