Identification of Host Biomarkers of EBV Latency IIb and Latency III

Abstract

Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral and host gene expression. Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of the immune suppressed. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs) serving as a model of these lymphomas. In previous studies, our lab has described a temporal model for immortalization with an initial phase characterized by expression of the Epstein-Barr Nuclear Antigens (EBNAs), high c-Myc activity, and hyper-proliferation in the absence of the Latent Membrane Proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly the NF{kappa}B-activating LMP1, defining latency III. LCLs, however, express a broad distribution of LMP1 such that a subset of these cells expresses LMP1 at levels seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA-Seq on early EBV-infected latency IIb cells and latency III LCLs sorted by NF{kappa}B activity. We found that latency IIb transcriptomes clustered independently from latency III independent of NF{kappa}B. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-FISH targeting EBV EBNA2, LMP1, and human CCR7. This study defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.\n\nIMPORTANCEEBV is a ubiquitous pathogen with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction express the membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more efficacious therapies in the future.