No abstract

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.

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.Epstein-Barr virus (EBV) is a human herpesvirus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing continuous proliferation. In B-lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles; this is called latent infection. In the latent state, EBV maintains its 170-kb genome as complete, multiple copies of plasmids. Latent-phase viral replication appears to faithfully mimic cellular replicons: EBV genomes or small oriP/EBNA-1 plasmids are synthesized only once in each S phase by the host cell replication machinery, following the rules of chromosome replication (28).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 (7) and leads to an ordered cascade of viral gene expression: activation of early gene expression followed by the lytic cascade of viral genome replicat...

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.

Human cytomegalovirus (HCMV), a member of the betaherpesvirus family, infects most individuals asymptomatically. The virus persists in CD34ϩ hematopoietic progenitor cells, monocytes, and CD34 ϩ -derived dendritic cells from healthy seropositive individuals (22,36,37). Reactivation of HCMV is related to a variety of diseases such as pneumonitis, hepatitis, and retinitis (7, 25). The mechanisms underlying maintenance of the latent viral genome and the switch from the latent to lytic forms of infection remain unclear.The host range of HCMV is narrow in cell culture. The virus replicates productively in terminally differentiated cells such as fibroblasts, epithelial and endothelial cells, and monocyte-derived macrophages (17,18,27,41,59,60,65). The major immediate-early (MIE) genes of HCMV play a key role in determining the efficiency of viral replication. IE1 (UL123) and IE2 (UL122) encode pIE72 and pIE86 proteins important for regulation of subsequent viral gene expression. The pIE72 protein contributes to efficient viral replication at low multiplicity of infection (MOI) (19,21). The IE86 protein is essential for early viral gene expression and autoregulates transcription of the IE1 and IE2 genes (12,43,47,56).The region upstream of the HCMV MIE promoter is divided into three regions: the modulator, the unique region, and the enhancer (52, 61). The modulator has no effect on MIE transcription and on viral replication in cell culture (51). The unique region also has no effect on transcription from the MIE promoter, but one or more cis-acting elements repress transcription from the divergent early viral UL127 promoter (39, 45). The enhancer is divided into distal and proximal components (50). Without the distal enhancer the recombinant virus replicates slowly and has a small-plaque phenotype in human fibroblast cells (50). Proximal and distal chimeras of the human and murine CMV enhancers replicate less efficiently at low MOIs and demonstrate the small-plaque phenotype (29). The entire enhancer region of human CMV is required for robust MIE gene expression.In a previous report, we demonstrated a direct correlation between the extent of the proximal enhancer and the amount of MIE gene transcription and the level of infectious virus replication (30). Deletion of the enhancer from nucleotide position Ϫ636 to Ϫ39 resulted in no replication in human foreskin fibroblasts (HFFs). In contrast, a recombinant virus with a deletion from position Ϫ636 to Ϫ67 replicated independently. This recombinant virus contained a single GC box upstream of the MIE promoter that binds the Sp1 transcription factor (30).The Sp1 family of transcription factors is composed of four proteins (Sp1, Sp2, Sp3, and Sp4) (64). In addition to a highly conserved DNA-binding domain, all four cellular proteins have glutamine-rich activation domains adjacent to serine/ threonine-rich stretches in their N-terminal regions. Sp1, Sp2, and Sp3 are ubiquitously expressed, but expression of Sp4 is limited to the brain (63). Sp1 and Sp3 both recognize GC-rich se...

A number of replication initiation sites that are present in the genome of eukaryotic cells are utilized in a temporal order during the DNA synthesis (S) phase of the cell cycle. Reinitiation of DNA replication is prevented, and only a single round of DNA replication is performed in a cell cycle. This DNA replication by the so-called replication licensing system is regulated by the loading of the minichromosome maintenance (MCM) complexes on chromatin DNA and their phosphorylation (37,50,51).During the G 1 phase of the cell cycle, replication origins in DNA are licensed by the assembly of prereplicative complexes (pre-RC) comprising the origin recognition complex (ORC), Cdc6, Cdt1, and the MCM complex (47, 51). The ORC binds to origins of DNA replication and remains bound during most of the cell cycle (30,40,48). Cdc6 and Cdt1 then bind to the complex and facilitate the loading of the MCM2-MCM7 (MCM2-7) complex. Cdt1 itself is regulated by geminin, which blocks the binding of the MCM complex to the pre-RC (39,46,54). Activation of the pre-RC occurs at the G 1 /S boundary after licensing and is mediated by the action of S-phase cyclin-dependent kinases (CDKs), primarily cyclin A/CDK2, cyclin E/CDK2, and Cdc7/Dbf4 (3, 10, 42, 45), which trigger a chain of reactions that lead to the binding of Cdc45 to the origin and phosphorylation of Cdc6 and the MCM complex. As a result, the DNA duplex unwinds, facilitating loading of the DNA polymerase machinery (24, 41, 52, 59). The phosphorylation of key components of this process by the CDKs leads to initiation of replication and at the same time helps to prevent rereplication during the S and G 2 /M phases of the cell cycle (6,7,23,55).All of the members of the MCM protein family contain highly conserved DNA-dependent ATPase motifs in the central domain (3, 44) and form several stable subassemblies, including 49,56). DNA helicase activity has been identified in the MCM4-6-7 complexes of human, mouse, and fission yeast (Schizosaccharomyces pombe) (19,35,36,56), while MCM2 and MCM3-5 are known to inhibit this activity by converting the double-trimer structure into a heterotetramer or a heteropentamer (43, 56). MCM4-6-7 proteins form trimers or hexamers to function as DNA helicases in vitro (37). Such DNA helicase activity is not processive under standard conditions of a DNA helicase assay. During S phase, MCM proteins are released from origins of replication after initiation of DNA replication and move with replication forks, where they are thought to function as DNA helicases. The mechanisms ensuring replication of DNA only once per cycle involve release of MCM proteins from chromatin after firing of the origins of replication and prevention of reloading (2, 11). Moreover, the phosphorylation of MCM4 with CDK2/cyclin A is associated with inactivation of the DNA helicase (unwinding) activity of the

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. Mismatch repair (MMR)2 systems play a primary role in mutation avoidance by removing base-base and small insertion-deletion mismatches that arise during DNA replication (1). Prokaryotes and eukaryotes have evolved similar systems for repair (2) and in Escherichia coli MMR is initiated when MutS binds to mismatched DNA, possibly through its interaction with the ␤-clamp accessory protein that is required for processive DNA replication (3-6). MutL binds to MutS to form MutS-MutL-DNA complexes that stimulate MutH binding and cleavage of unmethylated DNA strands at GATC sequences, either 5Ј or 3Ј of recognized mismatches. Exonucleases then chew away at the DNA beyond the mismatch site so that highly accurate DNA polymerase III can correctly re-synthesize the strand.In eukaryotes, mismatch recognition is accomplished by MSH2 (MutS homolog 2) forming a heterodimer with either MSH3 or MSH6 to bind to distinct but overlapping spectra of mismatches (7). In both the yeast Saccharomyces cerevisiae and humans, the repair of base-base mismatches appears to be solely dependent on MSH2-MSH6, whereas both MSH2-MSH6 and MSH2-MSH3 can participate in the repair of small (1 to 12-nucleotide) loop insertions. Currently, it is thought that MSH heterodimer binding to a mismatch triggers ATP-dependent steps that allow interactions with MLH (MutL homolog) heterodimers composed of MLH1-Pms1 or MLH1-MLH3 (7,8). No MutH homolog, however, has been identified in eukaryotes, and the exact details of strand discrimination and error removal are not known, although in both yeast and humans a number of other proteins have been implicated in MMR, including proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and DNA polymerases ␦ and ⑀ (9 -16).PCNA was originally characterized as a DNA sliding clamp for replicative DNA polymerases, but subsequent studies have revealed its striking ability to interact with multiple partner...

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.