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Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.

Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Reactivation of the Epstein-Barr virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found here that Transducer of Regulated cAMP-response Element-binding Protein (CREB) (TORC) 2 enhances Zp activity 10-fold and more than 100-fold with co-expression of the BZLF1 protein. Mutational analysis of Zp revealed that the activation by TORC is dependent on ZII and ZIII cis elements, binding sites for CREB family transcriptional factors and the BZLF1 protein, respectively. Immunoprecipitation, chromatin immunoprecipitation, and reporter assay using Gal4-luc and Gal4BD-BZLF1 fusion protein indicate that TORC2 interacts with BZLF1, and that the complex is efficiently recruited onto Zp. These observations clearly indicate that TORC2 activates the promoter through interaction with the BZLF1 protein as well as CREB family transcriptional factors. Induction of the lytic replication resulted in the translocation of TORC2 from cytoplasm to viral replication compartments in nuclei, and furthermore, activation of Zp by TORC2 was augmented by calcium-regulated phosphatase, calcineurin. Silencing of endogenous TORC2 gene expression by RNA interference decreased the levels of the BZLF1 protein in response to 12-O-tetradecanoylphorbol-13-acetate/ionophore. Based on these results, we conclude that Epstein-Barr virus exploits the calcineurin-TORC signaling pathway through interactions between TORC and the BZLF1 protein in reactivation from latency.

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.

The mismatch repair (MMR) system, highly conserved throughout evolution, corrects nucleotide mispairing that arise during cellular DNA replication. We report here that proliferating cell nuclear antigen (PCNA), the clamp loader complex (RF-C), and a series of MMR proteins like MSH-2, MSH-6, MLH1, and hPSM2 can be assembled to Epstein-Barr virus replication compartments, the sites of viral DNA synthesis. Levels of the DNA-bound form of PCNA increased with progression of viral productive replication. Bromodeoxyuridine-labeled chromatin immunodepletion analyses confirmed that PCNA is loaded onto newly synthesized viral DNA as well as BALF2 and BMRF1 viral proteins during lytic replication. Furthermore, the anti-PCNA, -MSH2, -MSH3, or -MSH6 antibodies could immunoprecipitate BMRF1 replication protein probably via the viral DNA genome. PCNA loading might trigger transfer of a series of host MMR proteins to the sites of viral DNA synthesis. The MMR factors might function for the repair of mismatches that arise during viral replication or act to inhibit recombination between moderately divergent (homologous) sequences.

When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication.

We determined cytotoxic T lymphocyte (CTL) epitopes through screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro–reactivated polyclonal Epstein-Barr virus (EBV)–specific CD8+ T cells as responders. In addition, to confirm that the epitopes were generated after endogenous processing and presentation of the EBV proteins, a novel T-cell receptor (TCR) down-regulation assay was introduced, in which a fluorescent tetrameric major histocompatibility complex (MHC)/peptide complex was employed for detecting TCR down-regulation after stimulation with the epitope presented on antigen-presenting cells. Through such screening, 3 HLA A*2402–restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24–positive fibroblast cells infected with recombinant vaccinia viruses expressing LMP2. Furthermore, ELISPOT assays with an epitope-specific CTL clone demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with interferon-γ. The epitope was presented on transporters associated with antigen processing (TAP)–negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating that the presentation is TAP independent. In conclusion, the 3 epitopes thus defined could be useful for studying EBV-specific CD8+ T-cell responses among populations positive for HLA A*2402.

p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection., Author Summary Inhibition of p53-mediated transactivation is essential for regulating the cellular environment advantageous for viral infection. Specially, DNA viruses target p53 for inactivation through the ubiquitin-proteasome pathway. The E6 protein of the high-risk human papillomaviruses and the cellular ubiquitin-protein ligase E6AP form a complex which causes ubiquitination and degradation of p53. The adenovirus E1B 55-kDa protein binds to both p53 and E4orf6, and recruits a Cullin-containing complex to direct the ubiquitin-mediated proteolysis of p53. However, in comparison with the effects of the smaller DNA viruses, much less is known regarding the precise mechanisms whereby the Epstein-Barr virus (EBV) inhibits functions of p53. EBV possesses two alternative life cycles, latent and lytic replication. In latent phase, p53 is regulated by MDM2 ubiquitin ligase while after induction of lytic replication p53 is phosphorylated and the level of activated p53 is regulated by a novel system independent of MDM2. This report describes a unique functional role of the BZLF1 protein encoded by EBV in the modulation of activated p53. In this pathway, BZLF1 protein serves as an adaptor molecule for both Cul2- and Cul5-containing E3 ubiquitin ligase complexes to stimulate the ubiquitination and degradation of p53 for inhibiting apoptosis, indicating redundancy in the EBV machinery to downregulate p53 level. Therefore, it would be possible that the complexes regulate not only p53 but also various proteins that interact with BZLF1 protein.

The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

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) can choose between two alternative lifestyles; latent or lytic replication. In the latent state, the EBV genomic DNA, which exists as a closed circular plasmid, appears to behave just like host chromosomal DNA and it has been demonstrated recently that replication of OriP-containing plasmids is indeed dependent on the chromosomal initiation factors, ORC2 and Cdt1. On the other hand, in the viral productive cycle, the EBV genome is amplified 100- to 1000-fold by the viral replication machinery. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments and the lytic programme arrests cell cycle progression and changes the cellular environment greatly. It has been revealed recently that the EBV lytic programme promotes an S-phase like cellular condition, which most favours viral lytic replication. This review describes recent advances regarding the molecular basis of EBV DNA replication during latent and lytic infections and then refers to cellular circumstances after induction of the lytic replication of EBV. Based on the molecular mechanism for the EBV lifestyle, purposeful induction of the lytic form of EBV infection is now advocated as one of the strategies for specific destruction of Epstein-Barr virus (EBV)-associated malignancies where the virus is latently infected.

The induction of lytic replication of the Epstein-Barr virus (EBV) completely arrests cell cycle progression, in spite of elevation of S-phase cyclin-dependent kinase (CDK) activity, thereby causing accumulation of hyperphosphorylated forms of retinoblastoma (Rb) protein (A. Kudoh, M. Fujita, T. Kiyono, K. Kuzushima, Y. Sugaya, S. Izuta, Y. Nishiyama, and T. Tsurumi, J. Virol.77:851-861, 2003). Thus, the EBV lytic program appears to promote specific cell cycle-associated activity involved in the progression from G1to S phase. We have proposed that this provides a cellular environment that is advantageous for EBV productive infection. Purvalanol A and roscovitine, inhibitors of S-phase CDKs, blocked the viral lytic replication when cells were treated at the early stage of lytic infection, while well-characterized inhibitors of enzymes, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C, known to be involved in BZLF1 gene expression did not. Inhibition of CDK activity resulted in the accumulation of the hypophosphorylated form of Rb protein and inhibition of expression of EBV immediate-early and early proteins. Cycloheximide block-and-release experiments clearly demonstrated that even in the presence of enough amounts of the BZLF1 protein, purvalanol A blocked expression of lytic viral proteins at transcription level. Furthermore, reporter gene experiments confirmed that BZLF1-induced activation of early EBV promoters was impaired in the presence of the CDK inhibitor. We conclude here that the EBV lytic program promotes specific cell cycle-associated activity involved in the progression from G1to S phase because the S-phase-like cellular environment is essential for the expression of immediate-early and early genes supplying the viral replication proteins and hence for lytic viral replication.

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G 0 /G 1 by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21 WAF-1/CIP-1 and p27 KIP-1 , followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21 WAF-1/CIP-1 , and p27 KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G 1 /S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G 1 to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

The Epstein–Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.