Identification of Host Biomarkers of Epstein-Barr Virus 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 gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. 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, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation 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 NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. 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-fluorescencein situhybridization (RNA-FISH) targeting EBVEBNA2orLMP1and humanCCR7orMGST1. This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCEEBV 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 of the cells express 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.

Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

“…The advent of high-throughput, single-cell based methods has revealed a more complex picture of herpesvirus infection, from latent infection (Messinger et al, 2019; Oko et al, 2019; Shnayder et al, 2018), to lytic infection (Drayman et al, 2019; Oko et al, 2019; Wyler et al, 2019), reactivation from latency (Adang et al, 2006; Oko et al, 2019) and viral tropism (Sen et al, 2014). Recently, we identified unanticipated heterogeneity between infected cells during lytic infection, with wide variance in viral gene expression, from cells with robust viral gene expression to cells with little to no detectable expression of either viral non-coding RNAs or mRNAs (Oko et al, 2019).…”

Epstein-Barr virus promotes survival through germinal center light zone chromatin architecture

“…Our lab has found that the early-and late-phases of EBV are transcriptionally distinct (Messinger et al, 2019) and utilize unique strategies to promote apoptosis resistance (Price et al, 2017). Apoptosis is regulated by complex protein-protein interactions between pro-apoptotic and anti-apoptotic members of the BCL2 protein family.…”

Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines

“…Indeed, we recently described a gene expression program having low expression of LMP1 and NFκB targets which was unique to early infection (Latency IIb) relative to an otherwise identical population of LCLs. 30 The wide distribution in LMP1 and NFκB target expression levels within an LCL has been characterized and ascribed to the dynamic sampling of a distribution of immune evasive states, at the fringes of which growth and survival can be compromised. 31–33…”