RESUMO
Inflammation in the bone marrow (BM) microenvironment is a constitutive component of leukemogenesis in acute myeloid leukemia (AML). Current evidence suggests that both leukemic blasts and stroma secrete proinflammatory factors that actively suppress the function of healthy hematopoietic stem and progenitor cells (HSPCs). HSPCs are also cellular components of the innate immune system, and we reasoned that they may actively propagate the inflammation in the leukemic niche. In two separate congenic models of AML we confirm by evaluation of the BM plasma secretome and HSPC-selective single-cell RNA sequencing (scRNA-Seq) that multipotent progenitors and long-lived stem cells adopt inflammatory gene expression programs, even at low leukemic infiltration of the BM. In particular, we observe interferon gamma (IFN-γ) pathway activation, along with secretion of its chemokine target, CXCL10. We show that AML-derived nanometer-sized extracellular vesicles (EVAML) are sufficient to trigger this inflammatory HSPC response, both in vitro and in vivo. Altogether, our studies indicate that HSPCs are an unrecognized component of the inflammatory adaptation of the BM by leukemic cells. The pro-inflammatory conversion and long-lived presence of HSPCs in the BM along with their regenerative re-expansion during remission may impact clonal selection and disease evolution.
Assuntos
Vesículas Extracelulares , Leucemia Mieloide Aguda , Humanos , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea/metabolismo , Leucemia Mieloide Aguda/genética , Inflamação/metabolismo , Vesículas Extracelulares/metabolismo , Microambiente TumoralRESUMO
Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Esfingomielina Fosfodiesterase , Animais , Humanos , Camundongos , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Esfingolipídeos/metabolismoRESUMO
Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.
First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1Mut MS may be genetically distinct from NPM1Mut AML.NPM1Mut MS may have inferior overall survival compared to NPM1Mut AML.
Assuntos
Leucemia Mieloide Aguda , Sarcoma Mieloide , Humanos , Medula Óssea/patologia , Proteínas Nucleares/genética , Nucleofosmina , Sarcoma Mieloide/diagnóstico , Sarcoma Mieloide/genética , Sarcoma Mieloide/patologia , Estudos Retrospectivos , Estudos de Coortes , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação , PrognósticoRESUMO
Epstein-Barr virus (EBV) is a human herpesvirus that is associated with a multitude of cancers. The primary EBV oncogene latent membrane protein 1 (LMP1) is secreted from infected cancer cells in small extracellular vesicles (EVs). Additionally, the tetraspanin protein CD63 forms a complex with LMP1 and CD63 can be trafficked to EVs through a ceramide-dependent manner. Therefore, we hypothesize that ceramide is required for efficient packaging of LMP1 into small EVs. Following treatment with the neutral sphingomyelinase inhibitor GW4869, LMP1 cellular localization was disrupted and immunoblotting of EV lysates revealed a significant reduction in extracellular LMP1. NTA of EVs from the LCLs treated with GW4869 demonstrated a significant decrease in particle secretion. Additionally, ceramide inhibition resulted in enhanced LMP1-mediated NFkB activation in EV producing cells. Taken together, these data reveal a critical role for the lipid ceramide in LMP1 exosomal trafficking and the oncogenic signaling properties of the viral protein.
Assuntos
Infecções por Vírus Epstein-Barr , Vesículas Extracelulares , Humanos , Compostos de Anilina , Vesículas Extracelulares/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Ceramidas/metabolismo , Transporte BiológicoRESUMO
High-grade B-cell lymphomas with 11q aberrations (HGBL-11q) represent a World Health Organization-defined group of lymphomas that harbor recurrent chromosome 11q aberrations involving proximal gains and telomeric losses. Although a limited number of HGBL-11q cases evaluated thus far appear to show a similar course and prognosis as Burkitt lymphoma (BL), many molecular differences have been appreciated, most notably the absence of MYC rearrangement. Despite biological differences between BL and HGBL-11q, histomorphologic and immunophenotypic distinction remains challenging. Here, we provide a comparative whole proteomic profile of BL- and HGBL-11q-derived cell lines, identifying numerous shared and differentially expressed proteins. Transcriptome profiling performed on paraffin-embedded tissue samples from primary BL and HGBL-11q lymphomas was additionally performed to provide further molecular characterization. Overlap of proteomic and transcriptomic data sets identified several potential novel biomarkers of HGBL-11q, including diminished lymphoid enhancer-binding factor 1 expression, which was validated by immunohistochemistry staining in a cohort of 23 cases. Altogether, these findings provide a comprehensive multimodal and comparative molecular profiling of BL and HGBL-11q and suggest the use of enhancer-binding factor 1 as an immunohistochemistry target to distinguish between these aggressive lymphomas.
Assuntos
Linfoma de Burkitt , Linfoma de Células B , Linfoma Difuso de Grandes Células B , Proteogenômica , Humanos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Fator 1 de Ligação ao Facilitador Linfoide , Proteômica , Linfoma de Células B/genética , Linfoma de Células B/patologia , Aberrações Cromossômicas , Biomarcadores , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Myeloid/lymphoid neoplasms (M/LN) with 13q12/FLT3 rearrangement have been suggested as candidates for possible inclusion in the World Health Organization classification group of M/LN with eosinophilia (M/LN-eo). We report 12 patients with confirmed FLT3 rearrangement, six with t(12;13)/ETV6-FLT3; one with ins(13;22)/BCR-FLT3; and five with an unconfirmed partner gene located on chromosome bands 2p16, 3q27, 5q15, 5q35, and 7q36. Disease presentations were heterogeneous, including lymphoblastic leukemia/lymphoma, myeloid sarcoma, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome. However, some common features were observed, such as extramedullary involvement (n = 7, 58%), associated eosinophilia in blood, bone marrow, or tissue (n = 8, 67%), multilineage involvement, either as biphasic myeloid/lymphoid neoplasms (n = 2) or mixed phenotype acute leukemia (n = 2). Mutations were detected in 4/8 (50%) patients by next-generation sequencing. None (0/10) had FLT3 or KIT mutations. Eleven patients received disease-based chemotherapy or hypomethylating agents, three received FLT3 inhibitors, and five patients proceeded to hematopoietic stem cell transplant. Together with a review of 16 cases published in the literature, it is apparent that M/LNs with FLT3 rearrangement show disease features reminiscent of members in the category of M/LN-eo with PDGFRA, PDGFRB, FGFR1, and PCM1/JAK2 rearrangement, characterized by a specific gene rearrangement, frequent eosinophilia, multi-lineage involvement and therapeutic benefit from kinase inhibitors.
Assuntos
Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Pré-Escolar , Feminino , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-Idade , Fusão Oncogênica , Adulto JovemRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are responsible for lifelong maintenance of hematopoiesis through self-renewal and differentiation into mature blood cell lineages. Traditional models hold that HSPCs guard homeostatic function and adapt to regenerative demand by integrating cell-autonomous, intrinsic programs with extrinsic cues from the niche. Despite the biologic significance, little is known about the active roles HSPCs partake in reciprocally shaping the function of their microenvironment. Here, we review evidence of signals emerging from HSPCs through secreted autocrine or paracrine factors, including extracellular vesicles, and via direct contact within the niche. We also discuss the functional impact of direct cellular interactions between hematopoietic elements on niche occupancy in the context of leukemic infiltration. The aggregate data support a model whereby HSPCs are active participants in the dynamic adaptation of the stem cell niche unit during development and homeostasis, and under inflammatory stress, malignancy, or transplantation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Vesículas Extracelulares/fisiologia , Hematopoese/fisiologia , HumanosRESUMO
Acquired aplastic anemia (AA) is a life-threatening bone marrow failure caused by an autoimmune cytotoxic T lymphocyte attack on hematopoietic stem and progenitor cells. Factors contributing to aberrant autoimmune activation in AA include a deficit of T regulatory cells and high levels of inflammatory cytokines. Several acquired conditions of immune dysregulation and genetic polymorphisms in inflammatory cytokines and human leukocyte antigen genes have been linked to an increased risk of AA. However, AA has not been reported in patients with Mendelian disorders of immune regulation. Here we report a patient with familial common variable immunodeficiency (CVID) caused by a pathogenic variant in NFKB1, who developed AA as an adult. The patient had a difficult clinical course and was unable to tolerate standard AA therapy with cyclosporine A and eltrombopag, with complications attributed in part to the effect of cyclosporine A on NF-κB signaling. Our case suggests a novel link between genetic disorders of immune regulation and AA and highlights the importance of recognizing inherited autoimmunity syndromes in AA patients for the selection of optimal therapy and prognostic counseling.
Assuntos
Anemia Aplástica/complicações , Anemia Aplástica/genética , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/genética , Haploinsuficiência/genética , Subunidade p50 de NF-kappa B/genética , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/patologia , Benzoatos , Medula Óssea/patologia , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunodeficiência de Variável Comum/patologia , Ciclosporina/uso terapêutico , Feminino , Predisposição Genética para Doença , Humanos , Hidrazinas , Pessoa de Meia-Idade , NF-kappa B , Pirazóis , Transdução de Sinais , Sequenciamento do ExomaRESUMO
BACKGROUND: Endosomal trafficking and amyloidogenic cleavage of amyloid precursor protein (APP) is believed to play a role in the neurodegeneration observed in Alzheimer's disease (AD). Recent evidence has suggested that packaging and secretion of APP and its amyloidogenic cleaved products into small extracellular vesicles (EVs) may facilitate uptake of these neurotoxic factors during disease progression. However, the molecular mechanisms underlying trafficking of APP into EVs are poorly understood. RESULTS: In this study, the mechanism and impact of APP trafficking into extracellular vesicles (EVs) were assessed by a series of inducible gene knockdowns. We demonstrate that vesicle-associated proteins Alix and Syntenin-1 are essential for proper subcellular localization and efficient EV secretion of APP via an endosomal sorting complexes required for transport (ESCRT)-independent pathway. The neurotoxic C-terminal fragment (CTFß) of APP is similarly secreted in association with small vesicles. These mechanisms are conserved in terminally differentiated neuron-like cells. Furthermore, knockdown of Alix and Syntenin-1 alters the subcellular localization of APP, sequestering the precursor protein to endoplasmic reticulum and endolysosomal compartments, respectively. Finally, transfer of small EVs containing mutant APP confers an increase in reactive oxygen species production and neurotoxicity to human induced pluripotent stem cell-derived cortical neurons and naïve primary neurons, an effect that is ameliorated by Alix and Syntenin-1 depletion. CONCLUSIONS: Altogether these findings elucidate a novel mechanism for understanding the intracellular trafficking of APP and CTFß into secreted extracellular vesicles, and the resultant potential impact on neurotoxicity in the context of Alzheimer's disease amyloidopathy.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Sinteninas/metabolismo , Precursor de Proteína beta-Amiloide/toxicidade , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hodgkin lymphomas (HLs) are lymphoid neoplasms uniquely characterized by a paucity of neoplastic cells embedded in a supportive heterogenous cellular microenvironment. Although first described in the 19th century, systematic biological understanding of HLs has been hindered due to the challenges presented in studying the complex tumor microenvironment and scarce tumorigenic cells. Recent advances in single-cell isolation and characterization, sensitive mutational analytic tools, and multiplex immunohistochemical strategies have allowed further advances in understanding the development and progression of HL. Here we provide a current update on the chromosomal and mutational abnormalities seen in HL, the impact of Epstein-Barr virus infection on driving a subset of HLs, and the possibility of disease monitoring via high-sensitivity detection of genetic aberrations. We also discuss recent developments in understanding the intricate microenvironment through intercellular cross-talk, and describe novel potential biomarkers to aid in distinction of HL from other overlapping entities.
Assuntos
Infecções por Vírus Epstein-Barr/patologia , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Microambiente Tumoral/fisiologia , Aberrações Cromossômicas , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Doença de Hodgkin/genética , Humanos , Mutação , Transdução de Sinais/genéticaRESUMO
The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and ß1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types.
RESUMO
Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.
Assuntos
Vesículas Extracelulares , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Proteínas/metabolismoRESUMO
EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.IMPORTANCE EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.
Assuntos
Exossomos/metabolismo , Herpesvirus Humano 4/fisiologia , Transporte Proteico , Proteínas da Matriz Viral/metabolismo , Análise Mutacional de DNA , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Proteínas da Matriz Viral/genéticaRESUMO
BACKGROUND: Alzheimer's disease (AD) is the major cause of dementia that has increased dramatically in prevalence over the past several decades. Yet many questions still surround the etiology of AD. Recently, extracellular vesicles (EVs) that transport protein, lipid, and nucleic acids from cell to cell have been implicated in the clearance and propagation of misfolded proteins. Investigation of EVs in AD progression, and their potential diagnostic utility may contribute to understanding and treating AD. However, the challenges of isolating brain-derived EVs have in part hindered these studies. NEW METHOD: Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis. RESULTS: We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry. CONCLUSIONS: Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.
Assuntos
Doença de Alzheimer/complicações , Encéfalo/citologia , Vesículas Extracelulares/patologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Fatores Etários , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Proteínas tau/genéticaRESUMO
Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Biotina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Exossomos/metabolismo , Exossomos/virologia , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas , Ligação Proteica , Mapeamento de Interação de Proteínas , Transdução de Sinais , Sinteninas/genética , Sinteninas/metabolismo , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells.IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication.