The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies. Over 90% of adults worldwide are infected with Epstein-Barr virus (EBV). While EBV infection is normally controlled by a healthy immune system, in immuno-compromised individuals, EBV can cause serious disease and/or cancer. During infection, EBV expresses viral microRNAs (miRNAs) and induces the expression of specific cellular miRNAs. In general, miRNAs inhibit target gene expression by binding to complementary regions on target messenger RNAs (mRNA). While cellular miRNAs regulate important biological processes such as cell growth and differentiation, and many miRNAs have been linked to cancer progression, the functions of EBV miRNAs are largely unknown. To identify targets of EBV miRNAs and cellular miRNAs in EBV-infected cells, we used a high-throughput method based on next-generation sequencing technology to give a global picture of miRNA-regulated gene expression. Our analysis showed that over 500 mRNAs can be regulated by viral miRNAs, many of which are directly relevant to EBV infection. This study provides a comprehensive survey of viral and cellular miRNA targets in B cells, which is a positive step towards identifying novel therapeutic targets for EBV-associated cancers.