The general problem in cancer treatment centres on finding agents that specifically affect cancer cells without damaging normal cells. The differences between cancer cells and normal cells are usually very subtle but about 15% of all human cancers involve a virus infection, for example the Epstein-Barr virus associated cancers. In these cancers, every tumour cell carries the virus in a latent infection but the number of normal cells infected is very low. So a treatment that could somehow cause the elimination of EBV infected cells would be very specific for the cancer in such cases. One potential approach could involve finding ways to reactivate the latent virus in cancer cells into the early part of the lytic cycle, impeding cell proliferation, targeting chemotherapeutic agents to the cancer and causing the cancer cells to become targets for immune surveillance. This review considers the mechanisms by which EBV reactivation is controlled and discusses possible therapeutic approaches.

A luciferase reporter system with stably transfected oriP plasmids in Akata Burkitt's lymphoma cells provides a quantitative assay for the BZLF1 Zp promoter in response to B-cell receptor (BCR) activation by cross-linking with anti-immunoglobulin. In this system, detailed kinetic studies of promoter activity are possible. Previously reported promoter elements upstream of ؊221 from the transcription start and the ZIIR sequence had little effect on the Zp promoter, but the ZI and ZIIIA elements were essential for early activation. The ZIIIB element mediates autoactivation. Mutation of the ZV repressor sequence greatly increased the induction of the promoter but did not make it constitutively active. Zp transcription in response to BCR cross-linking declined after a few hours; this decline was reduced and delayed by acyclovir or phosphonoacetic acid, indicating that viral DNA replication or a late viral gene can play a role in the switch off of the Zp promoter. Late expression of the LMP1 protein may account for this.

Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in cis, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between ؊49 and ؉30 are sufficient for late gene expression, which is dependent on ori lyt in cis. However, the TATT version of the TATA box found in many late genes was not essential for late expression.We recently described a system for studying Epstein-Barr virus (EBV) lytic gene regulation with plasmids stably transfected into the Akata Burkitt's lymphoma cell line (3). The plasmids contain the promoter being studied linked to the luciferase reporter gene and also have the EBV oriP plasmid origin of replication and a selectable marker (hygromycin resistance). Cell lines stably maintaining the plasmids, typically at about 10 copies per cell, can readily be isolated. Akata cells containing EBV can be induced into the lytic cycle by treatment with anti-immunoglobulin (anti-Ig), which cross-links the surface B-cell receptor, providing a rapid and physiologically relevant method for studying the EBV lytic cycle in cell culture (28). This system accurately reconstituted the regulation of the Zp immediate early promoter of EBV and was used to study its regulation in detail (3,5,14). The stably transfected reporter plasmid has an average copy number similar to that of the endogenous EBV genome present in the same cell, the plasmids are assembled into chromatin like EBV, and the reactivation of endogenous virus provides viral lytic gene products that may act in trans at their normal concentrations. These features make the system a good model for studying viral gene regulation, avoiding artifacts associated with transient transfection and incorrect expression levels of regulatory factors. In this study, we extended the use of the system to investigate the Rp promoter and EBV delayed early and late gene promoters.Zp and Rp have been considered to be the immediate early promoters of EBV, but controversy has surrounded the status of Rp, partly because of technical difficulties in mapping the 5Ј end of the RNA from this ...

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.