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Objectives: Although exosomes carrying Epstein-Barr virus-encoded small RNA-1 (EBER-1) are involved in the immunosuppressive tumor microenvironments of EBV-associated head and neck carcinomas, the effects of EBER-1-associated exosomes on tumor-infiltrating macrophages are poorly understood., Materials and Methods: The association between EBV infection and expression of indoleamine 2,3-dioxygenase (IDO) was assessed in 165 paraffin-embedded oral squamous cell carcinoma (OSCC) tissue samples. Using in vitro techniques, we investigated whether stimulation of the RIG-I/IL-6/TNF-α pathway by exosomes carrying EBER-1 is critical for IDO induction in macrophages. We performed a thymidine incorporation and a cell cytolytic assay to test for up-regulated IDO in macrophages that can block the proliferation and function of effector T cells., Results: Some infiltrated macrophages expressed levels of IDO higher than OSCC cells which was significantly associated with presence of EBV. The production of IDO, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in human monocyte-derived macrophages (MDMs) was induced by EBV-associated exosomes in vitro. Mechanistically, the retinoic acid-inducible gene I (RIG-I) pathway in MDMs was stimulated by EBV-encoded small RNA-1 (EBER-1) whereas the inhibition of these pathways by BX-795 almost abolished the production of these two cytokines and IDO induction. Also, the EBER-1-activated IDO in MDMs suppressed the proliferation of T lymphocytes and diminished the cytolytic activity of CD8 + T cells., Conclusion: Exosomes carrying EBER-1 could induce IDO expression in MDMs, considerably aided by an IL-6 and TNF-α-dependent mechanism via the RIG-I signaling pathway, which might create an immunosuppressive microenvironment affecting T-cell immune responses., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Epstein-Barr virus (EBV) encodes BamHIA rightward transcript (BART) microRNAs (miRNAs). These miRNAs are expressed at high levels in epithelial tumors, such as nasopharyngeal carcinoma (NPC). BART miRNAs play important roles in EBV-associated malignancies, however, the reason for their high expression in NPC is unclear. We performed multiple sequence alignment of six completely sequenced EBV strains: Akata, YCCEL1, SNU719, C666-1, Mutu I, and M81. A single-nucleotide deletion was identified at the promoter region of BART. The luciferase assay suggested that this single-nucleotide polymorphism (SNP) significantly increased BART promoter activity. In addition to deletion, substitution at the same site also increased BART promoter activity. Analysis of the 170 EBV genome sequences from NPC and EBV-associated gastric cancers revealed that the frequency of this SNP was associated with NPC incidence and this SNP was found to be accumulated in the BART promoter region. Overall, our results suggested that this SNP should enhance BART promoter activity and thus, might contribute to the development of EBV-associated epithelial malignancies., (Copyright © 2019 Elsevier Inc. All rights reserved.)