EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in Saccharomyces cerevisiae and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in S. cerevisiae, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in S. cerevisiae can occur through chromosome attachment.     

Reconstitution of Epstein-Barr virus-based plasmid partitioning in budding yeast

The EBNA1 protein of Epstein-Barr virus (EBV) mediates the partitioning of EBV episomes and EBV-based plasmids during cell division by a mechanism that appears to involve binding to the cellular EBP2 protein on human chromosomes. We have investigated the ability of EBNA1 and the EBV segregation element (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae. EBNA1 expression alone did not enable the stable segregation of FR-containing plasmids in yeast, but segregation was rescued by human EBP2. The reconstituted segregation system required EBNA1, human EBP2 and the FR element, and functionally replaced a CEN element. An EBP2 binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning, indicating that an EBNA1-EBP2 interaction is required. The results provide direct evidence of the role of hEBP2 in EBNA1-mediated segregation and demonstrate that heterologous segregation systems can be reconstituted in yeast.     

EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.     

The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes

During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.     

EBP2 plays a key role in Epstein-Barr virus mitotic segregation and is regulated by aurora family kinases

Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.     

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.     

Interaction between Basic Residues of Epstein-Barr Virus EBNA1 Protein and Cellular Chromatin Mediates Viral Plasmid Maintenance

The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 (amino acids 40–54) and CBD2 (amino acids 328–377) regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and -2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine-rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, whereas a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.     

Identification of a Novel Protein Interaction Motif in the Regulatory Subunit of Casein Kinase 2

Casein kinase 2 (CK2) regulates multiple cellular processes and can promote oncogenesis. Interactions with the CK2β regulatory subunit of the enzyme target its catalytic subunit (CK2α or CK2α′) to specific substrates; however, little is known about the mechanisms by which these interactions occur. We previously showed that by binding CK2β, the Epstein-Barr virus (EBV) EBNA1 protein recruits CK2 to promyelocytic leukemia (PML) nuclear bodies, where increased CK2-mediated phosphorylation of PML proteins triggers their degradation. Here we have identified a KSSR motif near the dimerization interface of CK2β as forming part of a protein interaction pocket that mediates interaction with EBNA1. We show that the EBNA1-CK2β interaction is primed by phosphorylation of EBNA1 on S393 (within a polyserine region). This phosphoserine is critical for EBNA1-induced PML degradation but does not affect EBNA1 functions in EBV replication or segregation. Using comparative proteomics of wild-type (WT) and KSSR mutant CK2β, we identified an uncharacterized cellular protein, C18orf25/ARKL1, that also binds CK2β through the KSSR motif and show that this involves a polyserine sequence resembling the CK2β binding sequence in EBNA1. Therefore, we have identified a new mechanism of CK2 interaction used by viral and cellular proteins.     

Epstein-Barr virus nuclear antigen 1 replication and segregation functions in nasopharyngeal carcinoma cell lines

Nasopharyngeal carcinomas (NPC) are usually Epstein-Barr virus (EBV) positive, but, with the exception of C666-1 cells, these cells lose the EBV genomes when grown in culture. Maintenance of EBV requires the viral EBV nuclear antigen 1 (EBNA1) protein, which ensures the replication and mitotic segregation of the genomes through interactions with OriP. Here we compare the abilities of C666-1 and NPC cells that have lost EBV genomes to replicate and segregate OriP plasmids. We found that either cell line can replicate and maintain OriP plasmids for extended periods under conditions where low levels of EBNA1 are expressed but that high EBNA1 levels selectively interfered with mitotic segregation.     

Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus.

RPA is the replicative single-strand DNA (ssDNA) binding protein of eukaryotic chromosomes. This report shows that human RPA interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus (EBV). RPA binds to EBNA1 both in solution, and when EBNA1 is bound to the EBV origin. RPA is a heterotrimer, and the main contact with EBNA1 is formed through the 70 kDa subunit of RPA, the subunit which binds to ssDNA. We propose that this interaction between RPA and EBNA1 is an early step in activation of the latent origin of EBV.     

Down-modulation of nucleoporin RanBP2/Nup358 impaired chromosomal alignment and induced mitotic catastrophe

Chromosomal missegregation is a common feature of many human tumors. Recent studies have indicated a link between nucleoporin RanBP2/Nup358 and chromosomal segregation during mitosis; however, the molecular details have yet to be fully established. Observed through live cell imaging and flow cytometry, here we show that RNA interference-mediated knockdown of RanBP2 induced G2/M phase arrest, metaphase catastrophe and mitotic cell death. Furthermore, RanBP2 down-modulation disrupted importin/karyopherin β1 as well as the expression and localization of the Ran GTPase activating protein 1. We found that N-terminal of RanBP2 interacted with the N-terminal of importin β1. Moreover, at least a portion of RanBP2 partially localizes at the centrosome during mitosis. Notably, we also found that GTPase Ran is also involved in the regulation of RanBP2–importin β1 interaction. Overall, our results suggest that mitotic arrest and the following cell death were caused by depletion of RanBP2. Our findings point to a crucial role for RanBP2 in proper mitotic progression and faithful chromosomal segregation.     

Neural Wiskott-Aldrich syndrome protein is required for accurate chromosome congression and segregation

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.