Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called “exosomes” that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC , an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human γ-herpesvirus EBV.

Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3' end adenylated miRNAs are relatively enriched in cells, whereas 3' end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3' end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.

Exosomes are small endosome-derived extracellular vesicles implicated in cell–cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB–PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB–PM fusion using live total internal reflection fluorescence and dynamic correlative light–electron microscopy. Quantitative analysis demonstrates that MVB–PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB–PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Exosomes are specialized membranous nano-sized vesicles derived from endocytic compartments that are released by many cell types. Microvesicles are distinctive from exosomes in that they are produc...

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)–associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P < .0001) despite heavy immune suppression and cytomegalovirus (CMV)-related disease. These findings illustrate the clinical importance of frequent EBV DNA load monitoring in LTx recipients. The increased EBV DNA loads in PTLD patients were restricted to the cellular blood compartment, as parallel serum samples were all below cut-off value, which indicates absence of lytic viral replication. EBV+ cells in PTLD patients have a very short doubling time, which can be as low as 56 hours, thereby creating the need for high screening frequency in high-risk patients. Furthermore, it is shown that EBV and CMV can reactivate independently in LTx recipients and that EBV DNA load monitoring may be useful in discriminating PTLD from rejection.

Relatively little is known about immune evasion during the productive phase of infection by the γ₁-herpesvirus Epstein–Barr virus (EBV). The use of a unique system to isolate cells in lytic cycle allowed us to identify a host shutoff function operating in productively EBV-infected B cells. This impairment of protein synthesis results from mRNA degradation induced upon expression of the early lytic-cycle gene product BGLF5. Recently, a γ₂-herpesvirus, Kaposi sarcoma herpesvirus, has also been shown to encode a host shutoff function, indicating that host shutoff appears to be a general feature of γ-herpesviruses. One of the consequences of host shutoff is a block in the synthesis of HLA class I and II molecules, reflected by reduced levels of these antigen-presenting complexes at the surface of cells in EBV lytic cycle. This effect could lead to escape from T cell recognition and elimination of EBV-producing cells, thereby allowing generation of viral progeny in the face of memory T cell responses.