Binding of EBNA-1 to DNA creates a protease-resistant domain that encompasses the DNA recognition and dimerization functions.

The Epstein-Barr virus nuclear antigen EBNA-1 is essential for replication of the viral DNA during latency. EBNA-1 binds as a dimer to palindromic recognition sequences within the plasmid origin of replication, ori-P. In this study, proteinase K susceptibility has been used to further characterize the DNA-binding domain of EBNA-1. Limited protease digestion of EBNA-1 (amino acids 408 to 641) generated a smaller DNA-binding species that had a degree of inherent protease resistance. When EBNA-1 was preincubated with a specific DNA probe, the protease resistance of the smaller binding species increased 100-fold, suggesting that the conformation of EBNA-1 changes on binding. The protease-resistant species comprised an 18-kDa polypeptide that was further cleaved at high levels of protease to 11- and 5.4-kDa products. A model of the proposed protease-resistant domain structure is presented. Constructions carrying serial, internal deletions across the 18-kDa domain were created. Each of the deletions perturbed dimerization ability and abolished DNA binding. These studies suggest that the DNA-binding and dimerization motifs of EBNA-1 lie within a conformationally discrete domain whose overall integrity is necessary for EBNA-1-DNA interaction.     

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.     

Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.     

The Epstein-Barr virus glycoprotein 110 carboxy-terminal tail domain is essential for lytic virus replication.

To investigate the importance of the Epstein-Barr virus (EBV) glycoprotein 110 (gp110) tail domain in the intracellular localization of gp110 and virus lytic replication, three carboxy-terminal truncation mutants of gp110 were constructed. Deletion of 16 amino acids from the carboxyl-terminal tail resulted in gp110 intracellular localization which was indistinguishable from that of wild-type gp110, whereas deletion of either 41 or 56 amino acids from the carboxyl-terminal tail of gp110 resulted in loss of retention of gp110 in the endoplasmic reticulum and nuclear membrane. None of the gp110 truncation mutants was able to complement EBV(gp110-)+ lymphoblastoid cell lines in transformation assays, indicating the importance of the gp110 tail domain in virus lytic replication. In electron microscopy analysis, no nucleocapsids or enveloped viruses were detected in EBV(gp110-)+ lymphoblastoid cell lines induced for lytic replication.     

Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.     

Nuclear localization of the PEP protein tyrosine phosphatase.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.     

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure.

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.     

Prediction and demonstration of a novel Epstein-Barr virus nuclear antigen.

The protein sequence predicted by the Epstein Barr virus (EBV) BERF4 open reading frame includes a tetrapeptide, Lys-Arg-Pro-Arg (KRPR), shown for other proteins to be a component of a signal for rapid nuclear localization. A subgenomic fragment of EBV DNA containing BERF4 has been incorporated into an expression vector, transfected onto primate cells and the nuclear distribution of the resulting protein established by immunofluorescence using EBV positive human sera. These sera contained high titres of antibodies to a fusion protein, produced in E. coli, consisting of beta-galactosidase and the C-terminal 167 amino acids of BERF4. Immunoaffinity purified antibodies reactive with the EBV component of the fusion show the molecular weight of this antigen in EBV immortalized B-cell lines to be about 160 kD. The demonstration that BERF4 contains an exon encoding a nuclear protein identifies a new EBNA gene (EBNA-6) and suggests that KRPR is a signal sequence common to a number of viral and cellular nuclear polypeptides which bind to nucleic acids and may therefore be of predictive value in identifying karyophilic proteins.     

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.