Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle

Cell lines and induction of the viral lytic cycle.

Sf9 cells, derived from the ovary of Spodoptera frugiperda, were used to propagate recombinant baculoviruses to produce the protein of interest. Sf9 cells were maintained in Sf-900 II SFM (Invitrogen) at 28°C. NPC-TW01 (TW01) is a human nasopharyngeal carcinoma cell line, derived from the tumor of a Taiwanese patient, that has lost the EBV genome (51). NA cells were established from TW01 cells infected with recombinant Akata EBV (52). TW01 and NA cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Biological Industries) and incubated at 37°C in 5% CO₂. In addition, the culture medium of NA cells was supplemented with the antibiotic G418 (Ameresco) at 400 μg/ml to maintain the EBV genome. For induction of the EBV lytic cycle, NA cells were treated with 40 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate (SB) (Sigma-Aldrich) (52).

Transfection.

Cells were transfected with plasmids or small interfering RNAs (siRNAs) by using transfection agents according to the manufacturer's instructions (Invitrogen). Briefly, for expression of the EBV BALF3 protein in a baculovirus expression system, Sf9 cells were cotransfected with 1 μg pBacPAK8-MTGFP-His-BALF3, a recombinant transfer vector, and 0.1 μg BD BaculoGold Linearized Baculovirus DNA (BD Biosciences) by using Lipofectamine 2000 (Invitrogen) at 28°C for 4 h, the culture medium was refreshed, and the cells were incubated at 28°C for 5 days to produce virions. To determine the subcellular localization of EBV BALF3, TW01 and NA cells were transfected with 0.8 μg pEGFP-C1 (Clontech Laboratories) or pEGFP-C1-BALF3 by using Lipofectamine 2000 at 37°C for 4 h, and the culture medium was refreshed prior to the addition of TPA and SB for 36 h of incubation. For the knockdown of EBV BALF3, NA cells were transfected with the indicated concentration of Stealth RNA interference siRNA specific to BALF3 or Negative Control Duplexes (Invitrogen) by siPORT NeoFX (Invitrogen) at 37°C for 24 h and then treated with TPA and SB for the indicated time after replacement of the culture medium.

Western blotting.

Cell extracts were harvested by radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% SDS [sodium dodecyl sulfate], 10 mM EDTA, 1% Igepal CA-630, protease inhibitor cocktail [Roche Applied Science]) for 20 min on ice and then centrifuged at 15,000 × g for 10 min at 4°C to collect the supernatants. The lysates were mixed with bromophenol blue buffer and then heated at 95°C for 5 min. The samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and then transferred onto a nitrocellulose membrane (GE Healthcare) at 300 mA for 90 min in a cold room. The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to His (GE Healthcare), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Biodesign), green fluorescent protein (GFP) (Clontech Laboratories), poly(ADP-ribose) polymerase 1 (PARP1) (Santa Cruz Biotechnology), α-tubulin (Millipore), EBV BMRF1 (88), or EBV viral capsid antigen (VCA) gp125 (GeneTex) at 4°C overnight prior to horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory) at room temperature for 1 h. The signal was detected by development with an enhanced chemiluminescence substrate (PerkinElmer) and exposure to X-ray film (Fujifilm).

Expression and purification of the EBV BALF3 protein.

pBacPAK8-MTGFP-His-BALF3 was generated by cloning the nucleotide sequence of His-BALF3 into pBacPAK8-MTGFP (53), a transfer vector, at the BglII cloning site. For the expression of the recombinant protein, the procedure was performed according to the manufacturer's instructions (Clontech Laboratories). Briefly, after cotransfection, the cell culture supernatant was collected and subjected to 10-fold serial dilution to select virus clones. For the examination of protein levels, the cells were infected with the selected clones and incubated at 28°C for 2 days, and then the cell extracts were harvested and subjected to Western blotting. Furthermore, the virus stock was amplified at 28°C for 5 days and the titer was calculated as the 50% tissue culture infective dose to determine the multiplicity of infection.

For purification of the recombinant protein, the procedure was performed according to the manufacturer's instructions (Qiagen). Briefly, cells infected with the recombinant viruses were extracted with lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 1% Igepal CA-630, protease inhibitor cocktail, pH 8.0) for 30 min on ice; centrifuged at 15,000 × g for 10 min at 4°C to collect the cell culture supernatants; and then mixed gently with Ni-nitrilotriacetic acid (NTA) resin on a rotary shaker at 4°C for 2 h. After incubation, the lysate-resin mixture was washed with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole, pH 8.0) and then the fractions containing the recombinant protein were harvested with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0).

In vitro nuclease activity assay.

The in vitro nuclease activity assay was carried out as described previously (20), with some modifications. pBS-TR, as a nuclease substrate, is a recombinant plasmid containing a 538-bp terminal repeat fragment of B95-8 EBV DNA (54, 55), generated by insertion at the EagI cloning site of pBlueScript (Stratagene). The protein of interest (2 μg/ml) was incubated with 0.5 μg plasmids in 10 μl of reaction buffer (10 mM Tris-HCl [pH 7.5] and the indicated concentration of MgCl₂, MnCl₂, or ATP) at 37°C for 1 h. After incubation, the samples were mixed with bromophenol blue/xylene cyanol FF buffer and then loaded into a 1% agarose gel for electrophoresis at 100 V. The signal was imaged and photographed by AlphaImage (Alpha Innotech) with ethidium bromide (EtBr) staining.

Quantitative reverse transcription-PCR (qRT-PCR).

RNA extracts were incubated with DNase I (Thermo Fisher Scientific) at 37°C for 30 min, and the nucleolytic reaction was stopped by incubation with 2.5 mM EDTA at 65°C for 10 min. The samples were mixed with 0.5 mM deoxynucleoside triphosphate (dNTP), 2.5 μM oligo(dT) primer, and 2 U RNaseOUT RNase inhibitor (Invitrogen) and incubated at 42°C for 2 min prior to RT with 10 U SuperScript III reverse transcriptase (Invitrogen) at 42°C for 50 min and then at 70°C for 15 min. After RT, the cDNA samples were used for PCR or quantitative PCR (qPCR). In PCR, the cDNA samples were mixed with 0.5 μM primers, 0.25 mM dNTP, and 0.125 U Taq DNA polymerase (Geneaid). PCR was performed on DNA Engine Thermal Cycler (Bio-Rad Laboratories) with the following cycling program: initial denaturation at 95°C for 3 min; 22 cycles of denaturation at 98°C for 10 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were mixed with xylene cyanol FF buffer and loaded into a 2% agarose gel for electrophoresis at 100 V, and then the signal was imaged and photographed by AlphaImage with EtBr staining. In qPCR, the cDNA samples were mixed with 1 μM primers and qPCR Master Mix (Kapa Biosystems). qPCR was performed using LightCycler (Roche Applied Science) with the following cycling program: initial denaturation at 95°C for 10 min and 50 cycles of denaturation at 95°C for 10 s, annealing at 50°C for 5 s, and extension at 72°C for 10 s. The primers used in this study were BZLF1-forward (5′-GAGTCAACATCCAGGCTTGG-3′) and BZLF1-reverse (5′-GCAGCACTACCGTGAGGTG-3′), BRLF1-forward (5′-CGGGAGACTTTCGAGTTCAT-3′) and BRLF1-reverse (5′-AAACTCCTATCCTCGTGTCC-3′), BALF5-forward (5′-CGGAGTTGTTATCAAAGAGGC-3′) and BALF5-reverse (5′-CGAGAAAGACGGAGATGGC-3′) (56), BMRF1-forward (5′-AGAGACACCCTCGCCTGC-3′) and BMRF1-reverse (5′-GCTTCTGCTTCTGCCTGG-3′), BALF3-forward (5′-CGTGAGGTAAAGCCCATCC-3′) and BALF3-reverse (5′-GCCGTCCTAACCGTGTCC-3′), and GAPDH-forward (5′-CCTGCCAAATATGATGACATCAAG-3′) and GAPDH-reverse (5′-ACCCTGTTGCTGTAGCCAAA-3′).

Indirect immunofluorescence staining.

Slide-cultured cells were fixed in 4% paraformaldehyde at room temperature for 20 min and then permeabilized in 0.1% Triton X-100 at room temperature for 5 min. For immunostaining, the samples were blocked in 4% fetal bovine serum at room temperature for 30 min and conducted with the anti-BMRF1 antibody at room temperature for 1 h prior to the incubation of Rhodamine Red-X-labeled goat anti-mouse IgG (Jackson ImmunoResearch Laboratory) at room temperature for another 1 h. DNA was stained with Hoechst 33258 at room temperature for 30 s. The slides were covered with mounting medium (Vector Laboratories) and photographed with a fluorescence microscope (Olympus) and a confocal microscope (Leica).

Subcellular fractionation.

The subcellular fractionation assay was carried out as described previously (57), with some modifications. Briefly, cells were harvested and resuspended in cytoplasmic extract buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.075% Igepal CA-630, protease inhibitor cocktail) for 5 min on ice and then centrifuged at 180 × g for 5 min. After centrifugation, the supernatants and pellets were collected and identified as the cytosolic and nuclear fractions, respectively. The nuclear fraction was washed twice with cytoplasmic extract buffer in the absence of the detergent prior to lysis by RIPA buffer. The extracts were subjected to Western blotting, and antibodies specific to α-tubulin and PARP1 are used as cytoplasmic and nuclear markers, respectively.

Detection and quantification of viral DNA.

Assays for the detection and quantification of viral DNA were carried out as described previously (56, 58), with some modifications. For the detection of released virions, cell culture supernatants were collected and treated with DNase I at 37°C for 1 h and the nucleolytic reaction was stopped by heating at 70°C for 10 min. The samples were digested with 1 mg/ml proteinase K (Invitrogen) at 50°C for 2 h and then inactivated at 70°C for 15 min. For the detection of intracellular viral genomic DNA, cell extracts were harvested in lysis buffer (10 mM Tri-HCl [pH 7.5], 2.5 mM MgCl₂, 1% Tween 20, 1% Igepal CA-630, 1 mg/ml proteinase K) at 50°C for 4 h and then inactivated at 70°C for 15 min. After proteolytic digestion, both samples were subjected to qPCR with primers corresponding to EBV BALF5. In this assay, Raji cells, an EBV-positive, nonproducing Burkitt's lymphoma cell line (59), were used as the standard for the amount of viral genomes (50 genome copies/cell).

Ultrathin sectioning and transmission electron microscopy.

Cells were prefixed in 2.5% glutaraldehyde for 1 h and then washed twice with phosphate-buffered saline (pH 7.0) prior to fixation in 2% osmium tetroxide for 1 h. After fixation, the samples were dehydrated in ethanol starting with a series of increasing concentrations: 50, 70, 80, and 95% for 10 min each, followed by 100% ethanol for 10 min three times. The dehydrated samples were embedded in Spurr's resin (Electron Microscopy Sciences) and placed at 68°C for 15 h for polymerization. Ultrathin sections were prepared with an ultramicrotome (Leica). The signal of the samples was imaged and photographed with a transmission electron microscope (Hitachi).

Purification of viral capsids.

The assay was carried out as described previously (89, 90) with some modifications. Briefly, cells were harvested by scraping, pelleted, and resuspended in a lysis buffer (0.01 M Tris, 0.5 M NaCl, 1 mM EDTA [pH 7.5], 1% Triton X-100, and protease inhibitor cocktail). The cells were then frozen, thawed, and centrifuged at 180 × g for 5 min to collect the nuclei. The pellets were resuspended in TNE buffer (0.01 M Tris, 0.5 M NaCl, 1 mM EDTA [pH 7.5]) and sonicated. The cell debris was removed by centrifugation at 15,000 × g for 10 min at 4°C and then the viruses were pelleted from the supernatants into a 5-ml 35% sucrose cushion prepared in TNE buffer by ultracentrifugation at 25,000 rpm for 1 h at 4°C using an SW41Ti rotor (Beckman Coulter). The pellets were resuspended in TNE buffer containing 1 mM dithiothreitol (DTT), and A-, B-, and C-type capsids were separated by density gradient ultracentrifugation on a 5-ml 20% to 50% sucrose gradient prepared in TNE buffer at 25,000 rpm for 1 h at 4°C using an SW41Ti rotor. After ultracentrifugation, the gradients were fractionated and subjected to Western blotting analysis.

Gardella gel analysis.

Gardella gel analysis was carried out as described previously (58, 60), with some modifications. Briefly, 2 × 10⁶ cells were suspended in 50 μl sample buffer (15% Ficoll, 0.5× Tris-borate-EDTA, 0.003% bromophenol blue, 400 μg/ml RNase A), and the cells were lysed by being loaded into the wells of a 0.8% agarose gel containing 2% SDS and 1 mg/ml proteinase K and run at 0.8 V/cm at 4°C for 3 h. The extracts were separated by being run in a 0.8% agarose gel at 4.5 V/cm at 4°C for an additional 40 h. After electrophoresis, the gel was used for Southern blotting with probe hybridization. In this study, a ³²P-radiolabeled random-primed probe corresponding to the EBV BamHI-W fragment was used for hybridization; in addition, chemically induced B95-8 cells, an EBV-producing marmoset B cell line treated with 40 ng/ml TPA and 3 mM SB (61), and Raji cells were used as the positive controls for linear and episomal EBV DNA, respectively.

Southern blotting and hybridization.

DNA samples in the agarose gel were depurinated in 0.25 N HCl for 30 min and then denatured by replacement with an alkaline solution (1.5 M NaCl, 0.5 M NaOH) for 20 min twice. After denaturation, the gel was soaked in transfer buffer (1.5 M NaCl, 0.5 M NaOH) for 15 min prior to transfer onto a nylon membrane (PerkinElmer) at room temperature overnight. The membrane was washed with 2× SSC buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), followed by UV exposure for cross-linking of the transferred DNA. Hybridization was performed by incubating the membrane with the ³²P-radiolabeled random-primed probe in Church buffer (0.5 M NaH₂PO₄ [pH 7.2], 7% SDS, 1% bovine serum albumin, 1 mM EDTA [pH 8.0]) at 65°C overnight, and then the membrane was washed with Church washing buffer (0.5 M NaH₂PO₄ [pH 7.2], 5% SDS, 1 mM EDTA [pH 8.0)] at 70°C. After hybridization, the signal was detected by exposure to X-ray film.

Expression and purification of EBV BALF3.

According to the recently updated amino acid sequence of EBV BALF3 available in NCBI GenBank (accession no. AFY97901) (62), the coding region of the BALF3 gene product contains 685 amino acids encoded by 2,058 nucleotides. Alignment of the sequences of BALF3 and its homologues from other HHVs, such as HSV-1 UL28 (GenBank accession no. AFP86392), HSV-2 UL28 (GenBank accession no. NP_044498), VZV ORF30 (GenBank accession no. AFO85607), HCMV UL56 (GenBank accession no. ABB71174), HHV-6 U40 (GenBank accession no. CAA58374), HHV-7 U40 (GenBank accession no. YP_073780), and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF7 (GenBank accession no. ACY00398), revealed that several regions were highly conserved, and these conserved regions have been described as putative functional domains for enzymatic activities (Fig. 1A). In the N-terminal half, BALF3 had the C173-X₂-C176-X₂₂-C199-H201 region, which is similar to the hypothetical zinc finger motif of HCMV UL56, which is considered to be involved in DNA binding (63). Otherwise, the ATP-binding motif of HCMV UL56 was likely to be in the region of amino acids 619 to 626, YNKTWGRS, in the C-terminal half of BALF3 (64). Supported by previous studies of bacteriophages, as well as herpesviruses, the terminase is believed to possess nuclease activity. In order to confirm this enzymatic property, the coding region of the EBV gene for BALF3, tagged with 6×His residues at the N terminus to enable purification, was cloned into transfer vector pBacPAK8-MTGFP and recombinant baculoviruses expressing the His-BALF3 protein were produced in Sf9 cells after cotransfection. Unlike the extract of cells mock infected or infected with the wild-type baculovirus, in Western blotting, that of cells infected with the recombinant His-BALF3 baculoviruses exhibited a clear major band and a minor band at approximately 75 and 38 kDa, respectively (Fig. 1B). According to the molecular weight predicted from the amino acid sequence, we consider that the protein with a mass of 75 kDa was the full-length His-BALF3 product. The His-BALF3 protein in the infected cell lysate was purified by interaction with nickel ions, and the lysate-resin mixture was washed by adding imidazole at a concentration of 50 mM to eliminate endogenous proteins with histidine residues. The soluble fractions were eluted by increasing the concentration of imidazole to 250 mM to dissociate the His-BALF3 protein from the resin, and proteins were visualized by SDS-PAGE and SYPRO Ruby staining (Fig. 1C).

Nuclease activity of EBV BALF3.

To analyze the biochemical properties of EBV BALF3, the nuclease activity of the His-BALF3 protein purified from the recombinant baculoviruses was assayed in vitro. The purified protein was incubated with pBS-TR containing a 538-bp terminal repeat fragment of the EBV genomic DNA, as the nuclease substrate, in the absence or presence of divalent cations, including Mg²⁺ and Mn²⁺, or ATP, and then separated by agarose gel electrophoresis. The nuclease activity of the terminase was measured by determining the amount of the open circular form of the plasmid converted from the supercoiled form, defined as a nicking reaction. In the presence of Mg²⁺, Mn²⁺, or ATP, the maximal rate of the nicking reaction was achieved at concentrations of 10, 0.1, and 20 mM, respectively (Fig. 2A to C). Comparison of the nicking levels showed that stimulation by ATP produced a greater amount of the open circular plasmid than either Mg²⁺ or Mn²⁺ (Fig. 2A to C), suggesting that ATP is more effective than the divalent cations in stimulating the nuclease activity of BALF3. Furthermore, the efficiency of the ATP-mediated nicking reaction was enhanced by Mg²⁺ at a concentration of 10 mM because the required concentration of ATP was lowered from 20 to 12 mM. This effect was not seen with Mn²⁺ at a concentration of 0.1 mM (Fig. 2D and E). Incubation with pBlueScript devoid of the terminal repeat fragment in the presence of 10 mM Mg²⁺ and 12 mM ATP led to the decreasing nucleolytic efficiency of BALF3, compared to that for the pBS-TR incubation (Fig. 2F), confirming that the terminal repeat of the EBV genomic DNA is the target substrate of BALF3. These results show that EBV BALF3 possesses endonuclease activity that is dependent on Mg²⁺, Mn²⁺, and ATP individually; in addition, the rate of the endonucleolytic reaction is synergistically increased by ATP together with Mg²⁺.

In addition, to determine whether contaminating proteins are present in the eluted fraction because proteins containing histidine residues are abundant in eukaryotic cells, including Sf9 cells, and endogenous proteins might interact with EBV BALF3, the coding region of the gene for BALF3 in pBacPAK8-MTGFP-His-BALF3 was mutated by replacement of the putative zinc finger and ATP-binding motifs with alanines, as shown in Fig. 1A, and the effects of these mutations on the nuclease activity were determined in vitro by a nuclease activity assay. Both the C173A C176A C199A H201A and G624A mutants exhibited significantly less endonuclease activity than the wild type (Fig. 2G and H), indicating the purity of the eluted BALF3 protein.

Expression of EBV BALF3 during the lytic cycle.

To determine whether EBV BALF3 is expressed in EBV-positive cells following induction of the lytic cycle, NA cell extracts were harvested at the times indicated within 72 h postinduction and subjected to qRT-PCR. During the lytic cycle, the BALF3 mRNA was detectable as early as 6 h postinduction and rose to the maximal level at 36 h postinduction (Fig. 3). Meanwhile, EBV lytic gene products, transactivator BZLF1 and DNA polymerase BALF5, were expressed in NA cells following TPA and SB treatment, confirming that the lytic cycle was activated (data not shown). According to a previous study with microarrays, the expression of the BALF3 gene in anti-IgG-induced Akata cells is reduced by less than 20% in the presence of phosphonoacetic acid (PAA), a viral DNA polymerase inhibitor, indicating that the expression of EBV BALF3 is relatively insensitive to PAA (65). In addition, analysis of the kinetic expression of mRNAs of lytic genes in TPA- and SB-induced NA cells shows that the immediate-early gene product BZLF1 increased clearly within 24 h postinduction, the early gene product BALF5 was expressed at the highest levels from 24 to 60 h postinduction, and the late gene product BLLF1 reached its maximal level at 60 h postinduction (data not shown). Accordingly, the results of the microarray analysis and the expression kinetics experiments suggest that EBV BALF3 may be classified as an early gene product.

Subcellular localization of EBV BALF3.

To observe the distribution of EBV BALF3 in host cells, TW01 and NA cells were transiently transfected with pEGFP-C1-BALF3 prior to induction of the lytic cycle for 36 h and visualized by indirect immunofluorescence staining. In TW01 cells with or without TPA and SB treatment, BALF3 was located in the cytoplasm, for the most part, and a similar pattern was seen in NA cells without induction. After induction, BALF3 could be detected in the nuclei of NA cells (Fig. 4A and B). Moreover, subcellular fractionation revealed that the amount of BALF3 in the nuclear fraction was greater at 36 h postinduction than without induction (Fig. 4C). These results show that EBV BALF3 is translocated from the cytoplasm into the nucleus in EBV-positive cells during the lytic cycle.

Contribution of EBV BALF3 to virion production.

To investigate what part EBV BALF3 plays during the lytic cycle, knockdown of BALF3 was achieved with a siRNA. According to the nucleotide sequence of the EBV genomic DNA reported in NCBI GenBank (accession no. AJ507799) (66), sense strands of siRNAs corresponding to the BALF3 mRNA were designed siBALF3-1 (5′-CACUGGCAUCUAGCCAGCAAAUUCU-3′), siBALF3-2 (5′-GCCGCCUUUGAGAAUUCCAAGUACA-3′), and siBALF3-3 (5′-GGGCAAGGCUGACUCACCUGUUUGA-3′). To determine the efficiency of knockdown by these siRNAs, NA cells were transfected with siBALF3-1, -2, or -3 prior to induction of the lytic cycle, resulting in the suppression of BALF3 expression in a concentration-dependent manner (Fig. 5A). In addition, there was no significant effect on the expression of other lytic gene products, including the transactivators BZLF1 and BRLF1, the DNA polymerase BALF5, and the DNA polymerase processivity factor BMRF1, in the presence of these BALF3 siRNAs (Fig. 5A). Previous studies have proven that the terminase participates in DNA cleavage, as well as packaging, which is required for lytic replication. To explore whether EBV BALF3 is involved in the production of infectious virions, NA cell supernatants were harvested after the transfection of each form of siBALF3 at 72 h postinduction, and the number of virions was determined by qPCR with BALF5 gene detection, showing that siBALF3-1, -2, and -3 were capable of reducing virus release by up to approximately 82, 66, and 57%, respectively, compared to the negative control (Fig. 5B). Furthermore, to observe the formation of intracellular capsids, cells containing each form of siBALF3 were ultrathin sectioned at 48 h postinduction and examined by transmission electron microscopy. Three types of capsids have been defined: the B-type capsid, which retains an inner shell that is constructed from scaffolding proteins and is devoid of viral DNA; the C-type capsid, which contains viral DNA and undergoes inner shell removal and outer shell structure stabilization; and the A-type capsid, in which the inner shell is eliminated but no viral DNA is packaged. B-type capsids had accumulated in the nucleus in the presence of each form of siBALF3, and the proportions of A- and C-type capsids were decreased (Fig. 6A and Table 1). In addition, three types of capsids were purified and separated by sucrose gradient ultracentrifugation (Fig. 6B), revealing that BALF3 knockdown caused a decrease in the amount of capsids (Fig. 6C) and the proportions of A- and C-type capsids to B-type capsid (Fig. 6D), which is consistent with the appearance of transmission electron microscopy. These results show that EBV BALF3 is involved in capsid maturation and the production of mature virions.

Contribution of EBV BALF3 to viral DNA replication.

To determine whether EBV BALF3 can affect viral DNA synthesis during lytic replication, NA cells transfected with each form of siBALF3 were harvested at 24 and 48 h postinduction and the EBV genome copy number was measured by qPCR with BALF5 gene detection. The intracellular EBV genome copy number decreased by up to approximately 92% for siBALF3-1, 89% for siBALF3-2, and 88% for siBALF3-3 at 24 h postinduction and approximately 93% for siBALF3-1, 87% for siBALF3-2, and 70% for siBALF3-3 at 48 h postinduction, compared to the negative control (Fig. 7B). In addition, these siBALF3 clones retained the property of gene silencing up to 48 h in this study (Fig. 7A). Furthermore, the use of Gardella gel analysis to detect the amount of episomal and linear EBV DNA in siBALF3-transfected NA cells at 48 h postinduction revealed that episomes were converted to concatemers after induction of the lytic cycle, and suppression of BALF3 expression led to reduced generation of concatemers (Fig. 7C). These results show that EBV BALF3 participates in viral DNA synthesis and may be involved in the process of rolling-circle replication.