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 EpsteinBarr 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 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...

The Epstein-Barr virus (EBV) BGLF4 gene product is a protein kinase (PK). Although this kinase has been characterized and several of its targets have been identified, its biological role remains enigmatic. We have generated and assessed a BGLF4 knockdown phenotype by means of RNA interference and report the following: (i) BGLF4-targeting small interfering RNA effectively inhibited the expression of its product, the viral PK, during lytic reactivation, (ii) BGLF4 knockdown partially inhibited viral DNA replication and expression of selected late viral genes, (iii) the absence of EBV PK resulted in retention of the viral nucleocapsids in the nuclei, and (iv) as a result of the nuclear retention, release of infectious virions is significantly retarded. Our results provide evidence that EBV PK plays an important role in nuclear egress of the virus and ultimately is crucial for lytic virus replication.

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Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however, along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task.

A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.