Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling

Abstract

Marek’s disease virus (MDV), an alphaherpesvirus of poultry, causes Marek’s disease and is characterized by visceral CD4+TCRαβ+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus–host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System

“…MDV-encoded pp38 is considered to be one of the best biomarkers of the latency-to-lytic switch in the LCL, where most of the MDV genome is held in a latent state, thought to be through epigenetic mechanisms [30]. However, previous studies have shown that between 1–10% of the MSB-1 population expressed pp38 at low levels [31,40]. Lytic replication in LCL can be induced with histone deacetylase (HDAC) inhibitors such as NaB or methylation inhibitor AZA [30].…”

“…Thus far, most of the data on MDV gene expression during the neoplastic stages of the disease have come from lymphoblastoid cell lines (LCL) derived from MD lymphomas. As clonal populations of transformed tumor cells with latent MDV genome and limited gene expression [29,30,31], LCLs provide an extremely valuable source to study the latency, reactivation, and transformation in situ. However, the manipulation of the viral and host genes in these cell lines hitherto has been challenging primarily because of the lack of availability of efficient tools.…”

The Transcriptional Landscape of Marek’s Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes

“…MDV encodes about 100 proteins that orchestrate the virus life cycle and/or contribute to pathogenesis [12,14]. Until now, analyses of the viral transcriptome has been limited to chicken fibroblasts that are not infected in chickens, ex vivo samples [15,16] and tumor cells [17]; however, the mRNA expression in the primary target cells of lytic replication in vivo remained elusive. This is mainly due to the short lifespan of B and T cells in culture and the low quantity of infected cells in lymphoid organs of chickens [7,18].…”

Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines

“…While the role of miR-M4 in the induction of MD lymphomas has been clearly demonstrated in these studies, it remains unclear whether continued high-level expression of miR-M4 is essential for maintaining the transformed phenotype of MDV-transformed tumor cells. As clonal populations of transformed tumor cells with latent MDV genome and limited gene expression (30–32), lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells. However, detailed investigations into the role of different viral and host determinants in these cells have been difficult due to the lack of tools for manipulation of viral/host genomes of these cells in situ .…”