Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with a variety of different human tumors (
1,
47,
81). Like all herpesviruses, EBV can infect cells in either the latent or lytic form (
13). The lytic form of infection is required for horizontal spread of the virus from cell to cell and from host to host. During the lytic form of viral replication, EBV uses a virally encoded DNA polymerase and the oriLyt replication origin to duplicate its genome (
24,
36,
51). The lytic form of EBV replication can be effectively inhibited
in vitro by the guanine nucleoside analogues, acyclovir (ACV) and ganciclovir (GCV) (
11,
16,
50,
56). Since acyclovir is significantly less toxic than ganciclovir in patients, acyclovir is generally used to treat diseases associated with lytic EBV infection, such as oral hairy leukoplakia (
3).
GCV and ACV cannot be incorporated into viral or cellular DNA unless they are phosphorylated and converted into nucleotides (
17,
29). Work in other herpesvirus systems has demonstrated that the first step in GCV or ACV phosphorylation is not performed efficiently by cellular nucleoside kinases but can be carried out by virally encoded enzymes in cells infected with various herpesviruses (
6,
17,
20,
25,
29,
75). Human herpes simplex virus 1 (HSV-1) and HSV-2 encode a viral thymidine kinase (TK) which mediates the first step in GCV and ACV phosphorylation in virally infected cells (
20,
21,
73), and ACV- and GCV-resistant HSV mutants isolated from patients commonly have mutations in the viral thymidine kinase gene (
5,
14,
46,
64) and less commonly within the viral DNA polymerase gene (
61). In contrast, the human cytomegalovirus (HCMV) does not encode a viral thymidine kinase protein. Instead, in HCMV-infected cells, the virally encoded protein kinase (UL97) mediates the first step of GCV phosphorylation (
53,
75) and (albeit much less efficiently) ACV phosphorylation (
76). Once made, the triphosphorylated form of ACV (and to a lesser extent GCV) is a much better substrate for herpesvirus-encoded DNA polymerases than cellular DNA polymerases (
17,
29) and thus inhibits viral DNA replication more effectively than cellular DNA replication.
EBV encodes both a thymidine kinase (EBV-TK, the product of the BXLF1 gene) (
18,
54) and a protein kinase (EBV-PK, the product of the BGLF4 gene) (
74). EBV-PK is a serine/threonine protein kinase that shares many of the same substrates as the UL97 cytomegalovirus (CMV) kinase (
28). Although it has been demonstrated that both GCV and ACV are activated and phosphorylated in lytically infected EBV-positive cells, it remains unclear whether GCV and/or ACV phosphorylation in EBV-infected cells is mediated primarily by the viral protein kinase, the viral thymidine kinase, or both.
All studies to date investigating this question have been performed outside the context of the viral genome, and different groups have reported conflicting findings, particularly in regard to the effects of EBV-TK. For example, one group reported that EBV-TK (expressed in bacterial lysates) phosphorylates GCV and ACV
in vitro (GCV more than ACV) (
52), and another group found that overexpression of EBV-TK in cells enhances GCV phosphorylation and sensitivity to the cytotoxic effects of GCV and ACV (GCV more than ACV) (
59). In contrast, two other groups reported that EBV-TK purified from bacterial lysates has a highly restricted substrate specificity in comparison to HSV-TK and phosphorylates ACV and GCV extremely poorly if at all (
33,
78). Furthermore, in one of these studies, overexpression of EBV-TK in cells did not result in GCV phosphorylation or GCV-mediated cell killing (
33).
DISCUSSION
Although GCV and ACV both inhibit the lytic form of EBV replication
in vitro and have been used to treat EBV infection in patients, it has remained unclear exactly how these drugs are converted to their active forms in lytically replicating EBV-infected cells. The conversion of these two nucleoside analogues to their monophosphorylated forms, which is the rate-limiting first step in their activation, is performed by the viral thymidine kinase in HSV-infected cells (
20) but mediated by the viral protein kinase (UL97) in HCMV-infected cells (
53,
75). Whereas HSV-1 TK is a polynucleoside kinase, UL97 is not known to be a nucleoside kinase at all, although it phosphorylates the acyclic purine analogs ACV and GCV. ACV and GCV are then subsequently further phosphorylated by cellular enzymes to become nucleotide triphosphates, which inhibit viral DNA polymerase-mediated viral DNA replication both as competitive alternative substrates for GTP and (in the case of ACV) as a DNA chain terminator (
17,
29). Since EBV encodes both a viral thymidine kinase and a viral protein kinase (homologous to UL97), one or both of these viral proteins might potentially be required for ACV and/or GCV activity against EBV. In this paper, we used a genetic approach to inhibit expression of either EBV-TK or EBV-PK in cells with lytic EBV infection and determined whether either protein is required for ACV and/or GCV antiviral activity. Our results show that EBV-PK but not the EBV-TK is required for the antiviral effects of both ACV and GCV against EBV.
The conflicting reports about the substrate specificity of EBV-TK with regard to GCV and ACV (
33,
52,
59,
78) have led to confusion in the field about whether EBV-TK and/or EBV-PK mediates ACV and/or GCV sensitivity in EBV-infected cells. We have demonstrated here that loss of EBV-TK activity in the context of the intact EBV genome does not in any way impair the ability of the virus to respond to either ACV or GCV (in fact, the IC
50s of both ACV and GCV are slightly lower in cells infected with the TKmut virus than in cells infected with the WT virus). Thus, EBV-TK is certainly not required for either ACV or GCV activation in cells with lytic EBV infection. One potential explanation for this result is that both EBV-TK and EBV-PK independently induce enough ACV and GCV phosphorylation in EBV-infected cells that loss of either viral protein alone does not affect ACV/GCV susceptibility. However, this possibility is made unlikely by the finding that cells infected with EBV PKmut (which can still express EBV-TK at a normal level) are highly resistant to ACV and GCV. Nevertheless, since EBV-TK and EBV-PK have been reported to directly interact in yeast two-hybrid assays (
8), consistent with the observation that EBV-TK is a substrate of EBV-PK (
87), it remains theoretically possible that only a PK-phosphorylated form of EBV-TK can enhance ACV/GCV phosphorylation in cells, since EBV TK phosphorylated by cellular enzymes is without activity (
33; J. D. Fingeroth, unpublished data).
Although TKmut did not show enhanced resistance to ACV/GCV (consistent with a previous report that GCV and ACV are not substrates for EBV-TK [
33]), it did confer some susceptibility to the antiviral effect of the thymidine analogue, BrdU, a known substrate of EBV-TK. These results indicate that as previously suggested, thymidine nucleoside analogs (rather than guanine nucleoside analogues, such as ACV/GCV) may prove useful as anti-EBV drugs that take advantage of the EBV-TK activity (
33). If so, EBV isolates that become resistant to ACV/GCV may not necessarily be resistant to thymidine nucleoside analogues, and the antiviral effects of the guanine nucleoside analogues and the thymidine nucleoside analogues together might prove synergistic for EBV and decrease the likelihood that resistance develops.
Our results here also suggest that the EBV-encoded serine/threonine protein kinase, like the HCMV UL97 kinase, induces enough GCV phosphorylation when expressed at a normal level in lytically infected cells to be clinically useful. Although the HCMV UL97 kinase is generally not thought to phosphorylate ACV efficiently enough for this drug to be used for treatment of HCMV infection in patients, ACV is currently considered by some to be the treatment of choice for lytic EBV infection in humans (
37). Notably, we found that it is EBV-PK, and not EBV-TK, which mediates the susceptibility of EBV to ACV. This result suggests that either EBV-PK phosphorylates ACV more efficiently than the HCMV UL97 kinase or EBV lytic replication is better inhibited than HCMV replication by very low levels of triphosphorylated ACV. A head-to-head comparison will clarify this issue.
Although EBV-PK has a relatively low sequence homology with UL97, EBV-PK can partially complement a UL97 defect in the HCMV genome (
67), and EBV-PK and HCMV UL97 may share many similar functions during lytic viral replication. The CMV UL97 kinase and EBV-PK are both required for nuclear egress of the viruses in most cell lines (
35,
62), and shared cellular substrates of these two kinases include nuclear lamin A/C (
35,
49), cellular elongation factor 1δ (
42-
44), and pRB (
39; C. V. Kuny, K. Chinchilla, M. Culbertson, and R. F. Kalejta, submitted for publication). The unusual ability of the EBV PKmut virus to efficiently produce infectious viral particles in 293T cells allowed us to study the role of EBV-PK in mediating ACV/GCV susceptibility. As reported in another article, we found that both the large and small forms of the SV40 T antigens contribute to the rescue of the PKmut virus in 293T cells (
57). Similarly, another group recently reported that an HCMV mutant unable to express the UL97 kinase can be partially rescued by the human papillomavirus E7 viral protein (
41). Interestingly, this paper also showed that the antiviral effect of maribavir (a drug which inhibits UL97 kinase activity) (
7) is highly attenuated in the presence of E7 (
41). Since maribavir has also been reported to inhibit lytic EBV replication in some cell types (
26,
30), it will obviously be of interest to determine if the PKmut virus is resistant to this drug in cells (such as 293T cells) where it is able to replicate.
Our results, as well as those of previous studies (
45), also raise the question of whether GCV might be a more effective drug than ACV for treating lytic EBV infection in patients. We found that the IC
50 of ACV for WT EBV was 4.1 μM, while the IC
50 of GCV was 1.5 μM. Previous studies using Southern blot methods to measure the amount of lytic EBV viral replication also reported that the IC
50 of ACV for WT EBV is considerably higher (7 to 10 μM) (
71,
86) than the IC
50 of GCV (found to be 1.0 μM) (
11,
56). The definition of
in vitro resistance of HSV isolates to ACV has varied in the literature from 4.4 to 13.2 μM, according to the method selected and various other factors (
14,
22,
23). The IC
50 for the EBV-PK mutant virus of acyclovir (36.4 μM) would clearly categorize it as resistant to ACV. Nevertheless, the relatively high IC
50 of ACV for WT EBV, although considerably less than that for HCMV (77 ± 2.1 μM), is much higher than that for HSV (IC
50 = 0.45 to 1.47 μM), raising the possibility that oral ACV might be only marginally active against lytic EBV infection in patients, particularly since the reported peak ACV serum level in patients receiving an 800-mg dose of oral acyclovir is only 7 μM (
37).
In the case of GCV, the reported peak serum level of drug following a dose of oral valganciclovir (900 mg) is 23 to 26 μM (
37). HCMV isolates are considered to be GCV sensitive at IC
50s of <6 μM (
12). Although the IC
50 of GCV for WT EBV is clearly within the range predicted for GCV susceptibility in patients, the high IC
50 of the PKmut EBV virus (19.6 μM) suggests that this mutant would be resistant to GCV therapy in patients.
A study directly comparing the ability of ACV versus that of GCV to treat lytic EBV infection in humans has not been performed. Interestingly, in most studies, ACV treatment has not been found to benefit patients with infectious mononucleosis (IM) (
77). In a number of these studies (
2,
79,
80), the peak level of ACV achieved in the serum was likely less than the IC
50 for EBV (since patients received doses of 600 to 800 mg orally five times per day), which could possibly explain the lack of clinical benefit in these studies. Nevertheless, the lack of clinical benefit from oral ACV treatment in patients with IM may reflect not only the relatively low susceptibility of EBV to this drug, as well as the low oral bioavailability of ACV, but the fact that many of the symptoms of IM are mediated by the host immune response to virally infected cells with the latent form of infection. We predict that either oral valacyclovir therapy, which results in much higher serum levels of acyclovir (up to 22 μM after a dose of 1,000 mg), or oral valganciclovir therapy would be considerably more effective than oral acyclovir for treating lytic EBV infection in patients in circumstances where it is indicated. A small study using valacyclovir to treat patients with IM suggested that it may improve clinical symptoms (
4). Valacyclovir was reported to effectively treat most cases of oral hairy leukoplakia (a lytic EBV infection on the tongue that occurs in immunosuppressed patients), although a few cases were resistant to this drug (
82). Our results suggest that a clinical study comparing the efficacy of valganciclovir with that of valacyclovir for treating patients who are severely ill with uncontrolled IM or who have unusual complications of primary EBV infection, such as viral meningoencephalitis, may be warranted. Nevertheless, in patients with less-severe forms of IM, antiviral therapy is probably not indicated.