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.

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.

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)-Replication protein A (RPA), the eukaryotic singlestranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6,14,21,32,38,41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G 1 /S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13,15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNAdamaging agents, have been suggested (33,54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5Ј ends of the DSB to generate 3Ј ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4).

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

During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi, J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, "TATAA." In contrast, the UL44 late viral promoter has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. The genome of HCMV is 240,000 bp in size with at least 150 known open reading frames (ORFs) (4). A majority of the ORFs are nonessential for viral replication in cell culture. Several ORFs are beneficial but not required for viral replication. However, approximately one-quarter, or 41 ORFs, are required for viral replication (39). The virus replicates productively in terminally differentiated cells, such as fibroblasts, epithelial and endothelial cells, and monocyte-derived macrophages (7,8,13,20,30,31,36).During productive infection, HCMV genes are expressed in a temporal cascade, designated immediate early (IE), delayed early, and late. The major IE genes UL123/UL122 (IE1/IE2) play a critical role in subsequent viral gene expression and the efficiency of viral replication (14,15,17,(22)(23)(24). The early viral genes encode proteins necessary for viral DNA replication (26). Following viral DNA replication, delayed early and late viral genes that encode structural proteins for viral production are expressed. Several early genes of HCMV have the unusual property of three promoters: two that initiate transcription early and one late (2, 21).The UL44 protein (pUL44), which binds double-stranded DNA, is an essential protein for viral DNA replication and interacts specifically with the viral DNA polymerase encoded by UL54 (27,29). pUL44 increases the processivity of the viral DNA polymerase along the viral DNA template (6, 37, 40).pUL44 protein accumulates to strikingly high levels at late times after infection (9, 35). The HCMV UL44 transcription unit initiates at three distinct sites, which are separat...