Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Apr 6;107(14):6328-33.
doi: 10.1073/pnas.0914843107. Epub 2010 Mar 18.

Functional delivery of viral miRNAs via exosomes

D Michiel Pegtel  1 , Katherine Cosmopoulos, David A Thorley-Lawson, Monique A J van Eijndhoven, Erik S Hopmans, Jelle L Lindenberg, Tanja D de Gruijl, Thomas Würdinger, Jaap M Middeldorp
Affiliations

Functional delivery of viral miRNAs via exosomes

D Michiel Pegtel et al. Proc Natl Acad Sci U S A. .

Abstract

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 gamma-herpesvirus EBV.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Exosomes from EBV-infected lymphoblasts (LCL) are enriched in small RNA and EBV-m iRNAs. (A) EM image of an MVE of an LCL with ongoing inward budding of the limiting membrane (arrow). (B) Purified exosomes isolated from LCL culture medium. (Scale bar, 100 nm,). (C) Western blots for HLA-DR, CD63, and cytochrome C of cell lysates and lysates from purified exosomes (CD63 under nonreducing conditions). (D) Bioanalyzer results of equal amounts of cellular (LCL) RNA compared with RNA isolated from purified LCL exosomes treated with RNase A (10 ng/μL). Small RNA species (indicated by arrows) are highly enriched in exosomes. (E) Detection of EBV-encoded mature miRNAs by multiplex quantitative RT-PCR using dilution series of chemically synthesized oligonucleotides (13). Shown are the average copy numbers measured in 500 pg RNA from three independent B95-8 LCL exosome purifications/isolations. One sample was treated with 10 ng/μL RNase A, one was treated with 400 ng/μL RNase A, and one sample was untreated. (F) Comparison between individual cellular EBV-miRNA copy-numbers in ∼104 LCL (B95-8) and relative abundance of exosomal EBV-miRNA copy numbers. (G) EBV-miRNA copy-numbers measured in 10 ng exosomal RNA from the X50-7 LCL compared with total cellular RNA. Cluster 2 BART EBV-miRNAs are ∼1,000-fold less abundant in exosomes than expected from their individual cellular expression levels. Error bars (SD) are derived from triplicate experiments.
Fig. 2.
Fig. 2.
EBV-miRNAs are transferred from LCL to MoDC via exosomes. (A) Confocal image of purified PKH67-labeled LCL (RN) exosomes (green) captured by HLA-DR–specific Dynal beads (red). (Inset: Dynal bead with multiple LCL-derived exosomes captured on the surface.) (B) Primary MoDC incubated for 2 h with increasing amounts of PKH67-labeled purified exosomes. (C) Quantification of B by FACS showing an increase in MFI (black bars) and percentage (gray bars) of PKH67-positive cells. (D) Confocal image of primary (immature) MoDC incubated for 2 h with purified PKH67-labeled LCL exosomes. TO-PRO staining (red) indicates the nucleus. (E) Schematic of the transwell coculture model with PKH67-labeled LCL (producers) in the top well and primary MoDC (recipients) in the bottom well. A porous (1.0-μm) membrane allows transfer of fluorescent exosomes but precludes LCL migration. (F and G) FACS results representing CD86+/CD19− MoDC in the bottom chamber before and after 24-h coculture with PKH67-labeled LCL stimulated for 3 h with 10 μM monensin. (H) MoDC incubated for 2 h with purified PKH67-labeled exosomes added directly to the top chamber. (I) FACS results showing MFI of MoDC upon coculture with PKH67-labeled LCL. Error bars (SD) are derived from duplicate wells. (J) Quantitative RT-PCR for EBV-miRNAs in a subset (∼2 × 104) of EBV-negative primary MoDC cocultured with B95-8 LCL for 24 h, as shown in E (black bars), compared with the level of these miRNAs in 500 pg RNA from purified B95-8 exosomes (white bars). (K and L) Comparison of individual miRNA levels in a subset of MoDC cocultured for 24 (K) and 48 h (L) in the presence of B95-8 LCL.
Fig. 3.
Fig. 3.
Internalization of EBV-miRNA containing exosomes leads to gene silencing in recipient primary DC. (A) EM image of purified secreted exosomes from EBV-infected B95-8 LCL. (B) Flow cytometry results of HeLa cells incubated for 2 h with PKH-labeled purified LCL exosomes. (C) Relative luciferase activity in HeLa cells after 8 h of cotransfection with a 3′UTR-CXCL11 reporter construct without (control) and with 50 μL of purified B95-8 LCL exosomes (white bar). (D) Normalized luciferase activity in HeLa cells transfected with wild-type (WT) 3′UTR-CXCL11 reporter construct or a mutated construct with disrupted BHRF1-3 target sites, incubated with and without EBV-positive exosomes. (E) Relative luciferase activity in primary MoDC transfected with 3′UTR-CXCL11 incubated for 24 h with 2-fold increasing (25, 50, and 100 μL) amounts of purified EBV-miRNA–positive LCL exosomes. (F) Relative luciferase activity measured in MoDC transfected with 3′UTR-CXCL11 and cocultured for 24 h with B95-8 LCL (white bar) or EBV-negative Jurkat cells (gray bar). (G) EM image of purified EBV-miRNA–negative BJAB exosomes. (H) Flow cytometry results of purified PKH67-labeled BJAB or LCL exosomes (HLA-DR+) incubated for 2 h with MoDC, indicating comparable internalization. (I) Normalized luciferase activity measured in MoDC transfected with a 3′UTR-CXCL11 reporter after 24 h incubation with 2 × 100 μL purified LCL (EBV-miRNA BHRF1-3+) and BJAB (EBV-miRNA BHRF1-3−) exosomes, (*, P < 0.025 in a two-tailed student t test). (J) Luciferase activity in HeLa cells with a 3′UTR-LMP1 luciferase and an empty vector or cluster 1 and cluster 2 BART miRNA expression vectors. (K) Dose–response as described in E with a 3′UTR-LMP1 reporter in MoDC. (L) As in I with 2 × 50 μL purified LCL exosomes and 2 × 100 μL BJAB-derived exosomes. *, P < 0.02, two-tailed student t test. Error bars (SD) in all graphs are derived from triplicates.
Fig. 4.
Fig. 4.
EBV-miRNAs are present in infected B cells and noninfected non-B cells in asymptomatic patients. (A) PBMCs from patients with various EBV loads were fractionated in non-B-cell, B-cell, and T-cell populations. Quantitative EBV-DNA PCR was performed. Shown are the number of EBV-DNA copies/104 cells. (B) Quantitative RT-PCR using RNA from fractioned B cells and non-B cells for BART1-5p, BART2-p, and BART3* EBV-miRNAs. Represented on the x axis are samples in which one or more EBV-miRNAs were detected in B cells (filled squares) or non-B cells (filled diamonds). The y axis shows the corresponding viral DNA loads (copies/mL blood). In 40% of the non-B-cell samples we were unable to detect any EBV-miRNAs (open diamonds). (C) Quantitative multiplex RT-PCR for three EBV-miRNAs in purified B-cell and non-B-cell fractions (2 × 104 cells) of four patients with varying EBV-DNA loads (copies/mL blood): 23,000 (patient 1), 10,000 (patient 2), 5,000 (patient 4), and 300 (patient 3). Shown are the copy numbers in log-scale for BART1-5p (black bars), BART2-p (gray bars), and BART3* (white bars) individually in each of the two cell fractions for each patient.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
    1. Cullen BR. Viral and cellular messenger RNA targets of viral microRNAs. Nature. 2009;457:421–425. - PMC - PubMed
    1. Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer. 2006;6:259–269. - PubMed
    1. Dolken L, et al. Mouse cytomegalovirus microRNAs dominate the cellular small RNAs profile during lytic infection and show features of post-transcriptional regulation. J Virol. 2007;81(24):13771–13782. - PMC - PubMed
    1. Mitchell PS, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA. 2008;105:10513–10518. - PMC - PubMed

Publication types