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.