*Purpose: Exosomes are nanometric (50-150nm) membrane vesicles that are released, into blood and other body fluids, by most cell types. Exosomes, which are generated from the inward budding of endosomal membranes, form multi-vesicular bodies that can transfer cytosolic proteins and nucleic acids. Studies have demonstrated that exosomes contain and transfer microRNAs (miRs) between cells that allow for local and distant intercellular communication. MicroRNAs are short non-coding RNA molecules that can post-transcriptionally regulate messenger RNA transcripts, resulting in translational repression. We demonstrated that Epstein-Barr virus (EBV) infection markedly alters the expression of host B cell microRNA. EBV is the causative virus in the majority of post-transplant lymphoproliferative disorders (PTLD), a potentially fatal complication of transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression.

*Methods: The microRNA profiles of: 1) EBV+ B cell lymphoma lines from patients with PTLD (n=6), 2) tissue sections of EBV+ PTLD tumors (n=10), 3) in-vitro generated EBV+ lymphoblastoid cell lines (n=5), 4) EBV- cell lines (n=2), 5) tissue sections of EBV- PTLD tumors (n=5), and 6) normal healthy B cells (n=5) were determined. One-way ANOVA analysis was used to identify the microRNAs differentially modulated between the groups. In all of these studies, and subsequent validation by RT-PCR, the microRNAs miR-17, miR-19a, miR-106a, miR-155 and miR-194 emerge as the group of miRNA differentially regulated. Further, we demonstrated that miR-194 is uniquely suppressed in EBV+ B cell lines from PTLD patients and that the 3’ untranslated region of IL-10 is targeted by miR-194 suggesting that EBV co-opts the host B cell microRNA network and specifically suppresses microRNA-194 to override control of IL-10 expression.

*Results: To determine if EBV+ cell lines secrete exosomes containing these microRNAs we cultured EBV+ B cell lymphoma lines from patients with PTLD (n=5) and EBV- B cell lymphoblast cell lines (n=3) in exosome-free media, isolated and purified exosomes from the supernatant. RNA was isolated from both the parent cells and purified exosomes, and the microRNAs quantitated. In all cases exosomes mirrored the microRNA expression of the parent cells. In agreement with our profiling studies, miR-17 and miR-19a are increased in the exosomes from EBV- tumors. Further we demonstrate that miR-155 is significantly and specifically increased in EBV+ cells and exosomes whereas miR-194 is completely suppressed by EBV.

*Conclusions: Taken together, these studies demonstrate that EBV alters the microRNA network both in the local infected cell and potentially, via exosomes, at distant uninfected cells. Cell-free microRNAs hold promise in both the diagnosis and treatment of EBV+ and EBV- PTLD.

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