The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production

Abstract

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC ₅₀ ] = 1.5 μM) and ACV (IC ₅₀ = 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC ₅₀ = 19.6 μM) and ACV (IC ₅₀ = 36.4 μM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC ₅₀ = 1.2 μM) and ACV (IC ₅₀ = 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.