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Journal of Virology, February 2007, p. 1195-1208, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.01518-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication{triangledown}

Chih-Chung Lu,1 Ho-Ting Huang,1 Jiin-Tarng Wang,1 Geir Slupphaug,2 Tsai-Kun Li,1 Meng-Chuan Wu,1 Yi-Chun Chen,1 Chung-Pei Lee,1 and Mei-Ru Chen1*

Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan,1 Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway2

Received 17 July 2006/ Accepted 30 October 2006


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ABSTRACT
 
Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.


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INTRODUCTION
 
Uracil in DNA may be generated from misincorporation of dUMP instead of dTMP by DNA polymerase during replication, resulting in a relatively harmless U · A pair, or from spontaneous deamination of cytosine to uracil, which will create a mutagenic U · G mismatch. The frequency of cytosine deamination is expected to be on the order of 60 to 500 per mammalian genome per day (36). If such a change is not repaired prior to the next round of replication, an A · T transition will ensue with further rounds of replication. Base excision repair (BER) is the major pathway to remove a damaged or inappropriate base (31). The initial step in BER to remove inappropriate uracil is catalyzed by a uracil-DNA glycosylase (UDG). UDGs hydrolyze the N-glycosidic bond between the uracil and the deoxyribose sugar, leaving an abasic site (AP site) in the DNA. After incision of the AP site by AP endonuclease 1 (APE1), BER may follow two tracks. In short-patch BER, the 5'-deoxyribose phosphate is removed by DNA polymerase ß, which also inserts a C or T, depending on the template base. Finally, DNA ligase seals the nick. The alternative long-patch BER pathway largely uses replication proteins. In humans, polymerase {delta} or {varepsilon}, aided by proliferating cell nuclear antigen (PCNA) and replication factor C, inserts 2 to 8 nucleotides. The displaced "flap" containing the 5'-deoxyribose phosphate is removed by the flap endonuclease FEN-1, and the nick is sealed by DNA ligase I (32). The UDG superfamily is divided into four protein families. Although these share a common structural fold, they are surprisingly divergent at the amino acid level (1, 51). The uracil-N-glycosylase (UNG) family (family 1) of proteins are the most ubiquitous, share similar biochemical properties, and are highly conserved at both the amino acid and structural levels (61, 65, 81). UNG proteins are relatively small monomeric proteins that usually do not require cofactors or ions for their activity. They are also highly specific against uracil in both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), with a preference for ssDNA (64, 75).

The human UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) isoforms of the enzyme through a mechanism that comprises transcription from two different promoters and alternative splicing (47). In addition to DNA repair, UNG2 is also involved in the somatic hypermutation and class switch recombination that yield secretory, high-affinity antibodies in B lymphocytes. Mutations in both alleles of UNG result in a hyper-immunoglobulin M (IgM) syndrome with life-threatening infections (25). Furthermore, UDG has recently been demonstrated to be essential for translocation between c-myc and the IgH locus (Igh), which is a characteristic feature of Burkitt's lymphoma (57). Notably, UNG2 interacts with both PCNA and replication protein A and colocalizes with both proteins in cellular replication foci (29, 50).

Genome replication of DNA viruses is closely linked to the cellular DNA repair machinery. In herpes simplex virus type 1 (HSV-1), several DNA repair proteins are recruited to the viral replication compartments, presumably for participation in virus DNA replication or repair (70, 80). It is notable that PCNA, replication factor C, and a series of mismatch repair proteins are assembled precisely at viral replication compartments after induction of Epstein-Barr virus (EBV) lytic replication (11). A more recent study demonstrated up-regulation of BER activities such as UNG2 and APE1 that may be involved in viral replication in human cytomegalovirus (HCMV)-infected cells (59).

Interestingly, some herpesviruses as well as poxviruses also encode UDGs belonging to the UNG family. The vaccinia virus D4R gene, which encodes the viral UNG, is essential for replication in tissue culture, although the catalytic activity is dispensable (14), suggesting that vaccinia virus UNG may participate in the formation of multiprotein DNA replication complexes. Indeed, the interaction of vaccinia virus UNG with A20 (a stoichiometric component of the viral processivity factor), along with E9 (viral DNA polymerase), is necessary and sufficient for the processive polymerase holoenzyme (67). The UNG encoded by HCMV UL114 also was shown to associate with ppUL44 (viral DNA polymerase processivity factor), and UL114 functions as part of the viral DNA replication complex to increase the efficiency of both early- and late-phase viral DNA synthesis (53). Moreover, deletion of HCMV UNG delays and diminishes replication of the virus in serum-deprived primary human fibroblasts (10). HSV-1 UNG (UL2 product) was first reported to be dispensable for viral replication in tissue culture (46), but later evidence suggested that the protein is required for virus reactivation from latency and for efficient replication in nerve tissue, which contains very low levels of cellular UNG (19, 55, 74).

EBV, a gamma-1-herpesvirus, can establish lifelong persistent infections in its natural host and transform B cells in vitro. EBV infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis, Burkitt's lymphoma, and Hodgkin's disease, and also tumors of epithelial origin, such as nasopharyngeal and gastric carcinomas (60). According to the genomic organization and sequence homology with the HSV-1 UL2 gene, EBV BKRF3 was recognized as a putative uracil DNA glycosylase. In an earlier study, BKRF3 was shown to enhance oriLyt-dependent DNA replication about twofold in a cotransfection-replication assay (18). Despite that, the function of EBV BKRF3 has not been investigated. In analogy to the crucial roles of other viral and human UDGs in DNA repair, DNA replication, and pathogenesis, we wondered whether BKRF3 might play an important role in the lytic replication of EBV or its pathogenic mechanisms.

Here we demonstrate that EBV BKRF3 encodes a structurally and functionally conserved viral uracil DNA glycosylase of the UNG family, and we provide evidence that BKRF3 as well as the cellular UNG2 is involved in DNA replication and/or repair to ensure the replication fidelity of viral DNA and promote higher levels of viral DNA.


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MATERIALS AND METHODS
 
Constructs and strains. To construct BKRF3-expressing plasmids, PCR products were amplified with the 5' primer 5'-CGGGATCCATGGCATCGCGGGGGC (nucleotides [nt] 98065 to 98080) and the 3' primer 5'-CGGGATCCCTACAGCCTCCAATCTATC (nt 98832 to 98814) and Akata cell lysates as the template, subjected to BamHI digestion, and cloned into the BamHI site of pET15a (Novagen, Inc.) or pCMV-Tag2B (Stratagene) to generate pET15a-BKRF3 or pFlag-BKRF3. The sequences in boldface are derived from GenBank accession number AJ507799. pET15a-BKRF3 has a T7 promoter for in vitro translation using a TNT reticulocyte lysate (Promega) and six consecutive histidine residues at the N terminus for purification. To generate pET15a-BKRF3mt (with amino acids [aa] 84 to 93 [water-activating loop] deleted), an improved method for generating an internal deletion (44) was performed using the single primer 5'-TGCGACCCCTCTGATATTCACGGGGGTCAAGCAAA (nt 98298 to 98313 and 98344 to 98360) and pET15a-BKRF3 as the DNA template. To generate pTrc99A-BKRF3 and pTrc99A-BKRF3mt for expressing BKRF3 and BKRF3mt in an Escherichia coli mutator assay, the BamHI fragment from pET15a-BKRF3 or pET15a-BKRF3mt was cloned into the E. coli expression vector pTrc99A (Pharmacia), which has a strong trc promoter that can be induced by addition of IPTG (isopropyl-ß-D-thiogalactopyranoside). The plasmid pTrc99A-UNG{Delta}84 was described by Slupphaug et al. (64) and encodes the common catalytic C-terminal domain of UNG1 and UNG2 (49).

The Rta-expressing plasmid RTS15 (pSG5-Rta, a gift from Diane Hayward) was described by Ragoczy et al. (56). The siZta and siGFP plasmids are pSUPER (5)-based constructs, as described by Chang et al. (7). Small interfering RNA (siRNA)-expressing plasmids were constructed by cloning siRNA sequences into pSUPER via the BamHI and HindIII sites. An economical and efficient method of siRNA vector construction (6) was adapted to generate siRNAs targeted against BKRF3 and cellular UNG2, using the two top-ranked sequences predicted by an online program (siRNA Wizard; InvivoGen). The siRNA sequences were further subjected to a BLAST search against human genome and expressed sequence tag databases to ensure that no other human genes were targeted. siBKRF3-1 and siBKRF3-2 are directed against the BKRF3 sequences 5'-GGAAGAGGAAACAGGAGAT-3' (nucleotides 44 to 62 of the open reading frame [ORF]) and 5'-GGCTAGATTTCCTACAACT-3' (nucleotides 101 to 119 of the ORF), respectively. siUNG2-1 and siUNG2-2 are directed against the UNG2 sequences 5'-GGGACAGGATCCATATCAT-3' (nucleotides 452 to 470 of the ORF) and 5'-GGCAAGAAGCCCATTGACT-3' (nucleotides 909 to 927 of the ORF), respectively.

Plasmid pUgi-IRES-hrGFP was constructed by ligating the UNG inhibitor-encoding gene from the EcoRI-XhoI fragment of pZWtac1 (79) into an EcoRI-XhoI site of pIRES-hrGFP-1a (Stratagene).

E. coli strain NR8051 [{Delta}(pro-lac) thi ara] and its ung-1 derivative NR8052 [{Delta}(pro-lac) thi ara trpE9777 ung-1] (33) were used in the mutator assay.

Mutator assay. The mutator assay protocol was modified from that described by Olsen et al. (48). Briefly, E. coli strains NR8051 (wild type) and NR8052 (ung-1) were transformed with plasmid pTrc99A, pTrc99A-UNG{Delta}84, pTrc99A-BKRF3, or pTRC99A-BKRF3mt. Ten-milliliter aliquots of medium were inoculated with 0.5 ml of the overnight cultures of transformants and grown at 37°C until the optical density at 600 nm reached 0.4 to 0.5. IPTG was added to a final concentration of 1 mM to induce UNG{Delta}84, BKRF3, or BKRFmt expression. Rifr and Nalr colonies were selected on LB plates containing 100 µg/ml rifampin (an inhibitor of bacterial RNA polymerase ß subunit) (Sigma Co.) or 40 µg/ml nalidixic acid (an inhibitor of bacterial DNA gyrase) (Sigma Co.), respectively. Mutation to Rifr and Nalr occurs normally at low frequency by base substitution in the rpoB or gyrA gene. Mutation frequencies were measured by determining the number of colony-forming cells that survived antibiotic selection per 4 x 108 viable cells plated.

DNA glycosylase assay. Single-stranded LMRC-U (5'-AGC TAC CAT GCC TGC ACG AAU TAA GCA ATT CGT AAT CAT GGT CAT), which was 5' end labeled with [{gamma}-32P]ATP followed by purification and quantification as previously described (43), or duplex oligonucleotides, which were prepared by mixing equimolar amounts of the appropriate single-stranded oligonucleotides followed by heating for 2 minutes at 95°C and slowly cooling to room temperature, were incubated with either purified His-BKRF3, E. coli UNG enzyme (NEB), in vitro transcription/translation products, or cell lysates at 37°C for 10 min. The standard assay mixture for DNA glycosylase activity contained 0.2 pmol of the labeled single-stranded or duplex oligonucleotides in 20 µl buffer containing 1 mM EDTA, 1 mM dithiothreitol (DTT), and 20 mM Tris-HCl (pH 8.0). UDG activity was stopped at 95°C for 5 min. After glycosylase cleavage, abasic sites were incised by 0.1 mM NaOH treatment at 95°C for 5 min. Reaction products were analyzed by electrophoresis through denaturing 15% (wt/vol) polyacrylamide gels (7 M urea, 1x Tris-borate-EDTA), visualized using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and quantified using ImageQuant software. For uracil DNA glycosylase inhibitor (Ugi) assay, experiments were performed with 1 U of Ugi (NEB) in each reaction mixture.

Purification of bacterially expressed recombinant BKRF3. To express His-BKRF3 protein, pET15a-BKRF3 was transformed into BL21(DE3), and the bacteria were induced in the exponential phase with 1 mM IPTG at 37°C for 2 h. The cell extracts were homogenized in lysis buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM imidazole) with 1 tablet/10 ml of Complete mini-protease inhibitor mixture (EDTA free; Roche). The protein sample was then incubated with nickel nitrilotriacetic acid-agarose (QIAGEN) at 4°C with rotation for 1 h. The protein was eluted with a buffer (20 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10% [vol/vol] glycerol) containing increasing concentrations of imidazole. The fractions containing His-BKRF3 were pooled together and dialyzed against 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT, and 10% (vol/vol) glycerol for uracil DNA glycosylase activity assay.

Antibodies. To generate BKRF3-specific polyclonal antibodies, 6-week-old BALB/c mice were immunized with 50 µg His-BKRF3 protein in complete Freund's adjuvant subcutaneously, followed by three boosters with incomplete Freund's adjuvant at 2-week intervals. Other primary antibodies used for immunoblotting detection and indirect immunofluorescence included BRLF1 monoclonal antibody (MAb) 467, BZLF1 MAb 4F10 (71), and BMRF1 MAb 88A9 (72); rabbit antisera against BGLF4 (77) and BALF5 (39); and cellular UDG2 (PU059) rabbit polyclonal antibody (64).

Cell culture and induction of viral lytic cycle. Akata and Raji are EBV-positive Burkitt's lymphoma cell lines (54, 68). NPC-TW01 is an NPC cell line lacking the EBV genome (35), and NA was derived from recombinant Akata EBV-converted NPC-TW01 (9). For lytic cycle induction, NA cells were treated with 40 ng 12-O-tetradecanoylphorbol-13-acetate ml–1 and 3 mM sodium butyrate (Sigma) or transfected with Rta-expressing plasmid RTS15. Akata cells were induced with 0.5% (vol/vol) goat anti-human IgG antibody (Cappel Inc.).

Indirect immunofluorescence. To detect intracellular expression of BKRF3, HeLa, TW01, or NA cells were slide cultured, harvested at the indicated time points, air dried, and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Immunostaining was conducted with anti-BKRF3 polyclonal antibody (1:50). Rhodamine red-conjugated anti-mouse IgG antibodies (1:200; Jackson) were used as secondary antibodies. After being washed with PBS, cells were stained with Hoechst 33258 at room temperature for 1 min and covered with mounting medium (H1000; Vector) for fluorescence (Axioskop 40 FL; Zeiss) microscopy and confocal laser scanning (Leica).

Subcellular fractionation. The subcellular fractionation protocol was adapted from that described by Krajewski et al. (30). Briefly, cells were covered or incubated with hypotonic buffer (5 mM Tris-HCl [pH 7.4], 5 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride) on ice for 1 h, harvested by scraping, and homogenized by passage through 27-gauge needles 15 times. Cell lysates were subjected to centrifugation at 500 x g for 5 min at 4°C. The resulting pellet was the nuclear fraction. The supernatant was then mixed with 3 volumes of LSB buffer (50 mM Tris-HCl [pH 7.5], 25 mM KCl, 5 mM MgCl2) and further centrifuged at 150,000 x g at 4°C for 2 h. The resulting pellet contained heavy membrane (rough endoplasmic reticulum, mitochondria, peroxisomes, Golgi apparatus, and lysosomes) and light membrane (plasma membrane) fractions. The cytosolic fraction in the supernatant was further concentrated for gel analysis.

Transfection. Cells were transfected with different plasmids by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To suppress Zta or BKRF3 expression, NA cells were pretransfected with 4 µg siRNA-producing plasmid for 24 h and then cotransfected with 2 µg siRNA-producing plasmid and 2 µg Rta-expressing plasmid. For suppression of cellular UNG2 expression, NA cells were pretransfected with 5 µg siUNG2 plasmid for 24 h, reseeded, and transfected again with 5 µg siUNG2 plasmid for 24 h before cotransfection with 3 µg siUNG2 plasmid and 2 µg Rta-expressing plasmid. To identify Rta-responsive gene expression, 50 µg of Rta-expressing plasmid RTS15 or vector control pSG5 (Stratagene Co.) was transfected into 107 Raji cells in 0.2 ml RPMI 1640 medium by eletcroporation at 200 V and 950 µF with an ECM630 system (BTX Co.).

Detection and quantification of EBV DNA. Cells were lysed, digested with proteinase K, and then subjected to the following PCR analysis as described previously (8). For quantification of EBV DNA, real-time PCR was performed according to the manufacturer's instructions (Applied Biosystems). The detection target of real-time PCR was the EBNA1 region of the EBV genome, and details on the primers and probes were provided in a previous study (40). We used H2B4 cells harboring one EBV genome per cell to generate a standard curve for quantification (8), and the EBV copy number was calculated by comparison with the standards. All samples were tested in duplicate.


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RESULTS
 
EBV BKRF3 encodes a putative conserved uracil-DNA glycosylase. According to the newly revised EBV sequence (12) (GenBank accession number AJ507799), the predicted coding sequence of BKRF3 contains 765 nt and encodes a protein of 255 amino acids. The predicted amino acid sequence of BKRF3 was aligned with different eukaryotic and prokaryotic Ung sequences (Fig. 1A) and showed identities of 48%, 42%, 47%, and 52% with UNG enzymes of HCMV, HSV-1, human, and E. coli, respectively. The short N terminus of BKRF3 is distinct from those of most of the UNG proteins described so far. The BKRF3 sequence is highly conserved through the various UNG domains that are involved in catalysis and binding, such as the conserved motifs GQDPYH (water-activating loop) and HPSPLS (DNA intercalation loop). To explore the possible function of BKRF3, UDG activity was determined using a 5'-32P-labeled synthetic uracil-containing 45-mer DNA as a substrate (Fig. 1B). Uracil excision by UDG generates an apyrimidinic site in the DNA oligomer, which is sensitive to alkaline conditions and will result in two fragments, one of which (32P labeled) is detected as a faster-migrating band (20-mer) upon gel electrophoresis. Because NaOH treatment alone did not generate fast-migrating oligonucleotides, this indicates that there were no detectable AP sites in the substrate prior to UNG treatment (Fig. 1D, lane 2). As for the purified E. coli UNG enzyme (NEB Co), the incubation of substrate with the in vitro transcription/translation product of BKRF3 resulted in the generation of the cleaved 20-mer. Furthermore, deletion of aa 84 to 93, encompassing the conserved catalytic motif of BKRF3, abolished the UDG enzyme activity, indicating that BKRF3 is a highly conserved enzyme (Fig. 1C and D).


Figure 1
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  		FIG. 1. Sequence alignment of uracil-DNA glycosylases encoded by EBV, HSV-1, HCMV, human, and E. coli. (A) The amino acids are numbered according to the EBV BKRF3 sequence, and identical residues are depicted in red, while similar residues are shown in blue. The conserved motifs are indicated. (B) Flow chart for the DNA glycosylase activity assay. 5'-32P-labeled 45-mer oligonucleotides containing a uracil residue at position 21 were incubated with UNG at 37°C for 10 min. Reaction products were treated with 0.1 N NaOH to reveal abasic sites generated by DNA glycosylase. Final products were displayed by 15% PAGE with 7 M urea. (C) Wild-type BKRF3 or mutant protein was synthesized in the TNT lysate-coupled transcription/translation system with [35S]methionine labeling and resolved by 12% SDS-PAGE. (D) 5'-32P-labeled LMRC-U was incubated with E. coli UNG (used as a positive control) (NEB Co.) or 5 µl of in vitro transcription/translation products of BKRF3 and BKRF3mt. Control, LMRC-U probe only. No UNG, LMRC-U probe treated with 0.1 N NaOH.

EBV BKRF3 complements an E. coli ung mutant in vivo. Uracil-DNA glycosylases are known to be responsible for removing uracil from DNA in the BER pathway to ensure DNA integrity. In addition to the UDG activity of BKRF3 observed in vitro, we sought to determine whether BKRF3 may contribute to the maintenance of genome integrity in vivo. To that end, a plasmid (pTrc99A-BKRF3) containing the EBV BKRF3 open reading frame expressed under the control of a trp/lac (tac) hybrid promoter was transformed into the ung mutant E. coli strain NR8052 (ung-1), and the effect of BKRF3 on mutation frequency to rifampin resistance was measured (Fig. 2). Mutation to Rifr occurs normally at a low frequency by base substitutions in the rpoB gene. As expected, in the presence of the transcriptional inducer IPTG, the wild-type E. coli strain NR8051 generated Rifr colonies at a low frequency (46.9 Rifr colonies per 4 x 108 cells) (Fig. 2A and C and Table 1). In contrast, the ung mutant E. coli strain NR8052 and the vector-transformed controls (pTrc99A) resulted in a 9.8-fold increase of Rifr colonies (Fig. 2A and Table 1). Similar to the effect of positive control human UNG{Delta}84 (hUNG), induction of BKRF3 reduced the mutation frequency 10-fold. The mutator assay was also performed with wild-type strain NR8051 harboring hUNG, BKRF3, and BKRF3mt, giving virtually the same number of resistant colonies as wild-type cells, indicating that exogenous expression of UDG cannot reduce the mutation frequencies further (Fig. 2A and C and Table 1). The efficiency of plating showed that E. coli growth was not influenced by the expression of BKRF3, BKRF3mt, or hUNG (Fig. 2B). These data demonstrate that BKRF3 is able to suppress the mutator phenotype of the E. coli ung mutant in vivo. This complementation was not restricted to the selection applied, being also evident when mutation to resistance to an inhibitor of bacterial DNA gyrase, nalidixic acid, was monitored (Fig. 2D and Table 1). Mutation to Nalr results from base substitutions in the gyrA gene. The variation in the reduction of mutation frequency observed in different selections may reflect differences in the types and abundances of mutations that allow E. coli growth. Because BKRF3 ({Delta}aa 84 to 93) lost the ability to complement the ung mutant, we conclude that BKRF3 complementation is dependent on UDG activity. These results not only demonstrate that BKRF3 may participate in a BER pathway in vivo but also confirm that UNG proteins are highly conserved through prokaryotes, eukaryotes, and viruses.


Figure 2
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  		FIG. 2. EBV BKRF3 can complement an E. coli ung mutant in mutator assays. (A) Mutation frequencies were examined by plating equal amounts of wild-type E. coli NR8051 or ung mutant E. coli NR8052 harboring pTrc99A-hUNG, pTrc99A-BKRF3, pTrc99A-BKRF3mt, or vector control pTrc99A. Representative rifampin plates demonstrate the mutation frequencies relative to the wild-type control as indicated. (B) Serial dilutions of the indicated bacteria were spotted on LB agar plates containing or not containing ampicillin to demonstrate the equal efficiency of plating. (C and D) Frequency of Rifr (C) or Nalr (D) mutations per 4 x 108 cells. Each point represents the mutation frequency of an independent culture. (E) In vitro UDG activity assay of individual cell lysates with single-stranded uracil-containing substrate. Cell lysates containing 1 µg of total protein were used in this experiment. (F) One unit of UNG inhibitor protein, Ugi, was added to the DNA glycosylase assay.


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  		TABLE 1. Mutation frequency of BKRF3-transformed UNG-deficient Escherichia coli

To confirm that BKRF3-encoded UDG activity in the E. coli ung mutant correlated with the mutator assay, cell extracts of the wild-type E. coli NR8051 and ung mutant E. coli NR8052 carrying a plasmid expressing BKRF3, BKRF3mt, hUNG, or the vector control were assayed for UNG enzyme activities (Fig. 2E). Indeed, the extracts from wild-type E. coli and from hUNG and BKRF3 transformants displayed measurable uracil excision activity, in contrast to extracts from ung mutant E. coli NR8052 with vector or BKRF3mt (Fig. 2E). We then examined the sensitivity of BKRF3 to the natural UNG inhibitor Ugi, which is expressed by phages PBS-1 and -2 and allows these uracil-containing DNA phages to survive and replicate in their genotypically ung+ hosts. Ugi can function in DNA mimicry that specifically and irreversibly inhibits E. coli and human UNG (45, 62). As shown in Fig. 2F, addition of Ugi resulted in the inhibition of the UDG activities of E. coli UNG, hUNG, and BKRF3 in vitro. These results demonstrate that BKRF3 encodes a conserved uracil-DNA glycosylase that can function in vitro and in vivo.

Expression and purification of BKRF3 UNG. The UNGs characterized so far are relatively small monomeric proteins that do not require cofactors for enzyme activity and have a preference for uracil in ssDNA over dsDNA in vitro (64). To examine further the biochemical properties of BKRF3 UDG activity, the coding region of the BKRF3 gene was cloned into the expression vector pET-15a, which encodes six consecutive histidine residues at the N-terminal end to facilitate purification. After transformation into E. coli BL21(DE3), a 28-kDa protein was induced by IPTG as displayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. 3A). The soluble fraction of the His-BKRF3 protein was purified using an affinity Ni2+ column, and the target protein was recovered by elution with buffers containing step gradients of 0 to 250 mM imidazole. In this way, a homogeneous purified protein was obtained, as judged by SDS-PAGE and Coomassie blue staining (Fig. 3A).


Figure 3
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  		FIG. 3. UDG activities of BKRF3 on single- or double-stranded substrates containing uracil. (A) Recombinant His-BKRF3 recovered by elution with buffer containing step gradients of 0 to 250 mM imidazole was displayed on SDS-PAGE. (B) One unit of Ugi was added to a DNA glycosylase assay to inhibit the enzyme activities of purified E. coli UNG or His-BKRF3 protein. (C and D) Titration of UDG activities of His-BKRF3 (C) or E. coli UNG (D) with single- and double-stranded oligonucleotides containing uracil. (E and F) Effect of MgCl2 (E) and MnCl2 (F) on the activity of the His-BKRF3 proteins. A single-stranded oligonucleotide containing uracil was incubated with 0.5 nM His-BKRF3 in the presence or absence of MgCl2 and MnCl2. The radioactivities of the products were scanned with a phosphorimager and quantified with the ImageQuant program and are indicated as excision percentage.

BKRF3 prefers uracil-containing ssDNA substrates. The substrate specificity of the recombinant His-BKRF3 protein was then characterized. The kinetics of uracil excision was measured in single-stranded and double-stranded oligonucleotides containing a mismatch of U · G, U · C, U · A, or U · T (Fig. 3C and Table 2). 5'-32P-labeled oligonucleotides were incubated with increasing concentrations of either His-BKRF3 or E. coli UNG. In these experiments, 0.1 nM His-BKRF3 excised about 50% of the uracils from ssDNA, whereas 1.0 nM His-BKRF3 was needed to achieve a similar degree of excision from dsDNA. Furthermore, the specificity constant (kcat/Km) with single-stranded substrate was about 10 times higher than that with the U · G, U · C, U · A, or U · T duplex substrate (Table 2). This result indicated that the His-BKRF3 preferentially excises uracil from ssDNA rather than dsDNA.


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  		TABLE 2. Kinetic constants of BKRF3 and E. coli UNG protein for excision of uracila

Several studies showed that the UDG activity of human UNG2 was stimulated 10- to 27-fold in the presence of 10 mM Mg2+ or Mn2+ (28, 29). To investigate possible effect of Mg2+ and Mn2+, UDG activity of BKRF3 was measured in the absence or presence of 0.5,1 2, 4, and 8 mM MgCl2 or MnCl2. Possibly because of minor contaminating nuclease activity, total amounts of labeled oligonucleotides were slightly reduced in the presence of 4 or 8 mM MgCl2 or MnCl2. However the uracil excision efficiency indicated that the UDG activity of BKRF3 was inhibited by about 80% in the presence of 8 mM MgCl2 (Fig. 3E) or MnCl2 (Fig. 3F).

According to sequence alignment (Fig. 1A), the BKRF3 protein has 52% sequence identity to E. coli UNG. Therefore, the kinetic constants of E. coli UNG acting on the same oligonucleotides were measured simultaneously for comparison (Fig. 3D and Table 2). The kinetic analyses demonstrated that the overall efficiency (kcat/Km) of BKRF3 was lower than that of E. coli UNG and that this was caused primarily by a much higher turnover (kcat) of the latter enzyme. Notably, BKRF3 had a markedly higher preference for ssDNA over dsDNA substrates (~10-fold) than E. coli UNG (<2-fold), and this was caused mainly by a low Km of BKRF3 against U in ssDNA. The single-strand preference of BKRF3 and the inhibition of its enzyme activity by Ugi are characteristics shared by the E. coli and human UNG proteins (28) and indicate that the active sites of the three enzymes are structurally conserved.

Expression of BKRF3 in EBV-positive cells upon induction of the lytic cycle. To determine whether BKRF3 is expressed in EBV-positive cell lines after induction of the lytic cycle, we used purified His-BKRF3 proteins to generate a BKRF3-specific mouse serum. The serum showing the strongest signal in detecting Flag-BKRF3 in transiently transfected 293T cells (Fig. 4A) was used to detect BKRF3 in chemically induced EBV-positive epithelial NA cells. BKRF3 became detectable at 24 h postinduction (hpi) and reached maximal expression levels at 36 hpi with an apparent molecular mass of 28 kDa, which is in agreement with the molecular mass predicted from the nucleic acid sequence. Simultaneously, two immediate-early transactivators, BZLF1 (Zta) and BRLF1 (Rta), and early antigen BMRF1 (EA-D) showed similar expression kinetics (Fig. 4B). These data were also in accordance with our and others' previous observation in microarray analysis that BKRF3 is expressed in the second cluster of EBV early genes (42, 82, 83). We then detected the expression of BKRF3 in anti-IgG-induced Akata cells and Rta-transfected Raji cells (Fig. 4C). Because Raji cells harbor an EBV genome with a deletion in the coding region of the major DNA-binding protein and are unable to complete viral DNA replication, the expression of BKRF3 protein in Raji cells indicates that its expression is independent of viral DNA replication. In addition, because it is known that exogenous expression of Rta in Raji cells does not induce expression of the other immediate-early transactivator, Zta, the detection of BKRF3 protein in Rta-induced Raji cells suggests that Rta alone is able to induce the expression of BKRF3.


Figure 4
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  		FIG. 4. Expression of EBV BKRF3 in EBV-positive cells upon induction of the lytic cycle. (A) 293T cells were transfected with a plasmid expressing Flag-BKRF3. Immunoblotting analysis of Flag-BKRF3 using anti-BKRF3 polyclonal antibody is shown. (B) EBV-positive NA cells were induced with 12-O-tetradecanoylphorbol-13-acetate-sodium butyrate and harvested at different time points as indicated. Immunoblotting analysis of BRLF1, BMRF1, BZLF1, BKRF3, and ß-actin was performed with specific antibodies. (C) Detection of BKRF3 expression in anti-human IgG-induced Akata cells at 24 hpi and in Rta-induced Raji cells at 48 hpi.

BKRF3 is expressed in both the nuclei and cytoplasm of EBV-positive and EBV-negative cells. The human UNG gene has been shown to encode both nuclear (UNG2) and mitochondrial (UNG1) isoforms of the enzyme. In addition to the conserved catalytic domains, the N-terminal extension found in human UNG is involved in its subcellular localization (47). We next determined the subcellular localization of BKRF3 by using indirect immunofluorescence. Following transient transfection, BKRF3 was detected in both the nuclei and cytoplasm of HeLa cells (Fig. 5A). BKRF3 also localized in both nuclei and cytoplasm at 36 hpi in chemically induced NA cells. Moreover, BKRF3 associated with viral factors in replication compartments such as BGLF4 (protein kinase) and BALF5 (DNA polymerase), as observed with fluorescence microscopy and confocal laser scanning (Fig. 5B). Subcellular fractionation of NA cells at 24 hpi further confirmed the localization of BKRF3 in the nucleus and cytoplasm but not in the heavy membrane fraction (Fig. 5C). A similar staining pattern was observed at 48 hpi, indicating that the subcellular localization of BKRF3 does not change with the progression of virus replication (data not shown).


Figure 5
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  		FIG. 5. Expression of BKRF3 in both the nuclei and cytoplasm of transiently transfected and EBV-replicating cells. (A) HeLa cells were transfected with plasmids expressing Flag-BKRF3. The expression of BKRF3 was detected with preimmune serum, specific mouse serum, or Flag antibody (Ab). (B) Expression of BKRF3 in NA cells was detected with specific mouse serum at 36 hpi, and the nuclei were stained with Hoechst 33258. Expression of BGLF4 and BALF5 in NA cells was detected with specific rabbit antiserum and observed with fluorescence microscopy (IFA) and confocal laser scanning as indicated. (C) Subcellular localization of BKRF3 in NA cells at 24 hpi fractionated into nuclear (N), heavy membrane (H), and cytosolic plus light membrane (C) fractions. After electrophoresis and blotting, BKRF3 was detected with specific mouse serum. PARP and {alpha}-tubulin were detected as nuclear and cytoplasmic markers.

BKRF3 contributes to EBV lytic replication. BKRF3 was reported previously to enhance oriLyt-dependent DNA replication about twofold in a cotransfection-replication assay (18). Moreover, HSV-1 UNG-negative mutants replicated and spread poorly in mice (55). More recent studies suggested that HCMV UNG excises uracil residues from replicating HCMV DNA to create a single nick for initiation of rolling-circle replication at a late stage in the infection process (10). To explore the possible biological function of BKRF3, experiments were carried out to determine whether BKRF3 contributes to EBV lytic DNA replication with a pSUPER-based system (5) to drive siRNA synthesis in Rta-transduced NA cells. Exogenous expression of Rta induced the expression of BZLF1, BMRF1, and BKRF3 proteins in NA cells, as detected by immunoblotting. The amplification of EBV DNA was detected simultaneously using a quantitative real-time PCR method (Fig. 6). Expression of BKRF3 was inhibited by double transfection of BKRF3-targeted siRNA (siBKRF3-1 or -2), and Zta expression was inhibited by siZta but was not affected by pSUPER or siGFP controls (Fig. 6A). As a relevant control, Zta-dependent expression of BMRF1 was blocked in the presence of siZta. Simultaneously, Rta-induced BKRF3 expression decreased moderately in the presence of siZta (Fig. 6A), indicating that the lytic induction of BKRF3 could be activated by Rta alone and probably could be enhanced synergistically by Zta and Rta. Regarding the Rta-induced amplification of viral genomes, expression of siZta completely blocked the viral DNA amplification in the lytic cycle (Fig. 6E). Meanwhile, siBKRF3-2 showed about a 20% inhibitory effect on the amplification of viral genomes at 48 hpi. Compared with RV-pSUPER controls, the UDG activity in a cell extract was increased by about 10% in Rta-induced NA cells (Fig. 6B and C). In addition, the total UDG activities of induced-NA cells were inhibited about 10% when the expression of BKRF3 was knocked down by siRNA (Fig. 6D). This implies that cellular UNG might partially complement the function of viral UNG when the latter is not expressed.


Figure 6
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  		FIG. 6. EBV DNA replication in NA cells expressing siBKRF3. NA cells were transfected with plasmids expressing siBKRF3-1, siBKRF3-2, siGFP, or siZta for 24 h and then transfected with Rta-expressing plasmids or control vectors (RV) for 24 h and 48 h. (A) Immunoblotting analysis of BRLF1, BMRF1, BZLF1, BKRF3, and ß-actin. (B) UDG activity assays using 1 µg total protein of cell lysates with single-stranded uracil-containing oligonucleotide substrates. (C and D) Serially diluted total extracts were used for UDG activity assay with single-stranded uracil-containing oligonucleotide substrates. The radioactivities of the products was scanned with a phosphorimager and quantified with the ImageQuant program and are indicated as excision percentage. (E) Quantification of EBV DNA at various time points by real-time PCR to detect EBNA-1 sequences. Error bars indicate standard deviations.

Cellular UDG2 contributes to EBV lytic replication. To investigate whether cellular UDG activity compensates for BKRF3 during virus replication, NA cells were transfected with UNG2-targeted siRNA (siUNG2-1 and -2). Expression of UNG2 was specifically inhibited by triple transfection of siUNG2-1 and was slightly inhibited by siUNG2-2 but not by the siGFP control in Rta-induced NA cells (Fig. 7A). The total UDG activities of induced NA cells were inhibited to about 50% by siUNG2-1 (Fig. 7B and C). In agreement with this, Rta-induced amplification of the viral genome was also repressed by 50% to 60% at 48 hpi (Fig. 7D).


Figure 7
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  		FIG. 7. EBV DNA replication in NA cells expressing siUDG2. NA cells were transfected with plasmids expressing siUNG2-1, siUNG2-2, or siGFP twice and then transfected with Rta-expressing plasmids or control vectors (RV) for 24 h and 48 h. (A) Immunoblotting analysis of BRLF1, BZLF1, BKRF3, UNG2, and ß-actin. (B) UDG activity assays using 1 µg total protein of cell lysates with single-stranded uracil-containing oligonucleotide substrates. (C) Serially diluted total extracts were used for UDG activity assay with single-stranded uracil-containing oligonucleotide substrates. The radioactivities of the products was scanned with a phosphorimager and quantified with the ImageQuant program and are indicated as excision percentage. (D) Quantification of EBV DNA at various time points by real-time PCR to detect EBNA-1 sequences. Error bars indicate standard deviations.

Requirement of UNG activity in EBV lytic replication. To further investigate the contribution of UNG activity to viral lytic replication, NA cells were cotransfected with Rta- and Ugi-expressing plasmids, which can inhibit both viral and cellular UNG activities (Fig. 2F). Indeed, the UDG activity could barely be detected in reactivated NA cells transfected with the Ugi-expressing plasmid (Fig. 8B). The weak residual activity could be caused by incomplete inhibition of UNG proteins or by the presence of other cellular UDG activities, such as SMUG1, that are not inhibited by Ugi. Interestingly, the copy numbers of EBV DNA in Rta-induced NA cells were reduced to about half in the absence of detectable UDG activity at various time points (Fig. 8C). However, the expression of early lytic genes was not affected by the presence of Ugi (Fig. 8A). This observation suggests that the enzymatic activity of UNG proteins is required for higher efficiency of viral DNA replication.


Figure 8
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  		FIG. 8. EBV DNA replication in NA cells expressing Ugi. NA cells were cotransfected with plasmids expressing UNG inhibitor (Ugi) and Rta. (A) Immunoblotting analysis of BRLF1, BMRF1, BZLF1, BKRF3, and ß-actin at 24 or 48 post transfection. (B) UDG activity assays using 1 µg cell lysates with single-stranded uracil-containing oligonucleotide substrates. (C) Quantification of EBV DNA at different time points by real-time PCR to detect the EBNA-1 sequence. Error bars indicate standard deviations.


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DISCUSSION
 
Several lines of evidence from the present study indicate that EBV BKRF3 encodes a structurally and functionally conserved uracil-DNA glycosylase of the UNG family. First, amino acid sequence alignment demonstrates a high degree of homology with other UNG proteins within the C-terminal catalytic domain and absolutely conserved catalytically important motifs. Notably, deletion of the water-activating group renders BKRF3 catalytically dead (Fig. 1). Second, expression of BKRF3 in E. coli can restore the normal phenotype of ung mutants to the same extent as human UNG (Fig. 2). Third, BKRF3 excises uracil in both dsDNA and ssDNA but has a preference for the latter (Fig. 3). Fourth, BKRF3 is inhibited by Ugi (Fig. 3), which is highly specific towards the UNG family of proteins and does not inhibit other UDGs (4, 45, 78).

UNG proteins have a well-documented function in base excision repair of uracil and may remove uracil close to the replication fork by association with replicative proteins or within nonreplicating DNA (28). In addition, some virally encoded UNGs may have a more direct function in viral DNA replication. In HSV-1, the deletion of UL2, which encodes UNG, affects the ability of the virus to replicate in mice, particularly in the central nervous system (55). Moreover, lack of viral UNG expression in HCMV infection has been demonstrated, leading to a delay in viral DNA synthesis and replication (10, 52). However, the viral UNG activity is dispensable for viral replication in cultured cells, presumably because adequate cellular activity is present. This also suggests that cellular UNG may complement viral UNG mutants in cultured cell lines (10, 46, 52). The cellular UNG2 activity is cell cycle associated, with maximal activity detected during late G1 and early S phases (16, 66), and is undetectable in adult neurons (19, 75). Thus, a role is implied for viral UNG in the replication of the virus in the host, particularly in nondividing cells (e.g., terminally differentiated cells), where levels of cellular UNG2 are believed to be low. Such a model is also supported by the present study. By using the Ugi-expressing plasmid to inhibit both viral and cellular UNG2 activities or by using siUNG2 to knock down UNG2, EBV DNA replication was suppressed to half of that with the vector control, whereas EBV lytic DNA replication was affected only slightly when BKRF3 expression was inhibited by siBKRF3. This indicates that the UNG activity is important for EBV lytic DNA replication and that cellular UNG2 may back up viral UNG in tissue culture cells. We hypothesize that the role of BKRF3 UDG activity in EBV replication is particularly important in terminally differentiated and nondividing cells. This likelihood is supported by several reports indicating that EBV lytic replication occurs in terminal differentiated cells. It is now generally believed that EBV persists in resting memory B cells in which only a small fraction of the infected cells are dividing in the peripheral blood (2, 24, 27). Moreover, using limiting-dilution reverse transcription-PCR to detect BZLF1 and the early gene BHRF1 demonstrated that about 10 to 20% of the EBV-infected plasma cells are undergoing lytic replication, compared to fewer than 0.1% of EBV-infected germinal center B cells and memory B cells (24). The authors thus suggested that the differentiated plasma cell is naturally responsible for replicating the virus and that the terminal differentiation of B cells into plasma cells may provide the signal that initiates EBV replication (24). The promoter of the immediate-early gene BZLF1 is activated only after differentiation of memory cells into plasma cells (34). In addition, acute infectious mononucleosis caused by EBV is accompanied by central nervous system disorders in 1 to 18% of patients (17, 21). It was also reported that cerebrospinal fluid samples from patients with infectious mononucleosis contained EBV DNA and EBV-specific anti-VCA and anti-EA antibodies in the neurological stage but not during convalescence (26). This suggested that EBV-associated neurological complications may be due to direct virus invasion of the central nervous system, although the pathogenesis is not completely understood (13, 26, 76). Considering these observations together, we postulate that EBV may replicate in terminally differentiated cells or nondividing cells such as plasma B cells and neuronal cells. Thus, the BKRF3 UNG appears to play an important role for replication in fully differentiated nonproliferating cells that probably contain low levels of cellular UNG2.

In cell culture, BKRF3 was found previously to increase about twofold the replication efficiency of an origin of lytic replication (oriLyt)-containing plasmid in a transient-transfection system (18). By using siBKRF3, total cellular UDG activities in the extract was barely affected (Fig. 6D); meanwhile, the viral DNA replication efficiency in cultured NA cells was reduced about 20% at the late stage by siBKRF3-2 (Fig. 6E). Previous studies have shown that UNGs from human, HCMV, and vaccinia virus interact with PCNA or its viral homolog and colocalize to the DNA replication foci (50, 53, 67). Moreover, the interaction of UNG with the processivity factor of vaccinia virus increased the processivity of the viral DNA polymerase (50, 53, 67). It is thus possible that cellular UNG2 activity may be sufficient at the early stage of viral DNA replication, whereas the viral enzyme is important in a later stage of viral DNA replication through interaction with viral replication factors, thereby enhancing the efficiency of viral replication.

Likewise, three possible mechanisms may account for the role of UNG activity in EBV lytic DNA replication: (i) UNG activity might participate in the DNA repair system to ensure the fidelity of viral DNA replication and therefore promote higher production of viral DNA. (ii) UDG might be involved in the initiation of rolling-circle replication, which can produce a burst of virus genomes. It is believed that herpesviral DNA replication is initiated with the origin-specific circular latent genome, which leads to an early theta-form replication and later undergoes a switch to rolling-circle DNA replication. In the process of switching, a single-stranded break needs to be introduced into the double-stranded circular-form DNA to generate a free 3'-hydroxyl group, serving as a primer for subsequent rolling-circle replication. UDG has been suggested to play a role in producing nicks for the initiation of rolling-circle DNA replication (10, 41, 42). This suggestion is partly supported by the observation in yeast that uracil is a critical source of AP sites in DNA which can be further converted into single-stranded breaks by AP endonuclease (22). (iii) UDG might enhance the interaction of the origin-binding protein (OBP) and oriLyt by removing uracil residues within the oriLyt region. In both EBV and HSV, the binding of OBP to the origin of lytic replication is absolutely required for viral DNA replication. In HSV, it has been demonstrated that uracil replacing either cytosine (mimicking cytosine deamination) or thymine (mimicking dUTP misincorporation) on site I of OriS diminishes the interaction between OBP and OriS in a gel shift assay (20). Thus, experiments to determine the effects of the binding of Zta to the uracil-containing Zta responsive element are awaited. No matter which mechanisms are involved, the UDG activity is important in EBV lytic DNA replication, as expression of Ugi suppressed the efficiency of viral replication. Additionally, recent studies with Kaposi's sarcoma herpesvirus identified human UNG2 as a protein interacting with latency-associated nuclear antigen. Depletion of UNG2 in Kaposi's sarcoma herpesvirus-positive cells by using siRNA reduced the number of viral genome copies and produced infection-deficient virus (73). Because UNG2 was previously found to interact with several cellular replication proteins, such as PCNA, PRA, and polymerase {alpha} (29, 50, 63), it would be interesting to study whether UNG2 can recruit these cellular machineries to the viral replication compartment for enhancing viral replication efficiency.

In addition to its role in viral DNA replication, a possible contribution of BKRF3 UDG activity to EBV pathogenesis is of concern. Cellular UNG2 is known to play a role in somatic hypermutation and class-switching recombination (CSR), which can generate high-affinity antibodies of different isotypes. CSR requires the up-regulation of the B-cell-specific enzyme activation-induced cytosine deaminase (AID). CSR is regulated tightly and usually requires up-regulation of CD40 ligand on antigen-activated CD4+ T cells (3). UNG and AID are required for the reciprocal translocation between IgH and the c-myc oncogene in vivo, which is the hallmark of B lymphoma (57, 58). Furthermore, combined loss of p53 function and DNA double-stranded break responses such as ataxia-telangiectasia mutated (ATM) or the nonhomologous end-joining pathway would lead to the accelerated appearance of AID-induced c-myc/IgH translocation (57). This places the expression of c-Myc under the control of IgH, which is expressed constitutively in B cells, suggesting its etiologic role in B-cell transformation. More than 90% of EBV-associated endemic Burkitt's lymphomas have undergone a c-myc/IgH chromosome translocation (69). Notably, EBV-encoded latent membrane protein 1 (LMP1) induces CD40-independent CSR from Cµ to multiple downstream C{gamma}, C{alpha}, and C{varepsilon} genes in B cells. This induction is associated with up-regulation of AID (23). Moreover, EBV infection of human B cells resulting in AID expression has been investigated, and increasing numbers of mutations in p53 were also seen in culture with time (15). It has been observed that expression of EBV LMP1 protein represses p53-mediated DNA repair and transcriptional activity through the activation of the NF-{kappa}B pathway. This suggests that the abrogation of DNA repair by LMP1 may induce genomic instability and contribute subsequently to tumorigenesis (37, 38). Here we propose that EBV BKRF3 may function in addition to LMP1 to contribute to genome instability, thus favoring AID/UNG-induced c-myc/IgH translocations.

In summary, although expression of BKRF3 transcripts has been observed upon induction of EBV-positive B cells and by microarray analysis of NK/T-cell lymphoproliferative cell lines (42, 82, 83), we demonstrated here for the first time that BKRF3 encodes a conserved and functional uracil DNA glycosylase of the UNG family. Because inhibition of both viral and cellular UDG activities results in suppression of EBV lytic DNA replication, we suggest that BKRF3 might play an important role in EBV lytic replication, particularly in terminally differentiated cells where the cellular counterpart is absent or limited in abundance. Future insights into the mechanism of BKRF3 function should allow a more complete understanding of EBV lytic cycle replication and the oncogenesis of EBV-associated malignances and may pave the way for the future design of specific BKRF3 inhibitors.


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ACKNOWLEDGMENTS
 
We thank Tim J. Harrison of the Royal Free and University College Medical School (University College London) for critical reading of the manuscript. We thank Samuel E. Bennett (Department of Environmental and Molecular Toxicology, Oregon State University) for providing pZWtac1. We thank Yu-Che Cheng (Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University) and Jinghua Tsai Chang (Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taiwan) for technical suggestions on enzyme kinetic analysis and siRNA construction. We also thank Albert Yan for his assistance with real-time PCR analysis.

This work was partly supported by the National Science Council, Taiwan (grants NSC-93-3112-B-002-018 and NSC-94-2320-B002-67).


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FOOTNOTES
 
* Corresponding author. Mailing address: No. 1, Jen-Ai Rd., 1st section, Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 8298. Fax: 886-2-23915293. E-mail: mrc{at}ha.mc.ntu.edu.tw. Back

{triangledown} Published ahead of print on 15 November 2006. Back


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Journal of Virology, February 2007, p. 1195-1208, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.01518-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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