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Comparative analysis of the Epstein-Barr virus encoded nuclear proteinsof EBNA-3 family

Surya Pavan Yenamandraa,b, Ramakrishna Sompallaec, George Kleina, Elena Kashubaa,b,∗aDepartment of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Box 280, S-17177 Stockholm, SwedenbIRIS (Center for Integrative Recognition in the Immune System), Karolinska Institutet, S-17177 Stockholm, SwedencDepartment of Cellular and Molecular Biology (CMB), Karolinska Institutet, Box 280, S-17177 Stockholm, Sweden

A R T I C L E I N F O A B S T R A C T

Article history:Received 5 February 2009Accepted 18 August 2009

Keywords:EBNA3 family proteinsSecondary structure analysisStonin homology domainPraline rich domainCharge clusters

It is known that the EBNA-3 family proteins (EBNA-3, -4 and -6, alternative nomenclature EBNA-3A, Band C correspondingly) show a limited sequence similarity.We have analyzed EBNA-3 proteins both at the primary sequence and secondary structure levels. EBNA-3and EBNA-4 were structurally more similar compared to other combinations with EBNA-6. We found“Stonin Homology Domain” profile in EBNA-4 and “Proline Rich Domain” in all EBNA-3 family of proteins.We have also found positive and negative charge clusters in all three proteins and mixed charge clustersin EBNA-3. Charged clusters are believed to play an important role in interactions with DNA or signalingproteins. Additionally, unique primary sequence repeats were found in all three proteins.

© 2009 Elsevier Ltd. All rights reserved.

1. Background

Epstein-Barr virus (EBV, HHV4) is a member of the human her-pesvirus family that consists of large DNA viruses. It is a lymphotropicgamma-herpesvirus that infects more than 90% of all human pop-ulations. EBV can cause infectious mononucleosis (IM) and B-celllymphomas in immunosuppressed hosts. It is one of the most highlytransforming viruses known [1]. Human B-cells are its natural hostcells. In the EBV-immortalized lymphoblastoid cells the virus ex-presses six nuclear proteins (EBNA1-6) and three latent membraneproteins (LMP1, LMP2A and B). This is referred as the latency III pro-gram. Six of the growth transformation associated proteins, EBNA-1,-2, -3, -5, -6 and LMP1 are essential for transformation (for reviewsee [2]). The mechanism of transformation is not well understood,but it is clear that the virus exploits the normal signaling pathwaysof the B-lymphocyte. The proteins expressed in latency III hijack thenormal B-cell growth pathways by mimicking constitutive growthpromoting and anti-apoptotic signals.

EBNA-3, EBNA-4 and EBNA-6 (EBNA-3A, -3B and -3C accordingto the alternative nomenclature) belong to the EBNA-3 family ofproteins. They are encoded by tandemly arranged genes and have a

∗ Corresponding author at: IRIS (Center for Integrative Recognition in the ImmuneSystem), Karolinska Institute, S-17177 Stockholm, Sweden. Tel.: +46852486259;fax: +468330498.

E-mail address: Elena.Kashuba@ki.se (E. Kashuba).

0010-4825/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.compbiomed.2009.08.006

similar genomic organization (reviewed in [2] and [3]). They containshort intron sequences that may be retained in the mRNA. The levelof the three proteins is differently regulated by cellular factors [4]. Allthree EBNA-3 family proteins associate with RBP-J� (NP_005340.2,Recombination signal binding protein for immunoglobulin kappa Jregion). This indicates that they may be involved in transcriptionalregulation [5–8]. They are expressed exclusively in immunoblastsof B-cell origin and bind to RBP-2N, the major isoform of RBP-J� inB-lymphocytes [9]. EBNA-6, EBNA-3 and EBNA-4 can also activatetranscription from the LMP1 promoter in vitro in the presence ofEBNA-2 [8,10]. EBNA-6 has been most extensively studied amongthe EBNA-3 family proteins. It associates with the co-repressorsCtBP (NP_001319.1, binding protein to the C-terminal half of E1A)[11], mSin3A (NP_001138829.1, homolog to the yeast SIN3), andNcoR (O75376, nuclear receptor co-repressor) [12]. This may ex-plain EBNA-6 ability to activate or to repress transcription from theLMP1 promoter in the presence of EBNA-2 [13–16] or in its absence[5,17,18]. Homologs of EBNA-6 encoded by the baboon and rhesusrelatives of EBV (BaLCV and RhLCV) were shown to bind RBP-J�and Spi1 (NP_001074016.1, spleen focus forming virus (SFFV) provi-ral integration oncogene, or PU.1) [19]. When bound to RBP-J�, theEBNA-6 homologs of both viruses could repress EBNA-2-mediatedtranscription in vitro and through Spi1 binding it activates EBNA-2-mediated transcription [19].

EBNA-3 can downregulate transcription of luciferase gene invitro, but a deletion mutant of EBNA-3 that lacked residues 100–364showed bifunctional activity: it repressed transcription at lowconcentration and activated at high concentration [20]. EBNA-3

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enhanced transcription of Arylhydrocarbon receptor (NP_001612)responsive genes; at a basal level of receptor and of receptor ac-tivated by a ligand (TCDD, for example) [21]. Binding of 3–5 foldoverexpressed EBNA-3 to RBJ-kappa in IB4 lymphoblastoid cell line(LCL) led to the donwregulation of c-myc (NP_002458.2), CD21(NP_001006659), CD23 (NP_001993.2) and to G0/G1 arrest [22].EBNA-3, however, for LCL proliferation is essential. In an LCL whereEBNA-3 was expressed from a 4-hydroxy-tamoxifen (HT) dependentpromoter, withdrawal of HT led to the loss of EBNA-3 expressionand subsequent growth arrest and cell death [23]. The level of c-mycwas unaltered in these cells.

EBNA-6 binds to histone deacetylases HDAC1 (NP_004955) andHDAC2 (NP_001518) [12,24], acetyltransferase p300 (NP_001420),and prothymosin alpha (NP_001092755.1) [25]. EBNA-6 was alsoshown to interact with the DEAD-box protein DP103 (NP_009135.3)[26] that has helicase activity [27] and the SMN1 (NP_000335.1,Survival of motor neurons) protein [28], a DP103 binding partner[29]. These reported interactions of EBNA-6 suggest that apart fromtranscription regulation it may also play an important role in chro-matin re-modeling. EBNA-6 is also believed to play a role in cellcycle control. Reportedly, it binds to pRb protein (NP_000312) invitro [30–32]. EBNA-6 transfection was found to decrease the levelof phosphorylated pRb and inhibit p27 protein (NP_004055) accu-mulation in primary rat fibroblasts [31]. The decrease of the p27level was later related to the finding that EBNA-6 binds cyclin A(NP_003905.1) [33,34] and the E3 ubiquitin ligase, Skp-Cullin-Fbox(SCF) complex [35]. It was proposed that the SCF complex is in-volved in the EBNA-6 coefficient degradation of pRb [32] and p27as well [35]. In LCL EBNA-6 is not responsible for the degrada-tion of pRb, however [36]. The level of total pRb and p27 did notchange upon inactivation of EBNA-6 in an LCL that carried a con-ditionally active (hydroxytamoxifen dependent) EBNA-6. The por-tion of lymphoblastoid cells in S and G2/M phases were reducedwithin one week after the inactivation of EBNA-6. In parallel, therewas an increase of p16 expression and a decrease in pRb phos-phorylation. We have shown recently that due to interaction withEBNA6, MRPS18-2 (NP_054765), a pRb binding protein, may in-hibit the association of pRb with E2F1 (NP_005216) competitivelyand thereby facilitate the entry of EBV infected B-cells into the Sphase [37].

Role of EBNA-3 proteins in the control of G2/M checkpointwas also indicated [38]. Binding of EBNA-3 to checkpoint kinasechk2/cds1 (NP_001005736) was shown. It was hypothesized thatthis binding could be responsible for G2/M checkpoint disruption.As discussed above, EBNA-3 family proteins exhibit partly similarfunctions, but they have distinct properties as well and they havedifferent binding partner spectra.

In this study we demonstrate that similarities on the secondarystructure to protein members of EBNA-3 family could possibly ac-counts to a correlation to their functions. Multiple sequence align-ment of the three proteins at the primary sequence level showedabout 30% similarity. This prompted us to compare for the first timethe secondary structure of EBNA-3, -4 and -6. Global and local align-ments were performed to explore novel motifs and other significantpatterns both at the primary and secondary structure levels of pro-teins.

2. Results

2.1. Secondary structure alignments of EBNA-3, EBNA-4 and EBNA-6

The global alignment scores that indicate the percentage similar-ity in secondary structure elements were 88.30, 71.52 and 71.81 forthe pairs EBNA-3/4, EBNA-3/6 and EBNA-4/6, respectively.

Fig. 1. (A) Secondary structure comparison between EBNA-3 and EBNA-4. The boxesrepresent the Helix-Coil-Helix (272–330 of EBNA3 and 275–348 of EBNA4) andStrand–Strand-Helix (residues 192–243 of EBNA-3 and 198–248 of EBNA-4) motifs.(B) The Blast alignment of the primary sequence of EBNA-3 (residues 104–337) andEBNA-4 (residues 110–348).

2.1.1. Local secondary structure comparison of EBNA-3 and EBNA-4The local alignment of the secondary structure elements con-

firmed that EBNA-3 and -4 are structurally more similar. The ob-tained pairwise local secondary structure alignment scores betweenEBNA-3 and -4, EBNA-3 and -6, and EBNA-4 and -6 were 84.5, 69,and 67, respectively. Fig. 1A shows the pair wise structural similaritybetween EBNA-3 and -4.

Position of amino acid from 98 to 330 of EBNA-3 and 106 to 341of EBNA-4 showed strong pairwise secondary structure similarities.But the rest of the region is followed by either random coil or loopregion in both the sequences.

Moreover, the blast alignment (Blast version 2.2.15) of primarysequence within the above specified regions of EBNA-3 and -4showed about 44% similarity (Fig. 1B).

A strong structural pattern of HELIX-COIL-HELIX (272–330of EBNA3 and 275–348 of EBNA4) and STRAND–STRAND-HELIX(residues 192–243 of EBNA-3 and 198–248 of EBNA-4) were ob-served.

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Fig. 2. (A) Secondary structure comparison between EBNA-3 and EBNA-6. The boxesrepresent the motifs Helix-Coil-Helix residues starting from 272–330 of EBNA3 and281–359 of EBNA6) and STRAND–STRAND-HELIX (residues 192–243 of EBNA-3 and201–254 of EBNA6). (B) The Blast alignment of the primary sequence of EBNA-3(residues 98–341) and EBNA-6 (residues 105–359).

2.1.2. Local secondary structure comparison of EBNA-3 and EBNA-6The local pairwise secondary structure alignment between EBNA-

3 and EBNA-6 showed that the positions 98–341 of EBNA-3 and105–359 of EBNA-6 had strong structural similarities with observedpatterns of HELIX-COIL-HELIX (residues starting from 272–330 ofEBNA3 and 281–359 of EBNA6) , STRAND–STRAND-HELIX (residues192–243 of EBNA-3 and 201–254 of EBNA6) motifs (Fig. 2A). Restof the region is followed by loop/coil structure in both EBNA-3 andEBNA-4 proteins compared.

The pairwise local alignment of primary sequence within thespecified positions of EBNA-3 and -6 showed about 38% of similarity(Fig. 2B).

2.1.3. Local secondary structure comparison of EBNA-4 and EBNA-6EBNA-4 and EBNA-6 showed local structural pairwise similari-

ties at the positions 106–341 of EBNA-4 and 105–349 of EBNA-6

Fig. 3. (A) Comparison of the secondary structures of EBNA-4 and EBNA-6 proteins.The boxes show HELIX-COIL-HELIX (residues 275–348 of EBNA4 and 281–359 ofEBNA6) and STRAND–STRAND-HELIX (198–248 of EBNA-4 and 201–254 of EBNA6)observed in both proteins. (B) The Blast alignment of the primary sequence ofEBNA-4 (residues 99–474) and EBNA-6 (residues 102–497).

with patterns of HELIX-COIL-HELIX (residues 275–348 of EBNA4 and281–359 of EBNA6) and STRAND–STRAND-HELIX (198–248 of EBNA-4 and 201–254 of EBNA6) motifs (Fig. 3A). Loop/coil region followedin both the proteins compared.

The local pairwise alignment of the primary sequences at theabove specified positions showed about 38% similarity betweenEBNA-4 and -6 (Fig. 3B).

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Fig. 4. Multiple alignment of secondary structure of EBNA-3, EBNA-4 and EBNA-6.

2.2. Multiple alignments of EBNA-3, EBNA-4 and EBNA-6 at secondarystructure level

Apart from pairwise secondary structure alignments, we tried tovisualize multiple alignment of EBNA-3 family of proteins at sec-ondary structure level. The Fig. 4 shows clearly Helix-Coil-Helix andStrand–Strand-Helix motifs in EBNA-3, EBNA-4 and EBNA-6 proteins.

2.3. Disorder prediction of EBNA-3 family of proteins

DisEMBL was run for the EBNA-3 family of proteins and the re-sults shows predicted Loop and non-Loop patterns. The Fig. 5A–C areEBNA-3, -4, and -6 protein sequences respectively, showing variousdisorder regions predicted by loop/coil predictor definition. Fig. 5Ashows loop/coil definitions predicted for EBNA-3 protein. The boldregions (black) represent predicted loop/coil regions and regions inregular font are more ordered in the proteins. Within the orderedregions red color (underlined) represents helix structure predictedby PSIPRED; green color (underlined) represents strand structurepredicted by PSIPRED. Similar representation can be visualized forEBNA-4 (Fig. 5B) and EBNA-6 (Fig. 5C) proteins.

2.4. Significant patterns in the primary sequences of the EBNA-3 family

2.4.1. Putative domains in EBNA-3 family of proteinsA further analysis was done on EBNA-3 family of protein se-

quences to explore the unique patterns or domains using Myhitsserver (http://myhits.isb-sib.ch/cgi-bin/motif_scan). This search forall known Prosite patterns and profiles gave a number of interestinghits (see the Table 1). Observed in silico domains included “Stoninhomology domain profile” (E-values = 5.7) located at 665–829 po-sitions near C-terminal region of EBNA-4. One important feature

Fig. 5. (A) Disorder regions (loops/coils) predicted by loop/coil predictor. Numericalnumber range shows predicted coil regions in EBNA-3 protein. Label Red underlinedrepresent Helix structures predicted by PSIPRED. Label Green underlined repre-sent Strand structures predicted by PSIPRED. (B) Disorder regions (loops/coils) pre-dicted by loop/coil predictor. Numerical number range shows predicted coil regionsin EBNA-4 protein. Label Red underlined represent Helix structures predicted byPSIPRED. Label Green underlined represent Strand structures predicted by PSIPRED.(C) Disorder regions (loops/coils) predicted by loop/coil predictor. Numerical num-ber range shows predicted coil regions in EBNA-6 protein. Label Red underlinedrepresent Helix structures predicted by PSIPRED. Label Green underlined representStrand structures predicted by PSIPRED. For interpretation of the references to colorin this figure legend, the leader is referred to the web version of this article.

identified was the long proline rich region in all EBNA-3 family pro-teins. This may be functionally important. The specified position ofthese proline rich domains in EBNA-3 (residues 417–794), EBNA-4(residues 526–753) and EBNA-6 (residues 391–899) showed strongexpectation values of 4.6e-14, 2.1e-23 and 7e-21 respectively.

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Table 1Patterns identified in EBNA-3 family proteins.

Pattern/profile Protein E-value Position

Stonin homology domain profile EBNA-4 5.7 665–829Proline rich domains EBNA-3, -4 and -6 4.6e-14, 2.1e-23 and 7e-21 417–794, 526–753 and 391–899

Table 2Novel patterns from SAPS analysis.

Property T-value Position

Positive charge clustersEBNA-3 5.25 376–402EBNA-4 4.93 152–185EBNA-6 a 78–84Negative charge clusterEBNA-3 4.05 and 5.89 16–60, 343–369EBNA-4 4.81 and 8.34 25–93, 355–378EBNA-6 4.48 367–393Mixed charge clusterEBNA-3 4.89 341–414

aEBNA-6 contains stretch of polyarginine repeat.

Fig. 6. Patterns and sequence elements present in EBNA-3, -4 and -6 proteins.Similar arrows represent the repetitive primary sequence structures of amino acids.Note: Figures are not scaled.

2.4.2. Primary sequence repeats and charge clusters in the EBNA-3family

We found charge clusters and repetitive structures of amino acidsin EBNA-3, -4 and -6 protein sequences, using SAPSmethod (Table 2).Fig. 6 depicts the presence of the positive charge clusters at positions376–402 in EBNA-3 (t-value: 5.25) and at positions 152–185 in EBNA-

4 (t-value: 4.93). Similarly, negative charge clusters were found atpositions 16–60 (t-value: 4.05) and at 343–369 (t-value: 5.89) inEBNA-3 protein sequence. Clusters of negatively charged regions arealso present in EBNA-4 at positions from 25 to 93 (t-value: 4.81)and from 355 to 378 (t-value: 8.34). The EBNA-6 primary sequencehas negatively charged clusters at positions 367–393 (t-value: 4.48).Mixed charge clusters were observed in EBNA-3 from positions 341to 414 (t-value: 4.89). Fig. 6 shows the existence of protein repeatsat the primary level of the EBNA-3 family. A unique polyArgininerepeats were located in EBNA-6 at the position 78–84 residues.

In addition to above findings, we also observed amino acid repeatsin EBNA3 family. Table 3 shows interesting repeats (both short andlong stretches of amino acid repeats) observed in EBNA3, EBNA4 andEBNA6. BLAST search against observed charged clusters and aminoacid repeats (Fig. 6) showed no significant hits to the non-redundantprotein database. All observed patterns were unique with no signifi-cant homology to any of the proteins in the non redundant database.The function of these unique patterns of charge clusters and repeti-tive structures has to be characterized further.

3. Discussion

3.1. Structural aspects of EBNA-3 family

Although multiple alignments or pairwise alignments work wellat the single base (residue) level they often fail to catch the under-lying structure. If the two proteins are evolutionarily distinct, theymay have diverged long ago and have undergone numerous mu-tations. As a consequence, secondary structure may be conservedwhile the primary sequences are less similar. EBNA-3, -4 and -6 areless similar at the primary sequence than at the secondary struc-ture level (Fig. 1A, 2A and 3A). Predicted secondary structures of allEBNA-3 proteins are rich in helices and contain at least two shortbeta stretches. However, their positions are shifted giving rise to lowscores in pairwise alignments.

Based on secondary structure pairwise and multiple alignmentsit is evident that EBNA-3, -4 and -6 are structurally similar at N-terminal region (Fig. 4). A strong supportive evidence for the sec-ondary structure similarities among EBNA-3 family of proteins canbe further obtained from the analysis of sequence disorder in theseproteins. Observations show that the helix or strand structures pre-dicted by secondary structure method (PSIPRED) were quoted as or-dered region by loop/coil predictor (colored red for helix and greenfor strand in Fig. 5A–C). The coil regions predicted by secondarystructure method for EBNA-3 family of proteins were quoted as dis-ordered regions by loop/coil predictor.

3.2. Tertiary structure of EBNA-3 family proteins

Based on secondary structure homology of EBNA-3, -4 and -6 wecan speculate that they may have alike structure, as the positionsof the alpha and beta chains are very similar (though not exactlyidentical). Short gaps between the regular structures could be ig-nored and long gaps probably take up a random coil structure whoselength does not matter. From the above analysis we expect the 3-Dstructures (in the ribbon model) of these proteins to be very similar.

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Table 3Aminoacid repeats in EBNA-3 family proteins.

Protein Position range Sequence repeated

EBNA-3 702−747, 755−800 QPQYFDIPLTEPINQGASAAHFLPQQPMEGPLVPEQWMFPGAALSQEBNA-3 667−683, 702−718, 755−771 QPQYFDLPLIQPISQGAEBNA-3 631−653, 725−747 PQQPMEGPLVPEQQMFPGAPFSQEBNA-3 634−653, 728−747, 781−800 PMEGPLVPEQQMFPGAPFSQEBNA-3 634−658, 781−805 PMEGPLVPEQQMFPGAPFSQVADVVEBNA-3 862−870, 891−899 LDLSIHGRPEBNA-3 875−885, 904−914 PEWPVQEEGGQEBNA-4 713−732, 733−752 VPPVPRQRPRGAPTPTPPPQEBNA-6 569−578, 579−588, 589−598, 599−600 GPPAAGPPAAEBNA-6 745−757, 758−770, 771−783, 784−784 PAPQAPYQGYQEP

Table 4EBNA-3 family binding proteins.

EBNA Interacting proteins

EBNA-3 RBP-J�; RBP-2N; CtBP; XAP2; �-subunit of TCP-1; UCKL1; AhR (DR); chk2/cds1 kinaseEBNA-4 RBP-J�; RBP-2NEBNA-6 RBP-J�; RBP-2N; DP103; ProT-�; SMN; NM23-H1; HDAC1; CtBP; Spi-1 (PU.1), Spi-B; Skp2; Roc-1; p50 of NF�B; c-fos; Sp1; Cycline A; S18-2 (pRb binding protein)

3.3. Significance of charge clusters and amino acid repeats

Based on previous studies, it is evident that G-protein activatorsand DNA binding proteins contain positive charge clusters in theirprotein sequences [39]. The negative charge clusters were observedin various transcription factors like c-myc, pRb and few other viralproteins (Varicella-zoster virus p62 and p63; herpes simplex virusICPO, ICP22, and ICP27 (but not VP16); polyoma virus middle-sizedtumor (T) antigen; papilloma virus E1 and E7 [39]. Mixed charge clus-ters consisting of both positive and negative charged regions havesignificant role in interacting with either DNA or other signaling pro-teins and plays crucial role in regulating various cellular processes[39]. We could expect similar kind of regulatory role for EBNA-3 fam-ily of proteins as these proteins contain strong clusters of positiveand negative charge residues along with mixed charge regions. Pro-line rich region is another interesting observation in these proteins.

Indeed, it was found that triplet repeats (poly Gly, Ser or Ala) ofamino acids play a strong role in transactivation function and dis-ruption of repeats leads to loss in function of hormone binding, nu-clear translocation and specific DNA binding [40,41]. As we discussedearlier, one of the major functions of EBNA-3 family proteins is theregulation of transcription. Similar functional studies are required toanalyze repeats observed in EBNA3 family of proteins.

Open question is whether EBNA-3 family proteins can bind di-rectly to DNA or not. Early reports gave a possibility to predict directprotein-DNA binding [42]. However, later on this hypothesis was ex-cluded from a consideration due to binding partners of EBNA-3 fam-ily proteins that could bind DNA directly (see the Table 4). We thinkthis hypothesis should be tested by new experimental methods, aschromatin immunoprecipitation (CHIP-assay) or new DNA bindingassay in vivo from Clontech. The presence of leucine zipper in EBNA-6 sequence [43,44] is an additional argument in favor of this idea.

Process of malignant B-cell transformation, induced by EBV, is avaluable model of cell transformation in general. Knowing the setof cellular proteins interacting with EBNAs enables to predict thecellular pathways that were used by virus during the transformation.Secondary structure prediction allows us to setup the new directionsof functional studies, concerning EBNA-3 family protein functions.

In conclusion, three EBNA-3 family proteins are broadly similarin their secondary structure. EBNA-3 and -4 are more similar while-6 is somewhat different (Fig. 6). Because of the considerable lengthof the proline rich region of 6, scores are lower for EBNA-3/6 andEBNA-4/6 at secondary structure level.

4. Methods

Sequences of EBNA-3, -4 and -6, encoded by EBV strain B95-8 were obtained from SWISS-PROT database (accession numbersare [GenBank: P12977, P03203 and P03204] correspondingly). Weimplemented the position specific iterative prediction (PSIPRED)method [45,46] for the secondary structures of proteins. We usedthe secondary structure element alignment (SSEA) method [47] toanalyze the structurally similar regions (both from global and localalignments) with in predicted secondary elements of EBNA-3 familyprotein sequences.

BLAST alignment was performed using Blast version 2.2.15, onprimary sequences of EBNA-3 family proteins.

DisEMBL softwarewas used to analyze disorder regions in EBNA-3family of proteins [48] (http://dis.embl.de/). Loop predictor definition[48] was used to check the regions predicted as loops–coils.

To explore specific domains and motifs, we applied motif scan[49,50] (http://myhits.isb-sib.ch/cgi-bin/motif_scan) on the proteinprimary sequences using a prosite parameter profile. Further we ap-plied SAPS (statistical analysis of protein sequences) method [51]to explore novel patterns, charged residue clusters and primary se-quence repeats. The test statistic t-value (standard deviations abovethe mean; to accommodate the multiple tests performed, the t-valuesignificance threshold is set to 4.0 for sequences up to 750 residues,to 4.5 for sequences of length 750–1500 residues, and to 5.0 forlonger sequences); is set for the predicted charge clusters.

Conflict of interest statement

Authors declare no conflict of interest.

Acknowledgments

We thank professor Chanchal K Mitra and Dr. Tatyana Sandalovafor a fruitful discussion of this work. Swedish Institute, Cancer Foun-dation, a matching grant from the Concern Foundation, Los Angeles,and the Cancer Research Institute, New York supported this work.

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