USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.

Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G 1 but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.The initiation of DNA replication in eukaryotic cells is a carefully regulated process requiring the orchestrated assembly of many proteins at origin sites, including the origin recognition complex and minichromosome maintenance (MCM) complex. The MCM complex consists of six subunits, MCM2 through MCM7 (MCM2-7), which form a hexamer. Studies in Saccharomyces cerevisiae, where MCM proteins were first identified (25), showed that each of the MCM subunits performs an essential function in the initiation and elongation of DNA replication (20,21). Genetic and biochemical studies conducted in yeast, Xenopus, Drosophila, and mammals point to probable roles of the MCM proteins in melting origin DNA and in functioning as the replicative helicase at replication forks (8,27).Biochemical analyses of the MCM complex have shown that MCM4, -6, and -7 are the most stably associated subunits, referred to as the helicase core (MCM4/6/7), with MCM2 and a dimer of MCM3 and MCM5 being more loosely associated with the core (13,23,30,36). MCM4, MCM6, and MCM7 on their own can form hexamers with weak but measurable DNA helicase activity. The addition of MCM2 to the MCM4/6/7 core complex disrupts the hexamer and inhibits DNA helicase activity (12, 23). The complete MCM2-7 complex has no detectable helicase activity in vitro (12, 23), but helicase activity has been reported for a larger complex containing MCM2-7, cdc45, and GINS (29). As expected, MCM complexes exhibit ATPase activity (6,23,35). ATPase activity has not been observed in individual MCM subunits but occurs when certain pairs of MCM proteins interact (6).Each of the six MCM subunits shares a region of homology referre...

The EBNA1 protein of Epstein-Barr virus (EBV) is essential for EBV latent infection in ensuring the replication and stable segregation of the EBV genomes and in activating the transcription of other EBV latency genes. We have tested the ability of four host proteins (Brd2, Brd4, DEK, and MeCP2) implicated in the segregation of papillomavirus and Kaposi's sarcoma-associated herpesvirus to support EBNA1-mediated segregation of EBV-based plasmids in Saccharomyces cerevisiae. We found that Brd4 enabled EBNA1-mediated segregation while Brd2 and MeCP2 had a general stimulatory effect on plasmid maintenance. EBNA1 interacted with Brd4 in both yeast and human cells through N-terminal sequences previously shown to mediate transcriptional activation but not segregation. In keeping with this interaction site, silencing of Brd4 in human cells decreased transcriptional activation by EBNA1 but not the mitotic chromosome attachment of EBNA1 that is required for segregation. In addition, Brd4 was found to be preferentially localized to the FR enhancer element regulated by EBNA1, over other EBV sequences, in latently EBV-infected cells. The results indicate that EBNA1 can functionally interact with Brd4 in native and heterologous systems and that this interaction facilitates transcriptional activation by EBNA1 from the FR element.As part of their life cycle, gammaherpesviruses and papillomaviruses establish persistent infections in proliferating cells in which their double-stranded circular DNA genomes are maintained at a constant copy number. Maintenance of copy number involves the doubling of the population of viral genomes each cell cycle and a segregation mechanism to ensure equal delivery of the genomes to the daughter cells during cell division. The mechanism of mitotic segregation is conserved in the gammaherpesviruses and papillomaviruses in that, in all cases, the viral genomes are tethered to the host mitotic chromosomes through the viral origin DNA binding protein, which binds directly to the viral segregation element and interacts with one or more host chromosomal proteins.The mechanism of papillomavirus genome segregation has been studied most extensively using the bovine papillomavirus (BPV). The BPV E2 protein tethers the viral genomes to host chromosomes through interactions with multiple E2 recognition sites in the minichromosome maintenance element (MME) (22,30,38,47). E2 binds the MME through its DNA binding and dimerization domain and interacts with mitotic chromosomes through the domain responsible for transcriptional activation (6, 47). The mechanism by which the E2 transactivation domain contacts mitotic chromosomes to mediate segregation has been the subject of several studies, and considerable evidence has implicated an interaction with the bromodomain protein Brd4 in this process. Brd4 was identified as a binding partner of E2 that colocalized with E2 on host mitotic chromosomes and was shown to enable E2 to maintain plasmids containing the MME in budding yeast (Saccharomyces cerevisiae) (9,34,62). Inte...

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 oriPplasmid maintenance but supported the transient replication oforiP 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.

by tethering the episomes to the cellular chromosomes in mitosis. A host nucleolar protein, EBNA1-binding protein 2 (EBP2), has been shown to be important for interactions between EBNA1 and chromosomes in metaphase and to associate with metaphase chromosomes. Here, we examine the timing of the chromosome associations of EBNA1 and EBP2 through mitosis and the regions of EBNA1 that mediate the chromosome interactions at each stage of mitosis. We show that EBP2 is localized to the nucleolus until late prophase, after which it relocalizes to the chromosome periphery, where it remains throughout telophase. EBNA1 is associated with chromosomes early in prophase through to telophase and partially colocalizes with chromosomal EBP2 in metaphase through to telophase. Using EBNA1 deletion mutants, the chromosome association of EBNA1 at each stage of mitosis was found to be mediated mainly by a central glycinearginine region, and to a lesser degree by N-terminal sequences. These sequence requirements for chromosome interaction mirrored those for EBP2 binding. Our results suggest that interactions between EBNA1 and chromosomes involve at least two stages, and that the contribution of EBP2 to these interactions occurs in the second half of mitosis.

The human EBP2 protein was found by two-hybrid analysis to interact with the Epstein-Barr virus nuclear antigen 1 (EBNA1). Homologs of human EBP2 can be found in Caenorhabditis elegans, Schizosaccharomyces pombe, and in Saccharomyces cerevisiae, and they all share a conserved 200 -300-amino acid block of residues at their C termini. To understand the cellular function of EBP2, we have begun to study the protein in S. cerevisiae. The yeast Ebp2 protein contains N-terminal, nucleolar-associated KKE motifs, and deletion analysis reveals that the C-terminal conserved region is required for the activity of the protein. The EBP2 gene codes for an essential protein that localizes to the nucleolus. Temperature-sensitive ebp2-1 mutants become depleted of ribosomes and cease to divide after several generations at the restrictive temperature of 36°C. This decline in ribosome levels is accompanied by a diminution in the levels of the 35 S-derived recombinant RNAs (rRNAs) (in particular the 25 S and 5.8 S rRNAs). Pulse-chase, Northern, and primer extension analysis of the rRNA biosynthetic pathway indicates that ebp2-1 mutants are defective in processing the 27 SA precursor into the 27 SB pre-rRNA.One approach for determining how viruses depend on and alter normal host cell functions is to identify and characterize host factors with which the viral proteins interact. Such an approach can potentially identify host cell proteins that are utilized for aspects of the viral life cycle as well as cellular targets of viral oncoproteins. Such a strategy has been applied to the Epstein-Barr virus, a ubiquitous human herpesvirus that is causally associated with infectious mononucleosis and several cancers (1). During latent infection of human host cells, EBV 1 genomes are maintained in the cell nucleus as doublestranded DNA episomes. Like the cellular chromosomes, the EBV episomes replicate once per cell cycle and segregate stably to the daughter cells (2-4). The maintenance of the EBV episomes requires the viral origin of DNA replication, oriP, and one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1) (3).EBNA1 binding to oriP is thought to activate EBV DNA synthesis and govern the segregation of the episomes by mediating interactions with host cell proteins. In mitosis, EBV genomes and the EBNA1 protein have been observed to associate with the host metaphase chromosomes (5, 6), and therefore, EBNA1 may govern the partitioning of EBV genomes by mediating interactions between the EBV episomes and a component of the condensed host chromosomes. Characterizing factors that interact with EBNA1 are also important because EBNA1 may play a direct role in the development of EBV-associated cancers (7).Previously, we used a yeast two-hybrid system to identify human proteins that specifically interact with EBNA1 (1). We isolated a cDNA molecule encoding a 35-kDa protein, termed EBP2 (EBNA1-binding protein), and verified its interaction with EBNA1 using three independent assays. The sequence of EBP2 is identical to the human nucleolar protein...

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11-and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2-and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.Membrane and secreted proteins that fail to properly fold or assemble are degraded in a process known as ER 2 -associated degradation or ERAD (1-4), in which aberrant proteins are retrotranslocated across the ER membrane into the cytoplasm and degraded by proteasomes (4 -8). Retrotranslocation in some instances appears to require Sec61 channels (2, 9 -12) but may involve Derlins for other ERAD substrates (13-15). Polyubiquitination of ERAD substrates occurs in the cytoplasm in a process coupled to export and likely provides directionality to retrotranslocation (16 -21). A complex of proteins, including p97 ATPase, ubiquitin fusion degradation-1 (Ufd1), and nuclear protein localization-4 (Npl4), forms an essential cytoplasmic component of the extraction and degradation machinery (18,(22)(23)(24)(25). Some ERAD substrates may also be extracted from the ER membrane by the proteasome (26 -29).Less is known about how ERAD substrates are recognized and targeted to the retrotranslocon. ER chaperones, including BiP, calnexin (CNX), and calreticulin (CRT) promote folding of ER proteins. However, unsuccessful folding or assembly may force these chaperones to withdraw from folding cycles and target substrates for retrotranslocation (3,8,30). One process that determines the duration of retention of a glycoprotein in the ER involves the monitoring of N-linked oligosaccharides. CNX and CRT bind immature monoglucosylated proteins and promote ER retention, folding, and assembly (31). However, protracted cycles of CNX and CRT binding and release leads to binding of ERAD enhancing ␣-mannosidase-like (EDEM) protein that accelerates ERAD (32, 33). The yeast osteosarcoma-9 protein (Yos9p) and protein disulfide isomerase also select misfolded lumin...

The Epstein-Barr virus (EBV) EBNA1 protein is important for the replication and mitotic segregation of EBV genomes in latently infected cells and also activates the transcription of some of the viral latency genes. A Gly-Arg-rich region between amino acids 325 and 376 is required for both the segregation and transcriptional activation functions of EBNA1. Here we show that this region is modified by both arginine methylation and serine phosphorylation. Mutagenesis of the four potentially phosphorylated serines in this region indicated that phosphorylation of multiple serines contributes to the efficient segregation of EBV-based plasmids by EBNA1, at least in part by increasing EBNA1 binding to hEBP2. EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5. Multiple arginines in the 325-376 region were methylated in vitro by PRMT1 and PRMT5, as was an N-terminal Gly-Arg-rich region between amino acids 41 and 50. EBNA1 was also shown to be methylated in vivo, predominantly in the 325-376 region. Treatment of cells with a methylation inhibitor or down-regulation of PRMT1 altered EBNA1 localization, resulting in the formation of EBNA1 rings around the nucleoli. The results indicate that EBNA1 function is influenced by both serine phosphorylation and arginine methylation.The EBNA1 protein of Epstein-Barr virus (EBV) enables the persistence of the episomal viral genome in latently infected, cycling B lymphocytes, which can lead to malignant transformation under some circumstances. EBNA1 makes several contributions to EBV latent infection. First, EBNA1 is required for the initiation of DNA replication from the EBV latent origin, oriP (69). This involves the binding of EBNA1 to the dyad symmetry (DS) element of oriP and recruitment of cellular replication initiation proteins (9, 13, 55). Second, EBNA1 is required for the stable segregation of the viral genomes during cell division. Segregation requires EBNA1 binding to the family of repeats (FR) element of oriP and attachment of EBNA1 to the host mitotic chromosomes through an interaction with the cellular hEBP2 protein present on the mitotic chromosomes (25,26,28,66). Third, through binding to the FR element, EBNA1 can activate the transcription of other EBV latency genes, although the mechanism of this activation is not known (19,52). Finally, EBNA1 can counteract the stabilization of p53 by USP7 that occurs in response to DNA damage, thereby decreasing apoptosis and increasing cell survival (30, 54).Several functionally important regions of EBNA1 have been defined. The replication, segregation, and transcriptional activation functions of EBNA1 all require the DNA binding and dimerization domain, located near the C terminus between amino acids 459 and 607 (6, 50, 68), in order to bind an 18-bp recognition site present in multiple copies in the oriP DS and FR elements (51) (Fig. 1A). However, this domain is not sufficient for any EBNA1 function (8,39,67). The replication function also requires sequences in the N-terminal half of EBNA1, which likely ...