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

William N Mwangi,Mick Watson,Lydia B Kgosana,Deepali Vasoya,Venugopal Nair

doi:10.1099/jgv.0.000744

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.

Highlights

Marek’s disease virus (MDV) is a widely prevalent alphaherpesvirus of poultry, associated with Marek’s disease (MD) and characterized by rapid-onset CD4+TCRab+T-cell tumours at high incidence in susceptible hosts
Compared to the multiple steps of fixation and permeabilization to demonstrate the virusencoded lytic proteins in the cytoplasmic compartment of conventional MDV-transformed lymphoblastoid cell lines (LCLs), NWB-s and 3867-k cell lines with the readily demonstrable enhanced green fluorescent protein (EGFP) marker were quicker for sorting of reactivating cells with minimal manipulation
The availability of infectious bacterial artificial chromosome (BAC) clones of MDV allowing the generation of recombinant viruses expressing fluorescent marker genes has enabled studies looking into the role of viral gene products in replication and pathogenesis

Summary

Introduction

Marek’s disease virus (MDV) is a widely prevalent alphaherpesvirus of poultry, associated with Marek’s disease (MD) and characterized by rapid-onset CD4+TCRab+T-cell tumours at high incidence in susceptible hosts. Initial infection and subsequent spread of MDV occurs through the inhalation of infectious virus in dust. The MDV lifecycle is completed by the virus replication in the feather follicle epithelium, the site from which infectious cell-free virus is shed into the environment through the dander [1, 2]. Neoplastic transformation of latently infected CD4+ lymphocytes in susceptible birds results in lymphomas. These tumours and the lymphoblastoid cell lines (LCLs) derived from them are mainly monoclonal with clonally restricted T cell receptor (TCR) profiles [3,4,5]

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Results

Conclusion