In Vivo Dynamics of EBNA1-oriP Interaction during Latent and Lytic Replication of Epstein-Barr Virus

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.The Epstein-Barr virus (EBV) 1 is a human herpes virus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing their continuous proliferation. In the B lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles, this being called latent infection. In the latent state, EBV maintains its 170-kbp genome as complete, multiple copies of plasmids. Latent phase viral replication appears to faithfully mimic cellular replicons; the EBV genomes or small EBNA1-oriP plasmids are synthesized only once in each Sphase by the host cell replication machinery, following the rules of chromosome replication (1).EBV-infected cell lines usually contain a small subpopulation of cells that have switched spontaneously from a latent stage of infection into the lytic cycle. The mechanism of switching is not fully understood, but one of the first detectable changes is expression of the BZLF1 gene product. The BZLF1 protein, together with the protein product of the BRLF1 gene, transactivates viral and certain cellular promoters (2) and leads to an ordered cascade of viral gene expression. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA polymerase; BMRF1, a polymerase processivity factor; BALF2, a single-stranded DNA-binding protein; and BBLF4, BSLF1, and BBLF2/3, predicted to be helicase-, primase-, and helicase-primase-associated proteins, respectively (3). All of these proteins except for BZLF1 conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome (4). Interestingly, viral lytic replication occurs in discrete sites in nuclei called replication compartments in which...

Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids

“…Immunofluorescence analysis with an anti-HA antibody revealed that HA-tagged EBNA1 localized to discrete, intranuclear punctate dots, which colocalized with FISH signals of EBV genomes. This punctate localization pattern of EBNA1 corresponded well with the localization of EBNA1 observed in latently infected B95-8 cells (Daikoku et al, 2004). Others have observed a diffuse pattern of EBNA1 localization (in interphase nuclei or on mitotic chromosomes) in other latently infected cells (Petti et al, 1990), and in cells overexpressing EBNA1 (Hung et al, 2001;Ito et al, 2002;Kanda et al, 2001a;Marechal et al, 1999).…”

Mitotic chromosome interactions of Epstein-Barr nuclear antigen 1 (EBNA1) and human EBNA1-binding protein 2 (EBP2)

“…Using biochemical fractionation, Kanda and colleagues (Kanda et al, 2001) observed that EBNA1 from interphase cells sedimented with the chromosomal pellet and was released by micrococcal nuclease, which is consistent with interaction of EBNA1 with chromosomes in interphase. By contrast, biochemical fractionation approaches used by Daikoku, Ritzi and colleagues (Daikoku et al, 2004;Ritzi et al, 2003) found little EBNA1 from interphase cells in the chromosomal pellet. Such divergent results might be due to the different buffer conditions used in each study.…”

“…For example, biochemical fractionation performed by Kanda and coworkers (Kanda et al, 2001) support the conclusion that EBNA1 is bound to chromatin, whereas those performed by Daikoku and colleagues (Daikoku et al, 2004) indicate that EBNA1 is largely soluble. However, these experiments were performed using different buffer conditions (salt and detergent concentrations), which might account for this discrepancy.…”

“…The supernatant (sample S) was removed and the pellet was resuspended in 50 l isotonic buffer (sample P). Where indicated, isotonic buffer was substituted with modified CSK buffer (mCSK: 10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl 2 , I mM EGTA, 1 mM DTT, 1 mM PMSF, 0.5% Triton X-100) (Daikoku et al, 2004) or hypotonic sucrose buffer (HSB: 10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.1% Triton X-100, protease inhibitors) (Kanda et al, 2001). Aliquots of 25 l of each of W, S and P samples were subjected to SDS-PAGE and western blotting using OT1x monoclonal antibody against EBNA1 and goat anti-mouse antibody conjugated with HRP (Santa Cruz Biotechnology).…”

“…In addition, it remains controversial as to whether or not EBNA1 binds chromosomes in interphase (Daikoku et al, 2004;Ito et al, 2002;Kanda et al, 2007;Kanda et al, 2001;Ritzi et al, 2003). In this study, we address the timing of the association of EBP2 and EBNA1 with host chromosomes through mitosis to try to understand how EBP2 contributes to EBNA1-mediated segregation and how relocalization of EBP2 from the nucleolus compares with that of other nucleolar proteins.…”