Abstract
The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.
Highlights
Epstein-Barr virus is a ubiquitous human herpesvirus that asymptomatically infects the vast majority of the human population, in the developing world, where primary infection typically occurs during the first few years of life, leading to lifelong EBV latency
We have investigated the role of an EBV protein (EBNA-LP) which is thought to support gene activation by the essential virus protein EBNA2
We have made an EBV in which the EBV nuclear antigens (EBNAs)-LP gene has been disrupted
Summary
Epstein-Barr virus is a ubiquitous human herpesvirus that asymptomatically infects the vast majority of the human population, in the developing world, where primary infection typically occurs during the first few years of life, leading to lifelong EBV latency. Taken together EBV is implicated in around 1-1.5% of worldwide cancer incidence [1]. These diverse malignancies likely arise due to defects at different stages of the virus life cycle, or perhaps infection of cell types not involved in the virus’s natural life cycle [2]. EBV infection activates B cells, transforming them into proliferating lymphoblasts. In vitro these continue to proliferate into lymphoblastoid cell lines (LCLs) whereas in vivo they can differentiate – probably via a germinal center – into resting memory B cells where the virus is quiescent, producing RNAs but no viral proteins [3,4]. LCLs express the ‘growth’ program of EBV genes (latency state III), comprising six EBV nuclear antigens (EBNAs), the latency membrane proteins (LMP1, LMP2A and LMP2B) and a number of EBV encoded RNAs, including the abundant nuclear RNAs EBER1 and EBER2
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