Pharmacologic induction of Epstein-Barr virus kinases: Sensitivity of kinase-expressing cells to nucleoside analogues

Epstein-Barr virus (EBV) is a widespread human herpesvirus that is associated with several malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and AIDS-associated non-Hodgkin's lymphomas. Because conventional approaches to the treatment of malignancy are often not curative or are associated with serious complications, new, specific approaches to treatment are needed. The presence of EBV in the tumor cells of some EBV-associated malignancies may provide a target to selectively kill these tumor cells. Treatment of an EBV(+) Burkitt's lymphoma, cell line with an inducer of lytic gene expression, including expression of the viral thymidine kinase (TK) and phosphotransferase, was associated with sensitization to ganciclovir and a variety of other nucleoside analogues. Induction of EBV kinases and lytic replication was demonstrated by treatment of several EBV(+) tumor cell lines with TPA, bryostatin, butyrate, and 5-azacytidine. To better characterize the EBV TK with regard to the use of various nucleoside analogues as substrates, the EBV TK was expressed in stable and transient transfection systems. TK-expressing clones were more sensitive to killing by GCV, bromovinyldeoxy uridine, and azidodeoxythymidine, and were shown to phosphorylate these compounds more efficiently than control cells. The phosphorylation of these nucleoside analogues by the EBV TK and the phosphorylation of ganciclovir by the EBV phosphotransferase were directly linked to the sensitivity of kinase-expressing cells to these analogues. The phosphorylation of azidodeoxythymidine and bromovinyldeoxyuridine by the EBV TK was due to the demonstrable TK and thymidylate kinase activities of the enzyme. These enzymatic activities could not be separated by site-directed mutagenesis at a conserved residue in the nucleoside/nucleotide binding site of the enzyme. The treatment of an EBV(+) Burkitt's lymphoma cell line with 5-azacytidine resulted in expression of the viral TK and phosphotransferase enzymes, sensitized cells to treatment with ganciclovir, penciclovir, bromovinyideoxyuridine, and azidodeoxythymidine, and resulted in greater levels of activated ganciclovir and azidodeoxythymidine than in control cells. Induction of EBV kinases in combination with a suicide substrate is an alternative strategy to gene therapy for selective killing of EBV infected cells.