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 microM) and ACV (IC(50) = 4.1 microM). 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 microM) and ACV (IC(50) = 36.4 microM). 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 microM) and ACV (IC(50) = 2.8 microM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

FIG. 1.

Creation of stable 293T cell clones latently infected with WT, PKmut, and TKmut viruses. (A) 293T cells latently infected with EBV WT or PKmut viruses were transfected with vector control or BZLF1 and BRLF1 expression vectors (“Z”) to induce lytic gene expression, and extracts were analyzed by immunoblotting for EBV-PK, BZLF1, and β-actin. (B) 293T cells latently infected with EBV WT or PKmut viruses were transfected with vector control or BZLF1 and BRLF1 expression vectors in the presence or absence of a cotransfected EBV-PK expression vector, as indicated. Extracts were analyzed by immunoblotting for BZLF1, BMRF1, and β-actin. The hyperphosphorylated (pp-BMRF1) and phosphorylated (p-BMRF1) forms of the BMRF1 protein are indicated; the hyperphosphorylated form of BMRF1 requires expression of EBV-PK (10, 30, 31). (C) 293T cells infected with WT or TKmut viruses were transfected with vector control or the BZLF1 (Z) and BRLF1 expression plasmids. Extracts were prepared and analyzed by immunoblotting for expression of the lytic EBV proteins, BMRF1, BZLF1, EBV-TK, and cellular β-actin.

FIG. 2.

The PKmut and TKmut viruses are not defective for viral production in 293T cells. (A) 293T cells infected with WT, PKmut, or TKmut viruses were transfected with the BZLF1, BRLF1, and gp110 expression vectors to induce lytic infection. Viral titers were quantitated by infecting Raji cells with various amounts of the supernatant 72 h posttransfection and counting the number of GFP-positive Raji cells using a fluorescence microscope. The number of GFP⁺ Raji cells per ml (GRU/ml) is shown. Results from three independent experiments are shown. (B) Extracts from the 293T cells used to create virus for panel A were immunoblotted with antibodies against BZLF1 and β-actin.

FIG. 3.

TKmut has sensitivities to GCV and ACV similar to those of WT EBV. 293T cells infected with WT EBV or TKmut EBV were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic replication in the presence or absence of acyclovir (25 μg/ml) (A) or GCV (5 μg/ml) (B). Viral titer and viral replication were examined 48 h later as described in the legend for Fig. 2. The average viral titer produced by the 293T WT and TKmut cells in the absence of drug is set as 100% for each cell line (± standard deviation). (C) 293T cells infected with WT EBV or TKmut virus were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic replication in the presence or absence of different concentrations of BrdU (10, 5, or 1 μg/ml) for 48 h. Viral titers were quantitated as described in the legend for Fig. 2. The average viral titer produced by the 293T WT and TKmut cells in the absence of drug is set as 100% for each cell line (± standard deviation).

FIG. 4.

PKmut has reduced susceptibility to acyclovir and ganciclovir compared to WT EBV. 293T cells infected with WT EBV or PKmut EBV were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic replication. Following transfection, cells were treated with no drug or different doses of GCV (10, 8, or 5 μg/ml) (A) or ACV (50, 25, or 10 μg/ml) (B) for 48 h. Viral titers were examined 48 h later. The average viral titer produced by the 293T WT and PKmut cells in the absence of drug is set as 100% for each cell line (± standard deviation). Results from two independent experiments are shown.

FIG. 5.

Expression of EBV-PK in trans restores susceptibility of PKmut to ganciclovir and acyclovir. 293T cells infected with WT or PKmut viruses were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic replication in the presence or absence of a cotransfected EBV-PK expression vector, as indicated. Following transfection, cells were treated with no drug or GCV (5 μg/ml) (A) or ACV (25 μg/ml) (B) for 48 h, and viral titers were quantitated. The average viral titer produced for each transfection condition in the absence of drug is set as 100% for each cell line (± standard deviation). The average viral titer of cells treated with GCV or ACV is normalized to the average viral titer of cells in the absence of drug.

FIG. 6.

GCV and ACV are not toxic to 293T cells at the doses used to inhibit EBV replication. 293T EBV-negative cells were transfected with BZLF1/BRLF1/gp110 expression vectors and then treated with no drug or different doses of GCV (A) or ACV (B). Forty-eight hours later, cells were stained with trypan blue. The percentage of viable cells without drug treatment is set as 100%.