Modulation of B-cell exosome proteins by gamma herpesvirus infection

Lineberger Comprehensive Cancer Center, Program in Molecular Biology and Biotechnology, and Department of Microbiology-Immunology, University of North Carolina, Chapel Hill, NC 27599.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2013; 110(31). DOI: 10.1073/pnas.1303906110
Source: PubMed


The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.

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    • "However, there are multiple benefits in transplanting exosomes rather than in transplanting the whole " factory, " the cell, into the body. In contrast to exogenously administered cells delivered systemically, exosomes, given their nano dimension may readily enter the brain and easily pass through the blood brain barrier (BBB) (Alvarez-Erviti et al., 2011; Kooijmans et al., 2012; Anthony and Shiels, 2013; Gheldof et al., 2013; Meckes et al., 2013). Exogenously administered MSCs may have many adverse effects, i. e. tumor modulation and malignant transformation. "
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    ABSTRACT: Cell-based therapy, e.g., multipotent mesenchymal stromal cell (MSC) treatment, shows promise for the treatment of various diseases. The strong paracrine capacity of these cells and not their differentiation capacity, is the principal mechanism of therapeutic action. MSCs robustly release exosomes, membrane vesicles (~30-100 nm) originally derived in endosomes as intraluminal vesicles, which contain various molecular constituents including proteins and RNAs from maternal cells. Contained among these constituents, are small non-coding RNA molecules, microRNAs (miRNAs), which play a key role in mediating biological function due to their prominent role in gene regulation. The release as well as the content of the MSC generated exosomes are modified by environmental conditions. Via exosomes, MSCs transfer their therapeutic factors, especially miRNAs, to recipient cells, and therein alter gene expression and thereby promote therapeutic response. The present review focuses on the paracrine mechanism of MSC exosomes, and the regulation and transfer of exosome content, especially the packaging and transfer of miRNAs which enhance tissue repair and functional recovery. Perspectives on the developing role of MSC mediated transfer of exosomes as a therapeutic approach will also be discussed.
    Frontiers in Cellular Neuroscience 11/2014; 8:377. DOI:10.3389/fncel.2014.00377 · 4.29 Impact Factor
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    • "This hypothesis is indeed plausible, considering that the EBER-binding protein La has also been reported to be excreted via exosomes [36]. Moreover, exosomes are now believed to be an important mechanism of transport of numerous small RNA and protein molecules and a means of intercellular communication [37], [38]. In this study we show that both EBER-1 and EBER-2 are present in culture supernatants of EBV-infected cells and are excreted out of the cells in a form that is protected by RNase. "
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    ABSTRACT: Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies of epithelial and lymphoid origin. However, the mechanism of oncogenesis is unclear. A number of viral products, including EBV latent proteins and non-protein coding RNAs have been implicated. Recently it was reported that EBV-encoded small RNAs (EBERs) are released from EBV infected cells and they can induce biological changes in cells via signaling from toll-like receptor 3. Here, we investigated if these abundantly expressed non-protein coding EBV RNAs (EBER-1 and EBER-2) are excreted from infected cells in exosomal fractions. Using differential ultracentrifugation we isolated exosomes from three EBV positive cell lines (B95-8, EBV-LCL, BL30-B95-8), one EBER-1 transfected cell line (293T-pHEBo-E1) and two EBV-negative cell lines (BL30, 293T-pHEBo). The identity of purified exosomes was determined by electron microscopy and western blotting for CD63. The presence of EBERs in cells, culture supernatants and purified exosomal fractions was determined using RT-PCR and confirmed by sequencing. Purified exosomal fractions were also tested for the presence of the EBER-1-binding protein La, using western blotting. Both EBER-1 and EBER-2 were found to be present not only in the culture supernatants, but also in the purified exosome fractions of all EBV-infected cell lines. EBER-1 could also be detected in exosomal fractions from EBER-1 transfected 293T cells whilst the fractions from vector only transfectants were clearly negative. Furthermore, purified exosomal fractions also contained the EBER-binding protein (La), supporting the notion that EBERs are most probably released from EBV infected cells in the form of EBER-La complex in exosomes.
    PLoS ONE 06/2014; 9(6):e99163. DOI:10.1371/journal.pone.0099163 · 3.23 Impact Factor
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    • "While still a relatively new field of study, experiments performed in regard to the effect of viral infections on host exosomes have begun to delineate the alterations of exosomal composition and determine the biochemical mechanisms driving variation in exosomal content. A recent proteomic analysis of B cells infected with either Kaposi's sarcoma herpes virus (KSHV), Epstein–Barr Virus (EBV), or a dual infection showed major changes to the exosomal protein composition as compared to exosomes from the uninfected parental cell line (Meckes et al., 2013). Specifically , a total of 345 proteins were identified by mass spectrometry to be uniquely incorporated into the exosomes released from the infected cells. "
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    ABSTRACT: Exosomes have recently been classified as the newest family members of 'bioactive vesicles' that function to promote intercellular communication. Long ignored and thought to be only a mechanism by which cellular waste is removed, exosomes have garnered a huge amount of interest in recent years as their critical functions in maintaining homeostasis through intercellular communication and also in different types of diseases has been demonstrated. Many groundbreaking studies of exosome functions have been performed in the cancer field and the infectious disease areas of study, revealing the importance and also the fascinating complexity of exosomal packaging, targeting, and functions. Selective packaging of exosomes in response to the type of infection, exosomal modulation of the immune response and host signaling pathways, exosomal regulation of pathogen spread, and effects of exosomes on the degree of pathogenesis have all been well documented. In this review, we provide a synthesis of the current understanding of the role of exosomes during infections caused by human pathogens, and discuss the implications of these findings for a better understanding of pathogenic mechanisms and future therapeutic and diagnostic applications. This article is protected by copyright. All rights reserved.
    Pathogens and Disease 01/2014; 71(2). DOI:10.1111/2049-632X.12135 · 2.40 Impact Factor
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