Extracellular vesicles from wild-type Epstein-Barr virus-transformed B-cells export host DNA and the viral lncRNA EBER1

Epstein-Barr virus (EBV) infection is nearly ubiquitous in humans and has been associated with multiple sclerosis (MS) and other immune-mediated diseases, yet mechanisms by which EBV-infected B-cells influence distal tissues remain incompletely understood. Extracellular vesicles (EVs) mediate intercellular communication during viral infection, but their integrated viral and host cargo has not been comprehensively defined in EBV-transformed B-cells generated from naturally infected individuals. Here, we performed an unbiased multiomic characterization of small EVs (sEVs) released from spontaneous lymphoblastoid cell lines (SLCLs) derived from normal donors and patients with stable or active MS, transformed ex vivo by endogenous wild-type EBV. Using surface profiling, quantitative proteomics, whole-genome sequencing of EV-associated DNA, total stranded RNA sequencing, droplet digital PCR, and super-resolution microscopy, we mapped the protein, DNA, and RNA cargo of these vesicles and determined their host and viral origins. SLCL sEVs contained canonical tetraspanins, B-cell markers, and were free of detectable virions. Proteomics identified over 6,000 shared proteins and revealed enrichment of nucleic acid-binding and chromatin-associated proteins. Whole-genome sequencing demonstrated abundant EV-associated DNA comprising two distinct compartments: high-molecular weight, DNase-sensitive DNA associated with the vesicle corona and DNase-resistant, nucleosome-sized ([~]130-150 bp) DNA protected within vesicles. In both compartments, DNA was overwhelmingly host-derived and broadly distributed across the genome, whereas EBV genomic DNA was minimal. RNA sequencing identified diverse EBV transcripts, with striking enrichment of the viral long noncoding RNA EBER1 across all lines. Super-resolution imaging and ddPCR confirmed EBER1 incorporation within individual vesicles. Notably, EBER1 has been detected in MS brain tissue in prior studies, and our findings provide a plausible vesicle-mediated mechanism for dissemination of this viral lncRNA from EBV-infected B-cells to distal sites. These findings establish a foundational multiomic profile of sEVs from wild-type EBV-transformed B-cells and reveal export of host DNA and EBER1, with broad implications for viral immunobiology and intercellular signaling in MS and beyond. SIGNIFICANCE STATEMENTEpstein-Barr virus (EBV) infects over 90% of adults worldwide and is strongly linked to multiple sclerosis (MS). How EBV-infected B-cells may communicate with distant tissues, including the central nervous system (CNS), remains unclear. We provide the first integrated multiomic profile of extracellular vesicles released from B-cells transformed by endogenous, wild-type EBV. These vesicles are enriched in nucleosome-associated host DNA and the viral long noncoding RNA EBER1 but contain minimal viral DNA. Because EBER1 has been detected in MS brain tissue, our findings suggest a vesicle-mediated mechanism by which EBV-infected B-cells could deliver viral RNA to the CNS independently of infectious virus. These results establish a framework for understanding how EBV latency reshapes intercellular communication in immune-mediated disease.