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J Virol. 2012 May; 86(10): 5412–5421.
PMCID: PMC3347263

SUMO Binding by the Epstein-Barr Virus Protein Kinase BGLF4 Is Crucial for BGLF4 Function


An Epstein-Barr virus (EBV) protein microarray was used to screen for proteins binding noncovalently to the small ubiquitin-like modifier SUMO2. Among the 11 SUMO binding proteins identified was the conserved protein kinase BGLF4. The mutation of potential SUMO interaction motifs (SIMs) in BGLF4 identified N- and C-terminal SIMs. The mutation of both SIMs changed the intracellular localization of BGLF4 from nuclear to cytoplasmic, while BGLF4 mutated in the N-terminal SIM remained predominantly nuclear. The mutation of the C-terminal SIM yielded an intermediate phenotype with nuclear and cytoplasmic staining. The transfer of BGLF4 amino acids 342 to 359 to a nuclear green fluorescent protein (GFP)-tagged reporter protein led to the relocalization of the reporter to the cytoplasm. Thus, the C-terminal SIM lies adjacent to a nuclear export signal, and coordinated SUMO binding by the N- and C-terminal SIMs blocks export and allows the nuclear accumulation of BGLF4. The mutation of either SIM prevented SUMO binding in vitro. The ability of BGLF4 to abolish the SUMOylation of the EBV lytic cycle transactivator ZTA was dependent on both BGLF4 SUMO binding and BGLF4 kinase activity. The global profile of SUMOylated cell proteins was also suppressed by BGLF4 but not by the SIM or kinase-dead BGLF4 mutant. The effective BGLF4-mediated dispersion of promyelocytic leukemia (PML) bodies was dependent on SUMO binding. The SUMO binding function of BGLF4 was also required to induce the cellular DNA damage response and to enhance the production of extracellular virus during EBV lytic replication. Thus, SUMO binding by BGLF4 modulates BGLF4 function and affects the efficiency of lytic EBV replication.


Epstein-Barr virus (EBV), first discovered in association with Burkitt's lymphoma (27), is linked to a variety of human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease (105). EBV infection results in either lytic replication or the establishment of viral latency. Both latent and lytic EBV gene products have been implicated in the development of cancer (28, 51, 72, 105). EBV can be reactivated from latency by various reagents, such as 5-bromodeoxyuridine (39, 46), phorbol esters (110), anti-Ig antibodies (23, 92, 97), sodium butyrate (71), methotrexate (28), bortezomib (33, 34), thapsigargin (88, 95), and arsenic trioxide (89). The transition from latency to lytic replication is mediated by two EBV immediate-early genes, BZLF1 and BRLF1. The encoded proteins, ZTA and RTA, function as transcriptional activators that regulate the expression of EBV lytic cycle genes and lytic viral DNA replication (16, 21, 22, 31, 36, 69, 70, 83, 86, 96). The lytic induction of EBV has been postulated as a therapeutic strategy for the treatment of virus-associated tumors (29, 30, 33, 77).

The small ubiquitin-related modifier (SUMO) was first identified as a posttranslational modifier of RanGAP1 (73, 75). Similarly to the ubiquitination pathway, SUMOylation involves a series of sequential enzymatic reactions. The SUMO precursor protein is first cleaved by sentrin-specific proteases (SENPs) to generate a C-terminal diglycine motif. This then forms an E1~SUMO thioester, which is transferred to the E2-conjugating enzyme UBC9. E2~SUMO directly transfers SUMO to the substrate at lysine residues to form an isopeptide bond. E3 SUMO protein ligases facilitate this process by recruiting E2~SUMO to specific substrates and by enhancing the transfer process. SUMOylated targets can be de-SUMOylated by the SENP removal of SUMO (37). SUMOylation has been implicated in a variety of cellular processes, including transcriptional regulation, cell cycle regulation, signal transduction, the DNA damage response (DDR), and the regulation of protein-protein interactions (38). Both latent and lytic EBV proteins interact with the SUMO system. EBNA3C is SUMOylated (84), while LMP1 modulates the SUMOylation processes by interaction with UBC9 (6). SUMOylation regulates the transcriptional activity of ZTA and RTA (10, 13, 14, 45, 47, 80). Noncovalent SUMO-protein interactions can also occur through a SUMO interaction motif (SIM) in the target proteins (3, 57, 90, 91, 93). EBNA3C contains a SIM motif and upregulates EBNA2-mediated gene activation by binding to a SUMOylated protein (84).

In this study, we used an EBV protein microarray to identify additional EBV proteins that bind to SUMO. One of the identified proteins was the conserved protein kinase BGLF4. BGLF4 is present in the virion and expressed at an early stage of the lytic cycle (40, 41, 99). BGLF4 phosphorylates multiple EBV proteins, including BMRF1 (18, 42), EBNA2 (106), EBNA-LP (55), ZTA (4), EBNA1, and virion proteins (108). BGLF4 also phosphorylates host proteins to create a cellular environment suitable for efficient viral replication. BGLF4 suppresses IRF3-mediated host immune responses by reducing the recruitment of active IRF3 to the IRF3-responsive element (48a, 98a). The BGLF4 phosphorylation of stathmin at Ser16/25/38 contributes to the alteration of cellular microtubule dynamics (19). BGLF4 induces the disassembly of the nuclear lamina by the phosphorylation of lamin A/C and facilitates EBV nuclear egress and virion production (43, 61, 62). The BGLF4 phosphorylation of cellular elongation factor 1-δ (EF1-δ) may play a role in viral protein translation (54, 56). The phosphorylation of MCM4 by BGLF4 blocks host chromosomal DNA replication in the infected cells (60). The mimicry of cyclin-dependent kinase activity by BGLF4 results in the phosphorylation of both retinoblastoma protein (Rb) and p27 and leads to a prolonged pseudo-S-phase environment suitable for efficient viral DNA replication (49, 61). An in vitro screen of human proteins phosphorylated by the herpesvirus conserved kinases identified more than 100 shared substrates with a statistical enrichment of proteins involved in the DDR (64). The BGLF4-dependent phosphorylation of several proteins in the DDR cascade, such as TIP60, ATM, and H2AX, is required for efficient viral replication (64, 94). In addition, BGLF4 phosphorylates the nucleoside analog drugs ganciclovir and acyclovir, and this activity may affect the drug-mediated inhibition of EBV lytic replication (40, 76). Finally, BGLF4 inhibits ZTA SUMOylation and enhances its transcriptional activity, although the underlying mechanism remains to be defined (45).

We identified SIMs in BGLF4 and found that several known BGLF4 functions were SIM dependent. SUMO binding activity by BGLF4 was required for the BGLF4 suppression of ZTA SUMOylation, the suppression of global cellular SUMOylation, the efficient dispersion of promyelocytic leukemia (PML) bodies, the induction of the cellular DDR, and the facilitation of EBV lytic replication.



The following antibodies were used: mouse anti-FLAG (M2)-horseradish peroxidase (HRP) (catalog no. A8592), rabbit anti-FLAG polyclonal (catalog no. F7425) and rabbit anti-β-actin polyclonal antibodies (catalog no. A5441) from Sigma-Aldrich; rabbit anti-TIP60 polyclonal antibody (catalog no. DR1041; Calbiochem); rabbit anti-TIP60 (phospho-Ser86) (catalog no. ab73207; Abcam); rat anti-hemagglutinin (HA) high-affinity antibody (catalog no. 11-867-431-001; Roche); anti-mouse Igκ light-chain-HRP antibody (catalog no. 559751; BD Biosciences); anti-phospho-ATM (Ser1981) (catalog no. 5883; Cell Signaling); anti-phospho-H2AX (Ser139) (γ-H2AX) (catalog no. 9947; Cell Signaling); anti-phospho-KAP1 (Ser824) (catalog no. 4127; Cell Signaling); anti-H2AX (catalog no. 2595; Cell Signaling); mouse-anti-SUMO1/GMP-1 (catalog no. 33-2400; Invitrogen); and murine anti-ZTA antibody (catalog no. 11-007; Argene). Rabbit polyclonal anti-PML, directed against amino acid positions 484 to 498 of the human 90-kDa PML isoform, was described previously (2), as was mouse anti-BGLF4 antibody (99).


Plasmids that have been previously described are HA-BGLF4, HA-BGLF4 (kinase dead [KD]), glutathione S-transferase (GST)-TIP60 (64, 108), ZTA and the ZTA mutant (mZTA; KF12/13AA) (86), and HA-SUMO2, GST-tagged SUMO1, SUMO2, and SUMO2 trimer expression vectors (109). Flag-tagged BGLF4 (mSIM-N), BGLF4 (mSIM-C), and BGLF4 (mSIM-NC) were generated using the QuikChange site-directed mutagenesis kit (Stratagene) and were expressed from the pSG5-Flag vector. Primers used were the following: mSIM-N (VI39/40DR), 5′-GCCTTTTTGGGGAAGGTGACTGACAGAGATTACTTCACCTTTCAGCAC-3′ and 5′-GTGCTGAAAGGTGAAGTAATCTCTGTCAGTCACCTTCCCCAAAAAGGC-3′; mSIM-C first-round primers (VV344/345ER), 5′-TTGCAATTTGCAGCCCCAAAGGAGAGGCTGTTGGAGGTGCTGTCGCAG-3′ and 5′-CTGCGACAGCACCTCCAACAGCCTCTCCTTTGGGGCTGCAAATTGCAA-3′; mSIM-C second-round primers (VL349/350ER), 5′-CCAAAGGAGAGGCTGTTGGAGGAGCGGTCGCAGATGTGGAACCTGAAC-3′ and 5′-GTTCAGGTTCCACATCTGCGACCGCTCCTCCAACAGCCTCTCCTTTGG-3′. The green fluorescent protein (GFP) reporter vector (GFP-NLS-PK; pGL22) for testing nuclear export sequences has been previously described (17). Two 57-mer synthetic oligonucleotides (forward, 5′-GATCCAAAGGTGGTGCTGTTGGAGGTGCTGTCGCAGATGTGGAACCTGAACTTAGAC-3′; reverse, 5′-GATCGTCTAAGTTCAGGTTCCACATCTGCGACAGCACCTCCAACAGCACCACCTTTG-3′) encoding BGLF4 amino acids 342 to 359 plus linker sequences were annealed and ligated into the BamHI site of the GFP reporter vector to create a GFP-NLS-PK-NES vector (pGL659).

Protein microarray SUMO binding assay.

SUMO2 monomer and trimer proteins were purified from bacteria as GST fusions and then cleaved from GST using acTEV protease (Invitrogen), which cleaves an engineered site in the polylinker of the Gateway vector (109). SUMO2 proteins were tagged with maleimide-Cy5 using a commercial kit (GE Healthcare) that chemically links Cy5 through maleimide to the single cysteine residue in SUMO2. EBV protein microarrays have been described previously (108). The EBV protein arrays were blocked for 3 h with 3% bovine serum albumin (BSA) in Superblock buffer (Pierce) and then incubated with Cy5-labeled SUMO2 at a final concentration of 40 nM in Superblock buffer at 4°C overnight. The chip was washed 3 times in TBST buffer (25 mM Tris-HCl [pH 7.4], 3 mM KCl, 150 mM NaCl, 0.05% Tween 20) for 5 min each time, rinsed with distilled water, and spun to dryness. The slides then were scanned with a GenePix 4000 scanner (MDS Analytical Technologies, CA), and the binding signals were acquired using GenePix software. EBV proteins, where both duplicate spots were positive in three independent SUMO binding assays, were scored as positive.

Cell lines and DNA transfection.

The Akata (EBV+)-vector-tetracycline (Tet)-, Akata (EBV+)-BGLF4 (WT)-Tet-, and Akata (EBV+)-BGLF4 (KD)-Tet-inducible cell lines were described previously (64, 108). The Akata (EBV+)-BGLF4 (mSIM-N)-tet cell line was constructed using previously described methods (108). Akata (EBV+) cell lines were maintained in RPMI 1640 with 10% fetal bovine serum. The medium for Tet-inducible cell lines was supplemented with 1.0 μg/ml puromycin and 1.0 mg/ml G418. 293T, Vero, and U2OS cells were maintained in Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum.

Protein expression and purification.

GST-tagged proteins were expressed and purified as described previously, with some modifications (65, 66). Briefly, Escherichia coli BL21 cells were transformed with the appropriate expression vectors (SUMO1, SUMO2, and SUMO2 trimer) and then cultured in LB medium at 37°C until the A600 reached 0.6. The bacteria were induced by adding 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 16°C for 12 to 16 h. Bacteria were harvested and then lysed by sonication. GST fusion proteins were purified by affinity chromatography using glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions.

GST affinity assay.

Cell lysate (500 μl) containing Flag-tagged wild-type (WT) or mutant BGLF4 was precleared with 50 μl of 50% glutathione-Sepharose 4B at 4°C for 1 to 2 h. Twenty μl of GST, GST-SUMO1, GST-SUMO2, or GST-(SUMO2)3 bound to glutathione-Sepharose 4B was added to the precleared lysate, rotated on a vertical wheel at 4°C for 2 h, washed 4 times with lysis buffer, and boiled in 2× SDS sample buffer. GST-(SUMO2)3 expresses 3 tandem copies of SUMO2 fused to GST (107). Proteins were separated on SDS-PAGE gels and detected by Western blotting using anti-Flag antibody.

In vitro phosphorylation assay.

Flag-tagged WT, mSIM-N, and KD mutants of BGLF4 were transfected into 293T cells. Transfected cells were harvested 48 h posttransfection using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% [vol/vol] NP-40, 1% [wt/vol] deoxycholate, 0.1% [wt/vol] SDS, 1 mM EDTA) containing protease inhibitors and phosphatase cocktail I and II (Sigma). Flag-tagged WT and mutants of BGLF4 were immunoprecipitated from the transfected 293T cells using anti-Flag-M2 agarose beads (catalog no. A2220; Sigma). The bound proteins were washed twice with lysis buffer and twice with kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 300 mM KCl, and 0.5% NP-40). Immunocomplexes then were incubated with 1 μg purified recombinant GST-TIP60 in kinase buffer with 40 nM ATP for 30 min at 30°C. The reaction was terminated by adding 2× SDS sample buffer and heating at 90°C for 5 min. Proteins then were separated on SDS-PAGE gels and checked by Western blotting using phospho-TIP60 (Ser86)-specific antibody.

Indirect immunofluorescence.

U2OS or Vero cells grown on 2-well slides were transfected using Lipofectamine 2000 (Invitrogen) to express the WT or the mSIM-N, mSIM-C, or mSIM-NC mutant of BGLF4. Two days after transfection, cells were fixed for 10 min in 1% paraformaldehyde, and slides were incubated in blocking buffer (2.5% normal goat serum, 0.3% Triton X-100, and 2% bovine serum albumen). Primary antibody (mouse anti-BGLF4 and rabbit anti-PML) was diluted 1:100 in blocking buffer and incubated with the cells for 2 h at room temperature. Slides were washed with phosphate-buffered saline (PBS) three times. Secondary antibody (fluorescein isothiocyanate [FITC] anti-mouse immunoglobulin or rhodamine anti-rabbit immunoglobulin [Jackson]) was used at a 1:500 dilution in blocking buffer and incubated with the cells for 1 h at room temperature. Slides were rewashed with PBS three times and mounted in Vectashield containing DAPI (4′,6′ diamidino-2-phenylindole) (Vector Laboratories). Vero cells were similarly transfected with pGL22 and pGL659 vectors. Cells were fixed and images captured using a Nikon E800 Eclipse fluorescence microscope.

Lytic induction of Akata (EBV+) cells and virus titration.

Akata (EBV+) cells were treated with 50 μg/ml of goat anti-human IgG (MP Biomedicals) for 48 h to induce the EBV lytic cycle. To measure EBV replication, virion-associated DNA present in the culture supernatant was determined by quantitative PCR (qPCR) analysis. The supernatant (180 μl) was treated with 4 μl RQ1 DNase (Promega) for 1 h at 37°C, and reactions were stopped by adding 20 μl of stop buffer and incubation at 65°C for 10 min; 12.5 μl proteinase K (20 mg/ml, Invitrogen) and 25 μl 10% (wt/vol) SDS then were added to the reaction mixtures, which were incubated for 1 h at 65°C. DNA was purified by phenol-chloroform extraction followed by isopropanol-sodium acetate precipitation and then resuspended in 200 μl nuclease-free water. qPCR reaction mixes (20 μl) contained primers (100 nM), 2 μl DNA, and 10 μl SYBR green PCR master mix (Applied Biosystems). Cycle threshold (CT) values were measured using a 7500 real-time PCR system (Applied Biosystems). Relative levels of virion DNA were normalized to supernatant virion DNA without lytic induction. The BALF5 primers used for quantitating EBV copy numbers were 5′-AGTCCTTCTTGGCTACTCTGTTGAC-3′ and 5′-CTTTGGCGCGGATCCTC-3′.


Screening for EBV proteins that bind SUMO.

To systematically identify SUMO binding proteins, we employed a proteomic approach using Cy5-labeled SUMO2 and a previously described protein array displaying 60 EBV proteins expressed as GST fusions and purified from yeast (108). We identified 11 EBV proteins, including the known SUMO binding partner EBNA3C (84), that reproducibly scored as SUMO2 binding in three independent assays. At least two consensus SIM motifs (V/I/L-X-V/I/L-V/I/L or V/I/L-V/I/L-X-V/I/L, where V is valine, I is isoleucine, L is leucine, and X is any amino acid), are present in each of these 11 proteins (Table 1). One of the newly identified SUMO binding proteins was the conserved protein kinase BGLF4. BGLF4 plays multiple roles in ensuring efficient EBV replication. We therefore focused on the interplay of BGLF4 and SUMO.

Table 1
SUMO binding proteins identified by proteomic screening

Mapping the SIM motifs in BGLF4.

To validate the interaction between BGLF4 and SUMO2, we first performed an in vitro pulldown assay using GST-SUMO2, a GST-SUMO2 tandem trimer [GST-(SUMO2)3], and lysate from Flag-BGLF4-transfected cells. We found that both GST-SUMO2 and GST-(SUMO2)3, but not the GST control, bound BGLF4 (Fig. 1A, upper). In addition, we noted that the kinase-dead BGLF4 (KD) also bound GST-SUMO2 (Fig. 1B), indicating that the interaction between BGLF4 and SUMO2 is independent of BGLF4 kinase activity.

Fig 1
BGLF4 interacts with SUMO1 and SUMO2. (A) BGLF4 interacts with SUMO2. (Upper) Western blot showing BGLF4 binding to GST-SUMO2 and the GST-SUMO2 trimer [GST-(SUMO2)3]. The amount of BGLF4 in the input lysate was 5% of that used in the pulldown assays. ...

Sequence analysis revealed two potential SIM motifs in BGLF4, one located at the N terminus (designated SIM-N) and another SIM at the C terminus (designated SIM-C) (Fig. 1C and Table 1). These motifs were mutated to generate: mSIM-N, mSIM-C, and mSIM-NC (in which SIM-N and SIM-C are mutated) (Fig. 1C). Each of these mutations disrupted the binding of BGLF4 to SUMO1 and to SUMO2, as evaluated by GST pulldown assays (Fig. 1D), confirming that these sequences represent functional SIMs.

Effect of SUMO binding on BGLF4 subcellular localization.

A previous study identified sequences that were necessary, but not sufficient, for BGLF4 nuclear localization (44). We investigated whether SUMO binding played any role in BGLF4 intracellular distribution. Immunofluorescence assays on transfected cells showed the expected nuclear localization for BGLF4 (Fig. 2A). The mSIM-N mutant had a predominantly nuclear localization with weak cytoplasmic staining (Fig. 2A). The mutation of the C-terminal SIM resulted in individual cells showing either nuclear or perinuclear plus cytoplasmic staining. The mutation of both N- and C-terminal SIMs led to BGLF4 adopting a cytoplasmic distribution (Fig. 2A). A second N- and C-terminal SIM mutant (mSIM-NC*) in which the C-terminal SIM mutation was restricted to the first two valine residues of the SIM then was generated. This BGLF4 SIM mutant also localized to the cytoplasm (Fig. 2A). The BGLF4 amino acid sequence immediately adjacent to the C-terminal SIM contains hydrophobic amino acids whose relative spacing is suggestive of a nuclear export motif. To test this possibility, sequences encoding BGLF4 amino acids 342 to 359 were transferred to a GFP-tagged nuclear protein, GFP-NLS-PK, that has previously been used to identify nuclear export signals (NES) in the EBV Mta protein (17). In transfected Vero cells, the GFP-NLS-PK protein had a strictly nuclear localization (Fig. 2B). The addition of BGLF4 amino acids 342 to 359 resulted in the relocalization of GFP-NLS-PK to the cytoplasm (Fig. 2B). Therefore, BGLF4 contains a functional nuclear export signal. The mutation of the adjacent C-terminal SIM is sufficient to cause a proportion of BGLF4 to display a cytoplasmic localization. However, the mutation of both N- and C-terminal SIMs is necessary for the complete abrogation of nuclear accumulation, suggesting that there is cooperation between the SIM motifs.

Fig 2
SIM mutation alters BGLF4 subcellular localization and uncovers a nuclear export signal (NES). (A) Merged immunofluorescence images showing the subcellular localization of the WT and SIM mutant BGLF4 proteins in transfected U2OS cells. Cells were stained ...

BGLF4 inhibits ZTA and global cellular SUMOylation in a SIM-dependent manner.

BGLF4 was previously shown to inhibit ZTA SUMOylation and regulate ZTA transcription activity (45). We asked whether SUMO binding by BGLF4 plays a role in the inhibition of ZTA SUMOylation. Since BGLF4 with the N-terminal SIM mutated lacked SUMO binding in vitro but still retained the wild-type BGLF4 nuclear localization phenotype, we selected this mutant for functional assays. ZTA or mZTA (KF12/13AA), HA-SUMO2, and WT or mSIM-N BGLF4 were transfected as indicated in Fig. 3A, and the presence of SUMOylated ZTA was examined by Western blotting. We found that ZTA was readily SUMOylated in transfected cells with or without cotransfected HA-SUMO2 (Fig. 3A, lanes 1 and 2). ZTA SUMOylation was abolished by cotransfection with WT BGLF4 but not by mSIM-N BGLF4 (Fig. 3A, lanes 3 and 4). Covalent SUMO modification has been shown to occur on K12 of ZTA (45). The SUMO-ZTA bands were not detected when the ZTA mutant KF12/13AA was transfected (Fig. 3A, lanes 5 to 8). To ensure that the results were not influenced by the overexpression of transfected HA-SUMO2, we repeated the assay to examine ZTA modification by endogenous SUMO and similarly found that WT BGLF4 abolished ZTA SUMOylation, while the mSIM-N BGLF4 mutant had no effect on ZTA SUMOylation (Fig. 3B, lanes 3 and 4). To address whether the kinase activity of BGLF4 played a role in abolishing ZTA SUMOylation, ZTA was cotransfected with the kinase-dead BGLF4 mutant. Interestingly, we found that the elimination of BGLF4 kinase activity also abolished the ability of BGLF4 to interfere with ZTA SUMOylation (Fig. 3B, lanes 5 and 6). To ensure that the mutation of the N-terminal SIM did not inadvertently abolish BGLF4 kinase activity, an in vitro kinase assay was performed using WT, mSIM-N, and KD BGLF4 immunoprecipitated from transfected cells. A known substrate of BGLF4 is the cell protein TIP60, which is phosphorylated at Ser86 (64). Using a pTIP60 S86-specific antibody, we demonstrated that both BGLF4 and mSIM-N BGLF4 phosphorylated TIP60 at Ser86, while the vector control and KD BGLF4 failed to phosphorylate TIP60 in vitro (Fig. 3C). Our results indicate that the SUMO binding and kinase activities of BGLF4 independently contribute to the inhibition of ZTA SUMOylation.

Fig 3
SIM- and kinase activity-dependent inhibition of SUMOylation by BGLF4. (A) SIM-dependent inhibition of ZTA SUMOylation by BGLF4. Immunoblot analysis of cell lysates from 293T cells cotransfected as indicated. ZTA and SUMO-modified ZTA were detected using ...

To address whether the BGLF4 inhibition of SUMOylation is confined to ZTA or is a more general phenomenon, we constructed an Akata (EBV+) cell line that carries a Tet-inducible SIM mutant BGLF4 (mSIM-N) and examined global SUMOylation upon the doxycycline induction of BGLF4. As a comparison, we also tested three previously constructed cell lines expressing either the vector control, WT BGLF4, or KD BGLF4 (64). The results showed that WT BGLF4 globally suppressed SUMOylation events, while neither the mSIM-N nor KD BGLF4 protein had any significant effect on SUMOylation compared to the vector control (Fig. 3D and E). The SIM- and kinase-dependent BGLF4 suppression of ZTA SUMOylation thus extends to the modulation of cell protein SUMOylation.

BGLF4 disrupts PML bodies in a SIM-dependent manner.

The ability of BGLF4 to disrupt promyelocytic leukemia (PML) bodies has been controversial (61, 85). We reexamined this function of BGLF4 in transfected Vero cells. We observed that the number of PML bodies per cell was reduced upon the expression of WT BGLF4 compared to the level for the control cells (Fig. 4A). This is consistent with the results reported by Kuny et al. (61). The mSIM-N BGLF4 mutant also reduced the PML body number, although less efficiently than WT BGLF4 (Fig. 4A). As expected, there was no significant difference in the number of PML bodies in cells transfected with the cytoplasmic mSIM-NC* mutant (Fig. 4A). Quantitative analysis revealed that cells expressing WT BGLF4 had 2.5-fold fewer PML bodies than the control cells, while mSIM-N-transfected cells reduced the PML body number by 1.6-fold. Cells expressing mSIM-NC* retained the same number (99%) of PML bodies as control cells (Fig. 4B). The dispersal of PML bodies by BGLF4 is therefore another function of BGLF4 that is SIM dependent.

Fig 4
BGLF4 disperses PML bodies in a SIM-dependent manner. (A) Immunofluorescence assays performed on Vero cells transfected with WT or SIM BGLF4 mutants. Cells were stained with anti-BGLF4 (upper) and anti-PML (middle) antibodies. Nuclei were visualized with ...

BGLF4 induction of the DDR and enhancement of viral lytic replication is SIM dependent.

BGLF4 contributes to EBV lytic replication by inducing the DNA damage response (DDR) through TIP60 phosphorylation (Fig. 5A) (64). To examine the role of SUMO binding by BGLF4 in the induction of the DDR, we compared the phosphorylation status of several proteins in the DDR cascade, namely, TIP60, ATM, KAP1, and H2AX. After the doxycycline treatment of the Akata cells, the induction of WT BGLF4 led to the phosphorylation of each these proteins (Fig. 5B). However, neither mSIM-N nor KD BGLF4 was capable of inducing the phosphorylation of TIP60, ATM, KAP1, or H2AX. The BGLF4 induction of the DDR is thus also SIM and kinase activity dependent.

Fig 5
BGLF4 induction of the DDR and enhancement of lytic EBV replication is SIM dependent. (A) Schematic illustration of BGLF4 function in the DDR and viral replication. P, phosphorylation; Ac, acetylation; S, SUMO. (B) SIM- and kinase activity-dependent activation ...

BGLF4 is required for efficient EBV replication. To determine whether the loss of SUMO binding by BGLF4 affects EBV lytic replication, we compared the extracellular virion DNA copy number in Akata B cells expressing WT, mSIM-N, or KD BGLF4. After a 6-h induction of these proteins, EBV lytic replication was activated via IgG cross-linking for 48 h, and then the supernatants were harvested to assay for extracellular virion DNA. As illustrated in Fig. 5C, the preinduction of WT BGLF4 enhanced the production of extracellular virus, while the preinduction of mSIM-N and KD BGLF4 had no effect above that of the vector control. Thus, eliminating the SUMO binding activity of BGLF4 affects the efficiency of lytic EBV replication to the same extent as the ablation of BGLF4 kinase activity.


High-throughput screening is a new tool for the investigation of pathogen-host interactions (9, 11, 32, 53, 59, 87). We previously identified viral and host phosphorylation targets of the BGLF4 protein kinase using EBV and host protein microarrays (64, 108). In this study, we used a similar proteomic approach to identify 11 EBV proteins that bind to SUMO. We focused on the protein kinase BGLF4 and evaluated the importance of SUMO binding for BGLF4 function.

Viruses interact with the host SUMOylation pathway by modulating key components of the SUMO conjugation machinery and by encoding proteins that are themselves targets for covalent SUMOylation and noncovalent SUMO binding (8, 102, 103). EBV ZTA is SUMOylated by SUMO1/2/3, and the EBV protein kinase BGLF4 counteracts ZTA SUMOylation through an unknown mechanism (1, 45, 80). Here, we link BGLF4 SUMO binding and BGLF4 kinase activity to the BGLF4-mediated inhibition of SUMOylation. The phosphorylation-mediated regulation of SUMOylation has been described for a number of proteins. KRAB-associated protein 1 (KAP1), a corepressor for kruppel-associated box zinc finger proteins, recruits proteins involved in transcriptional repression and chromatin condensation. The phosphorylation and SUMOylation of KAP1 are mutually exclusive. The ATM-mediated phosphorylation of KAP1 Ser824 represses its SUMOylation, and conversely, SENP1-mediated de-SUMOylation of KAP1 enhances its phosphorylation (67, 68). The Kaposi's sarcoma-associated herpesvirus (KSHV) protein kinase ORF36 also phosphorylates KAP1 and prevents KAP1 SUMOylation (50). The calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of liver X receptor (LXR) at Ser427 creates a docking site for SENP3 and promotes LXR de-SUMOylation (48). The loss of the SUMO modification of Elk-1 correlates with Elk-1 phosphorylation mediated by ERK pathway activation (104). The phosphorylation of IκBα inhibits its SUMO conjugation in vitro (24). The SUMOylation of ATF7 is abrogated by p38β2-mediated phosphorylation on Thr51 (12), and the SUMO modification of both c-Jun and p53 is downregulated by phosphorylation (79). ZTA has been shown to be phosphorylated at multiple sites (Thr14 and Ser 167/173/186/209) by cellular kinases and by the viral kinase BGLF4 (5, 26). It is plausible that BGLF4 binding to SUMOylated viral or host proteins enhances the phosphorylation of the targeted protein, and that this then either inhibits the continued SUMOylation of these targets or leads to the recruitment of SENPs and de-SUMOylation.

Several viral proteins have been shown to bind SUMO via SIMs. For example, EBV EBNA3C upregulates EBNA2-mediated gene activation by SIM-dependent binding to a SUMOylated repressor (84). The SIM in human cytomegalovirus (HCMV) IE2 is required for efficient IE2 SUMOylation at K175/180 and for its binding to SUMOylated TATA-binding protein (TBP)-associated factor 12 (TAF12) (7, 58). The KSHV b-ZIP protein is a viral E3 SUMO ligase, and the ligase activity is dependent on the presence of a SIM (15). The herpesvirus protein kinases Us3, UL97, ORF36, and BGLF4, encoded by herpes simplex virus type 2 (HSV-2) (52), HCMV (61, 82, 98), KSHV (61), and EBV (61), respectively, have a role in disrupting PML bodies. The kinase activity of Us3 and UL97 is required for this function. We now demonstrate that EBV BGLF4 requires SUMO binding for PML body dispersion. KSHV vIRF3/LANA2 and varicella-zoster virus (VZV) ORF61 also disrupt PML bodies in a SIM-dependent manner (74, 100).

BGLF4 localizes predominantly to the nucleus, although weak cytoplasmic and perinuclear staining also have been described (40, 99). We found that the mutation of both N- and C-terminal SIMs resulted in BGLF4 adopting a cytoplasmic distribution. We further demonstrated that the C-terminal SIM lies adjacent to a functional nuclear export signal. The data suggest that the accumulation of BGLF4 in the nucleus is dependent on the blocking of nuclear export through coordinate SUMO binding mediated by the two BGLF4 SIMs. Coordinated binding may increase the affinity of the SUMO interaction or may be required to mediate a change in BGLF4 protein conformation. The regulation of nucleocytoplasmic trafficking by covalent SUMOylation has been shown for a number of proteins (101), and several of these are covalently SUMOylated at sites in close proximity to nuclear export signals (25). SIMs have been associated with the regulation of the intranuclear localization of proteins, but we are not aware of previous reports implicating SUMO binding in nuclear-cytoplasmic distribution.

SUMOylation and SIM-dependent protein-protein interactions play an important role in the DDR and in double-strand break (DSB) repair (68, 90). SUMO, UBC9, and the SUMO E3 ligases PIAS1 and PIAS4 relocate to γ-H2AX foci after DNA damage (35, 78). The SUMOylation of TIP60 at lysines 430 and 451 is also necessary for the p53-dependent DDR (20). The cellular DDR triggered by viral infection or lytic induction plays an important role in viral life cycle (63). The EBV infection of primary B cells elicits a robust DDR that restricts EBV-induced B cell transformation (81). Conversely, aspects of the DNA damage pathway are selectively incorporated into the EBV lytic replication program and are required for efficient viral replication (63, 64). We demonstrate that the BGLF4-induced DDR requires the presence of a BGLF4 SIM. Therefore, BGLF4 appears to take advantage of the SUMO modification of DDR proteins to integrate into the complex formed at the site of DNA damage and to then facilitate the DDR. Overall, SUMO binding by BGLF4 was found to be as important as the kinase activity of BGLF4 for mediating functions that foster EBV replication.


We thank Mei-Ru Chen for anti-BGLF4 antibody and Gary S. Hayward for anti-PML antibody. We also thank Ijee Uzoma and Eric Cox for assistance with the array screening and Richard Roden for assistance with the immunofluorescence assays.

This work was funded by NIH grant R37CA42245 to S.D.H.


Published ahead of print 7 March 2012


1. Adamson AL, Kenney S. 2001. Epstein-Barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J. Virol. 75:2388–2399 [PMC free article] [PubMed]
2. Ahn JH, Brignole EJ, III, Hayward GS. 1998. Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING finger-dependent cryptic transactivator function of PML. Mol. Cell. Biol. 18:4899–4913 [PMC free article] [PubMed]
3. Arriagada G, Muntean LN, Goff SP. 2011. SUMO-interacting motifs of human TRIM5alpha are important for antiviral activity. PLoS Pathog. 7:e1002019. [PMC free article] [PubMed]
4. Asai R, et al. 2006. Epstein-Barr virus protein kinase BGLF4 is a virion tegument protein that dissociates from virions in a phosphorylation-dependent process and phosphorylates the viral immediate-early protein BZLF1. J. Virol. 80:5125–5134 [PMC free article] [PubMed]
5. Asai R, Kato A, Kawaguchi Y. 2009. Epstein-Barr virus protein kinase BGLF4 interacts with viral transactivator BZLF1 and regulates its transactivation activity. J. Gen. Virol. 90:1575–1581 [PubMed]
6. Bentz GL, Whitehurst CB, Pagano JS. 2011. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9. J. Virol. 85:10144–10153 [PMC free article] [PubMed]
7. Berndt A, Hofmann-Winkler H, Tavalai N, Hahn G, Stamminger T. 2009. Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection. J. Virol. 83:12881–12894 [PMC free article] [PubMed]
8. Boggio R, Chiocca S. 2006. Viruses and sumoylation: recent highlights. Curr. Opin. Microbiol. 9:430–436 [PubMed]
9. Brass AL, et al. 2009. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243–1254 [PMC free article] [PubMed]
10. Calderwood MA, Holthaus AM, Johannsen E. 2008. The Epstein-Barr virus LF2 protein inhibits viral replication. J. Virol. 82:8509–8519 [PMC free article] [PubMed]
11. Calderwood MA, et al. 2007. Epstein-Barr virus and virus human protein interaction maps. Proc. Natl. Acad. Sci. U. S. A. 104:7606–7611 [PubMed]
12. Camuzeaux B, et al. 2008. p38beta2-mediated phosphorylation and sumoylation of ATF7 are mutually exclusive. J. Mol. Biol. 384:980–991 [PubMed]
13. Chang LK, et al. 2004. Post-translational modification of Rta of Epstein-Barr virus by SUMO-1. J. Biol. Chem. 279:38803–38812 [PubMed]
14. Chang LK, et al. 2008. Enhancement of transactivation activity of Rta of Epstein-Barr virus by RanBPM. J. Mol. Biol. 379:231–242 [PubMed]
15. Chang PC, et al. 2010. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO E3 ligase that is SIM-dependent and SUMO-2/3-specific. J. Biol. Chem. 285:5266–5273 [PMC free article] [PubMed]
16. Chang YN, Dong DL, Hayward GS, Hayward SD. 1990. The Epstein-Barr virus Zta transactivator: a member of the bZIP family with unique DNA-binding specificity and a dimerization domain that lacks the characteristic heptad leucine zipper motif. J. Virol. 64:3358–3369 [PMC free article] [PubMed]
17. Chen L, Liao G, Fujimuro M, Semmes OJ, Hayward SD. 2001. Properties of two EBV Mta nuclear export signal sequences. Virology 288:119–128 [PubMed]
18. Chen MR, Chang SJ, Huang H, Chen JY. 2000. A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. J. Virol. 74:3093–3104 [PMC free article] [PubMed]
19. Chen PW, et al. 2010. Regulation of microtubule dynamics through phosphorylation on stathmin by Epstein-Barr virus kinase BGLF4. J. Biol. Chem. 285:10053–10063 [PMC free article] [PubMed]
20. Cheng Z, et al. 2008. Functional characterization of TIP60 sumoylation in UV-irradiated DNA damage response. Oncogene 27:931–941 [PubMed]
21. Chevallier-Greco A, et al. 1986. Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter. EMBO J. 5:3243–3249 [PubMed]
22. Countryman J, Jenson H, Seibl R, Wolf H, Miller G. 1987. Polymorphic proteins encoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency. J. Virol. 61:3672–3679 [PMC free article] [PubMed]
23. Daibata M, Humphreys RE, Takada K, Sairenji T. 1990. Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata. J. Immunol. 144:4788–4793 [PubMed]
24. Desterro JM, Rodriguez MS, Hay RT. 1998. SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol. Cell 2:233–239 [PubMed]
25. Du JX, Bialkowska AB, McConnell BB, Yang VW. 2008. SUMOylation regulates nuclear localization of Kruppel-like factor 5. J. Biol. Chem. 283:31991–32002 [PMC free article] [PubMed]
26. El-Guindy AS, Paek SY, Countryman J, Miller G. 2006. Identification of constitutive phosphorylation sites on the Epstein-Barr virus ZEBRA protein. J. Biol. Chem. 281:3085–3095 [PubMed]
27. Epstein MA, Achong BG, Barr YM. 1964. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet i:702–703 [PubMed]
28. Feng WH, et al. 2004. Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas. J. Natl. Cancer Inst. 96:1691–1702 [PubMed]
29. Feng WH, Hong G, Delecluse HJ, Kenney SC. 2004. Lytic induction therapy for Epstein-Barr virus-positive B-cell lymphomas. J. Virol. 78:1893–1902 [PMC free article] [PubMed]
30. Feng WH, Israel B, Raab-Traub N, Busson P, Kenney SC. 2002. Chemotherapy induces lytic EBV replication and confers ganciclovir susceptibility to EBV-positive epithelial cell tumors. Cancer Res. 62:1920–1926 [PubMed]
31. Fixman ED, Hayward GS, Hayward SD. 1995. Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. J. Virol. 69:2998–3006 [PMC free article] [PubMed]
32. Friedel CC, Haas J. 2011. Virus-host interactomes and global models of virus-infected cells. Trends Microbiol. 19:501–508 [PubMed]
33. Fu DX, et al. 2008. Bortezomib-induced enzyme-targeted radiation therapy in herpesvirus-associated tumors. Nat. Med. 14:1118–1122 [PMC free article] [PubMed]
34. Fu DX, et al. 2007. Virus-associated tumor imaging by induction of viral gene expression. Clin. Cancer Res. 13:1453–1458 [PubMed]
35. Galanty Y, et al. 2009. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462:935–939 [PMC free article] [PubMed]
36. Gao Z, et al. 1998. The Epstein-Barr virus lytic transactivator Zta interacts with the helicase-primase replication proteins. J. Virol. 72:8559–8567 [PMC free article] [PubMed]
37. Gareau JR, Lima CD. 2010. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat. Rev. Mol. Cell Biol. 11:861–871 [PMC free article] [PubMed]
38. Geiss-Friedlander R, Melchior F. 2007. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8:947–956 [PubMed]
39. Gerber P. 1972. Activation of Epstein-Barr virus by 5-bromodeoxyuridine in “virus-free” human cells (complement-fixing antigen-immunofluorescence-leukocytes). Proc. Natl. Acad. Sci. U. S. A. 69:83–85 [PubMed]
40. Gershburg E, Marschall M, Hong K, Pagano JS. 2004. Expression and localization of the Epstein-Barr virus-encoded protein kinase. J. Virol. 78:12140–12146 [PMC free article] [PubMed]
41. Gershburg E, Pagano JS. 2008. Conserved herpesvirus protein kinases. Biochim. Biophys. Acta 1784:203–212 [PMC free article] [PubMed]
42. Gershburg E, Pagano JS. 2002. Phosphorylation of the Epstein-Barr virus (EBV) DNA polymerase processivity factor EA-D by the EBV-encoded protein kinase and effects of the L-riboside benzimidazole 1263W94. J. Virol. 76:998–1003 [PMC free article] [PubMed]
43. Gershburg E, Raffa S, Torrisi MR, Pagano JS. 2007. Epstein-Barr virus-encoded protein kinase (BGLF4) is involved in production of infectious virus. J. Virol. 81:5407–5412 [PMC free article] [PubMed]
44. Gershburg S, Murphy L, Marschall M, Gershburg E. 2010. Key motifs in EBV (Epstein-Barr virus)-encoded protein kinase for phosphorylation activity and nuclear localization. Biochem. J. 431:227–235 [PubMed]
45. Hagemeier SR, et al. 2010. Sumoylation of the Epstein-Barr virus BZLF1 protein inhibits its transcriptional activity and is regulated by the virus-encoded protein kinase. J. Virol. 84:4383–4394 [PMC free article] [PubMed]
46. Hampar B, Derge JG, Martos LM, Walker JL. 1972. Synthesis of Epstein-Barr virus after activation of the viral genome in a “virus-negative” human lymphoblastoid cell (Raji) made resistant to 5-bromodeoxyuridine (thymidine kinase-virus antigen-immunofluorescence-herpesvirus fingerprints). Proc. Natl. Acad. Sci. U. S. A. 69:78–82 [PubMed]
47. Heilmann AM, Calderwood MA, Johannsen E. 2010. Epstein-Barr virus LF2 protein regulates viral replication by altering Rta subcellular localization. J. Virol. 84:9920–9931 [PMC free article] [PubMed]
48. Huang W, et al. 2011. Coronin 2A mediates actin-dependent de-repression of inflammatory response genes. Nature 470:414–418 [PMC free article] [PubMed]
48a. Hwang S, et al. 2009. Conserved herpesviral kinase promotes viral persistence by inhibiting the IRF-3-mediated type I interferon response. Cell Host Microbe 5:166–178 [PMC free article] [PubMed]
49. Iwahori S, et al. 2009. Phosphorylation of p27Kip1 by Epstein-Barr virus protein kinase induces its degradation through SCFSkp2 ubiquitin ligase actions during viral lytic replication. J. Biol. Chem. 284:18923–18931 [PMC free article] [PubMed]
50. Izumiya Y, et al. 2007. Kaposi's sarcoma-associated herpesvirus-encoded protein kinase and its interaction with K-bZIP. J. Virol. 81:1072–1082 [PMC free article] [PubMed]
51. Joab I, et al. 1991. Detection of anti-Epstein-Barr-virus transactivator (ZEBRA) antibodies in sera from patients with nasopharyngeal carcinoma. Int. J. Cancer 48:647–649 [PubMed]
52. Jung M, Finnen RL, Neron CE, Banfield BW. 2011. The alphaherpesvirus serine/threonine kinase us3 disrupts promyelocytic leukemia protein nuclear bodies. J. Virol. 85:5301–5311 [PMC free article] [PubMed]
53. Karlas A, et al. 2010. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463:818–822 [PubMed]
54. Kato K, et al. 2001. Epstein-Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1delta (EF-1delta): EF-1delta is universally modified by conserved protein kinases of herpesviruses in mammalian cells. J. Gen. Virol. 82:1457–1463 [PubMed]
55. Kato K, et al. 2003. Identification of protein kinases responsible for phosphorylation of Epstein-Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function. J. Gen. Virol. 84:3381–3392 [PubMed]
56. Kawaguchi Y, et al. 2003. Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta. J. Virol. 77:2359–2368 [PMC free article] [PubMed]
57. Kerscher O. 2007. SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep. 8:550–555 [PubMed]
58. Kim ET, Kim YE, Huh YH, Ahn JH. 2010. Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth. J. Virol. 84:8111–8123 [PMC free article] [PubMed]
59. Konig R, et al. 2010. Human host factors required for influenza virus replication. Nature 463:813–817 [PMC free article] [PubMed]
60. Kudoh A, et al. 2006. Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication. J. Virol. 80:10064–10072 [PMC free article] [PubMed]
61. Kuny CV, Chinchilla K, Culbertson MR, Kalejta RF. 2010. Cyclin-dependent kinase-like function is shared by the beta- and gamma-subset of the conserved herpesvirus protein kinases. PLoS Pathog. 6:e1001092. [PMC free article] [PubMed]
62. Lee CP, et al. 2008. Epstein-Barr virus BGLF4 kinase induces disassembly of the nuclear lamina to facilitate virion production. J. Virol. 82:11913–11926 [PMC free article] [PubMed]
63. Li R, Hayward SD. 2011. The Ying-Yang of the virus-host interaction: control of the DNA damage response. Future Microbiol. 6:379–383 [PMC free article] [PubMed]
64. Li R, et al. 2011. Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication. Cell Host Microbe 10:390–400 [PMC free article] [PubMed]
65. Li RF, et al. 2007. Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway. J. Mol. Biol. 374:777–790 [PubMed]
66. Li RF, Zhang F, Lu YJ, Sui SF. 2005. Specific interaction between Smad1 and CHIP: a surface plasmon resonance study. Colloids Surf. B Biointerfaces 40:133–136 [PubMed]
67. Li X, et al. 2007. Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression. J. Biol. Chem. 282:36177–36189 [PubMed]
68. Li X, et al. 2010. SUMOylation of the transcriptional co-repressor KAP1 is regulated by the serine and threonine phosphatase PP1. Sci. Signal. 3:ra32. [PMC free article] [PubMed]
69. Liao G, Wu FY, Hayward SD. 2001. Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments. J. Virol. 75:8792–8802 [PMC free article] [PubMed]
70. Lieberman PM, Hardwick JM, Sample J, Hayward GS, Hayward SD. 1990. The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions. J. Virol. 64:1143–1155 [PMC free article] [PubMed]
71. Luka J, Kallin B, Klein G. 1979. Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94:228–231 [PubMed]
72. Ma SD, et al. 2011. A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas. J. Virol. 85:165–177 [PMC free article] [PubMed]
73. Mahajan R, Delphin C, Guan T, Gerace L, Melchior F. 1997. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88:97–107 [PubMed]
74. Marcos-Villar L, et al. 2011. Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2. J. Gen. Virol. 92:188–194 [PubMed]
75. Matunis MJ, Coutavas E, Blobel G. 1996. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135:1457–1470 [PMC free article] [PubMed]
76. Meng Q, et al. 2010. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production. J. Virol. 84:4534–4542 [PMC free article] [PubMed]
77. Moore SM, Cannon JS, Tanhehco YC, Hamzeh FM, Ambinder RF. 2001. Induction of Epstein-Barr virus kinases to sensitize tumor cells to nucleoside analogues. Antimicrob. Agents Chemother. 45:2082–2091 [PMC free article] [PubMed]
78. Morris JR, et al. 2009. The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462:886–890 [PubMed]
79. Muller S, et al. 2000. c-Jun and p53 activity is modulated by SUMO-1 modification. J. Biol. Chem. 275:13321–13329 [PubMed]
80. Murata T, et al. 2010. Transcriptional repression by sumoylation of Epstein-Barr virus BZLF1 protein correlates with association of histone deacetylase. J. Biol. Chem. 285:23925–23935 [PMC free article] [PubMed]
81. Nikitin PA, et al. 2010. An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells. Cell Host Microbe 8:510–522 [PMC free article] [PubMed]
82. Prichard MN, et al. 2008. Human cytomegalovirus UL97 kinase activity is required for the hyperphosphorylation of retinoblastoma protein and inhibits the formation of nuclear aggresomes. J. Virol. 82:5054–5067 [PMC free article] [PubMed]
83. Rooney CM, Rowe DT, Ragot T, Farrell PJ. 1989. The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle. J. Virol. 63:3109–3116 [PMC free article] [PubMed]
84. Rosendorff A, et al. 2004. EBNA3C coactivation with EBNA2 requires a SUMO homology domain. J. Virol. 78:367–377 [PMC free article] [PubMed]
85. Salsman J, Zimmerman N, Chen T, Domagala M, Frappier L. 2008. Genome-wide screen of three herpesviruses for protein subcellular localization and alteration of PML nuclear bodies. PLoS Pathog. 4:e1000100. [PMC free article] [PubMed]
86. Sarisky RT, et al. 1996. A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator. J. Virol. 70:8340–8347 [PMC free article] [PubMed]
87. Shapira SD, et al. 2009. A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139:1255–1267 [PMC free article] [PubMed]
88. Shirley CM, et al. 2011. Bortezomib induction of C/EBPbeta mediates Epstein-Barr virus lytic activation in Burkitt lymphoma. Blood 117:6297–6303 [PubMed]
89. Sides MD, et al. 2011. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells. Virology 416:86–97 [PMC free article] [PubMed]
90. Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y. 2004. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. U. S. A. 101:14373–14378 [PubMed]
91. Song J, Zhang Z, Hu W, Chen Y. 2005. Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280:40122–40129 [PubMed]
92. Takada K. 1984. Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines. Int. J. Cancer 33:27–32 [PubMed]
93. Takahashi H, Hatakeyama S, Saitoh H, Nakayama KI. 2005. Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein. J. Biol. Chem. 280:5611–5621 [PubMed]
94. Tarakanova VL, et al. 2007. Gamma-herpesvirus kinase actively initiates a DNA damage response by inducing phosphorylation of H2AX to foster viral replication. Cell Host Microbe 1:275–286 [PMC free article] [PubMed]
95. Taylor GM, Raghuwanshi SK, Rowe DT, Wadowsky RM, Rosendorff A. 2011. Endoplasmic reticulum stress causes EBV lytic replication. Blood 118:5528–5539 [PubMed]
96. Taylor N, Countryman J, Rooney C, Katz D, Miller G. 1989. Expression of the BZLF1 latency-disrupting gene differs in standard and defective Epstein-Barr viruses. J. Virol. 63:1721–1728 [PMC free article] [PubMed]
97. Tovey MG, Lenoir G, Begon-Lours J. 1978. Activation of latent Epstein-Barr virus by antibody to human IgM. Nature 276:270–272 [PubMed]
98. Tower C, et al. 2011. Human cytomegalovirus UL97 kinase prevents the deposition of mutant protein aggregates in cellular models of Huntington's disease and ataxia. Neurobiol. Dis. 41:11–22 [PubMed]
98a. Wang JT, et al. 2009. Epstein-Barr virus BGLF4 kinase suppresses the interferon regulatory factor 3 signaling pathway. J. Virol. 83:1856–1869 [PMC free article] [PubMed]
99. Wang JT, et al. 2005. Detection of Epstein-Barr virus BGLF4 protein kinase in virus replication compartments and virus particles. J. Gen. Virol. 86:3215–3225 [PubMed]
100. Wang L, et al. 2011. Disruption of PML nuclear bodies is mediated by ORF61 SUMO-interacting motifs and required for varicella-zoster virus pathogenesis in skin. PLoS Pathog. 7:e1002157. [PMC free article] [PubMed]
101. Wang YE, Pernet O, Lee B. 2011. Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers. Biol. Cell. 104:121–138 [PubMed]
102. Weitzman MD, Lilley CE, Chaurushiya MS. 2011. Changing the ubiquitin landscape during viral manipulation of the DNA damage response. FEBS Lett. 585:2897–2906 [PMC free article] [PubMed]
103. Wimmer P, Schreiner S, Dobner T. 2012. Human pathogens and the host cell SUMOylation system. J. Virol. 86:642–654 [PMC free article] [PubMed]
104. Yang SH, Jaffray E, Hay RT, Sharrocks AD. 2003. Dynamic interplay of the SUMO and ERK pathways in regulating Elk-1 transcriptional activity. Mol. Cell 12:63–74 [PubMed]
105. Young LS, Rickinson AB. 2004. Epstein-Barr virus: 40 years on. Nat. Rev. Cancer 4:757–768 [PubMed]
106. Yue W, Gershburg E, Pagano JS. 2005. Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter. J. Virol. 79:5880–5885 [PMC free article] [PubMed]
107. Zhang XD, et al. 2008. SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis. Mol. Cell 29:729–741 [PMC free article] [PubMed]
108. Zhu J, et al. 2009. Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4. J. Virol. 83:5219–5231 [PMC free article] [PubMed]
109. Zhu S, et al. 2009. Protection from isopeptidase-mediated deconjugation regulates paralog-selective sumoylation of RanGAP1. Mol. Cell 33:570–580 [PMC free article] [PubMed]
110. zur Hausen H, O'Neill FJ, Freese UK, Hecker E. 1978. Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature 272:373–375 [PubMed]

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