Replication licensing of the EBV oriP minichromosome.

The latent EBV genome may persist in the integrated form as well as the circular episomal form. However, most of the latent viral DNA molecules are known to exist in the circular episomal form, which binds to host chromosomes during mitosis. The DS element of oriP in the circular episomal DNA functions as a replication origin. As it replicates once in a single S phase, it is possible that oriP is regulated by the cellular replication licensing mechanism including the MCM family of replication licensing factors. Transient replication analysis using the oriP plasmid and HeLa/EB1 cells revealed that the DS element requires early G1 phase for the next round of replication, the same cell-cycle window in which the replication licensing of cellular chromatin occurs. After this phase, the sedimentation velocity of the oriP minichromosome increases. MCM2 associates with the oriP minichromosome at late G1 but not at G2/M, and this association requires the DS element in the plasmid. The interaction of EBNA1 and the MCM proteins on the DS element was also suggested. These results suggested that the cellular licensing mechanism controls the replication from oriP. This also suggested a similarity in the replication machinery of the cellular chromatin and the latent EBV genome. In addition to DS-dependent replication, the EBV genome replicates in a manner independent of the DS element in several cultured cell lines. The DS-dependent replication is likely to be suppressed in these cell lines by the expression of other viral proteins. In contrast, EBV-positive Burkitt's lymphoma and circulating EBV-infected B cells express only EBNA1 or both EBNA1 and LMP2. DS-dependent replication may play a major role in these EBNA1-only cells, and the licensing regulation of oriP is important for maintenance of the EBV genome during this latent period of the viral life cycle. EBNA1 is required for efficient nuclear retention and partitioning of oriP-carrying plasmid by its binding to the FR element, thus providing stable persistence of the latent EBV genome during cell division. The copy number of latent EBV DNA molecules in B-cell lines remains fairly constant during multiple passage in culture. However, very little is known about the mechanism by which the viral DNA molecules are equally segregated into daughter cells. To understand the mechanisms responsible for stable nuclear retention and partitioning of the latent viral genome, it is essential to analyze the episomal and integrated viral DNAs at a single-cell level by FISH and other techniques.

Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 mitogen-activated protein kinase.

Latent Epstein-Barr virus (EBV) is maintained by the virus replication origin oriP that initiates DNA replication with the viral oriP-binding factor EBNA1. However, it is not known whether oriP's replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressed oriP activity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressed oriP activity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-induced oriP suppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitt's lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level of oriP activity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.

The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers.

In cell lines established from Marek's disease tumors, several viral transcripts are expressed and among them the products of pp38/pp24 mRNA and 1.8 kb-mRNA have been suggested to be involved in viral oncogenicity. The long inverted repeats of Marek's Disease virus serotype 1 (MDV1) genome contain closely located transcriptional promoters for phosphorylated protein pp38/pp24 and 1.8 kb-mRNA. These promoters initiate transcription in opposite directions and are separated only by a short enhancer region, which is likely to regulate both promoters simultaneously. We have analyzed the transcription activity of these promoters in MDV1 (Md5 strain) infected CEF by transient expression of CAT reporter genes and found that the promoters were in fact active in infected cells and the promoter for 1.8 kb-mRNA was more active than the pp38/pp24 promoter. Deletion analysis of the short enhancer region revealed that the 30 bp region overlapping the enhancer elements for 1.8 kb-mRNA was important for promoter activity for pp38/pp24. The gel shift analysis revealed that nuclear factor(s) actually bound to the overlapping 30 bp region. In addition, the activity of these promoters in infected cells varied with MDV strains. These results suggest that pp38/pp24 and 1.8 kb-mRNA promoters share a common regulatory sequence but a viral or a cellular factor(s) induced by viral infection regulates the promoter by distinct mechanisms.

Requirement of replication licensing for the dyad symmetry element-dependent replication of the Epstein-Barr virus oriP minichromosome.

Latent Epstein-Barr virus genome is maintained in cells by the viral oriP-binding factor EBNA1 and cellular replication factors. EBNA1 binds to the dyad symmetry (DS) element in oriP and initiates DNA replication once in a single S phase, but the mechanism by which this DS-dependent replication is initiated is unknown. Replication licensing of cellular chromatins occurs during early G1 phase. Because licensing is essential for the next round of replication in S phase, it facilitates once-in-a-cell-cycle replication of the cellular genome. Using the transient replication assay with HeLa/EB1 cell, we demonstrate that the oriP plasmid required a cell cycle window including early G1 phase for replication in the next S phase. The plasmid containing only the DS element had a similar requirement of early G1 phase for replication. Analysis using sucrose density gradient centrifugation revealed that the oriP minichromosome existed in two distinct states: one formed at late G1 and the other formed at G2/M. These results suggest that the DS-dependent DNA replication from oriP requires the replication licensing, implying a possible involvement of the cellular licensing factor MCM in the DNA replication from oriP.

Epstein-Barr virus (EBV)-infected cells were frequently but dispersely detected in T-cell lymphomas of various types by in situ hybridization with an RNA probe specific to EBV-specific nuclear antigen 1.

Association of Epstein-Barr virus (EBV) with T-cell lymphomas was examined by in situ hybridization (ISH) with an antisense probe specific to abundantly expressed EBV-encoded small RNA-1 (EBER1). In addition to EBER1, EBV-specific nuclear antigen-1 (EBNA-1) is commonly expressed in EBV-associated tumors and latently infected B-lymphocytes. We examined paraffin sections of T-cell lymphomas except those of nasal origin for expression of latent viral transcripts by ISH. Using ISH with improved antisense RNA probe specific to EBNA-1 mRNA, the virus was detected in 19 (59%) of 32 cases, whereas the EBER1 transcript was found in only 15 (47%) of 32 cases by conventional EBER-ISH, resulting in 21 EBV-positive cases (66%) by combining the two methods. Latent membrane protein 1 (LMP1) mRNA of EBV was detected in 15 of 32 cases (47%), while no EBNA2 expression was observed in any these tumors. Patients with these lymphomas positive for LMP1 expression showed lower survival rates than those without expression of the viral mRNA. These results indicate that, in addition to EBER-ISH, RNA-RNA ISH with EBNA1 probes could be useful for detection of EBV-infected cells in paraffin sections, and detection of LMP1 mRNA expression in tumor cells could be a useful prognostic factor for T-cell lymphoma.

Tumorigenicity of mouse BALB/c 3T3 fibroblast cells which express Epstein-Barr virus-encoded LMP1 and show normal growth phenotypes in vitro is correlated with loss of transforming growth factor-beta 1-mediated growth inhibition.

Latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) genome is known to induce loss of contact inhibition and the anchorage-independent growth in rodent fibroblasts and increased expression of cell-surface activation markers and cell adhesion molecules in human B lymphocytes. To analyze the role of LMP1 in tumorigenicity, we prepared BALB/c 3T3 clones (B3LP) expressing LMP1. These B3LP cells showed non-transformed phenotypes in vitro which were characterized by normal cell morphology, contact inhibition in growth and anchorage-dependent growth. The activity of NF-kappa B induced generally in several cell lines after transfer of the LMP1 gene was not detected in B3LP cells. However, B3LP expressing LMP1 at moderate levels lost sensitivity to growth arrest by transforming growth factor-beta 1 (TGF-beta 1) and formed tumors in severe combined immune deficiency mice. Cells expressing the truncated form of LMP1 and expressing LMP1 at low level were sensitive to TGF-beta 1-mediated growth arrest and did not form tumors in mice. Therefore, cells expressing LMP1 at moderate but not at low levels formed tumors in mice and lost sensitivity to TGF-beta 1. Our results suggest that loss of TGF-beta 1-mediated growth inhibition is an important event for the tumorigenicity of LMP1-expressing cells.

Major product pp43 of human cytomegalovirus U(L)112-113 gene is a transcriptional coactivator with two functionally distinct domains.

Human cytomegalovirus U(L)112-113 encodes four phosphoproteins, pp84, pp50, pp43, and pp34, with common amino-termini. A previous report by Kerry et al. (J. Virol. 70, 373-382, 1996) demonstrated that U(L)112-113 products activate U(L)54 promoter in cooperation with immediate-early (IE) proteins. In this study, we identified a domain required for transcriptional activation in the pp43 protein, which consisted of two distinct regions: domain I (amino acids 272-296) and domain II (amino acids 297-306). Domain I contained two long glycine stretches, and domain II was a short proline-containing region. Both of domains were required for IE2-dependent transcriptional activation. The pp43 mutant that had domain I but lacked domain II acted as a dominant negative mutant and suppressed most of the IE2-dependent activation, indicating the importance of coactivation by pp43 in this transcriptional activation. The major protein pp43 also weakly activated the promoter through IR1 element in a manner independent of IE2. Only domain I was required for this IE2-independent activation. These domains were common in pp84, pp50, and pp43 but did not exist in pp34, which did not activate transcription alone. These results suggest that the major product, pp43, of U(L)112-113 has two functionally distinct domains and plays an important role in mediating IE2-dependent transcriptional activation.

Identification of minimal oriP of Epstein-Barr virus required for DNA replication.

DNA replication from oriP of Epstein-Barr virus is mediated by the virus replication factor EBNA1 and cellular factors and occurs approximately once in each cell cycle. We have identified a minimal oriP element that is necessary and sufficient for DNA replication. We transfected plasmids containing several oriP fragments into HeLa cells expressing EBNA1 and analyzed their replication during four days after transfection using the methylation sensitive restriction endonuclease DpnI. All the oriP fragments containing the four EBNA1-binding sites known as the dyad symmetry sequence (DS) initiated DNA replication. The sequences flanking DS were not essential for DNA replication, but deletion of two or three EBNA1-binding sites in DS significantly reduced or totally abolished its replication activity. These results indicated that the four EBNA1-binding sites in DS constitute the minimal oriP element for DNA replication and suggest that DNA replication is initiated by recruitment of cellular replication factors onto or near the minimal oriP by EBNA1. We also found that the minimal oriP initiated DNA replication in mouse fibroblasts expressing EBNA1 but worked only at reduced efficiency, suggesting species specificity in DNA replication machineries.

The E12 inhibitory domain prevents homodimer formation and facilitates selective heterodimerization with the MyoD family of gene regulatory factors.

Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. However, the mechanism which ensures this selective dimerization is not understood at present. In our functional studies of various amino acid changes in the E12 HLH domain, we found that a single substitution in E12 helix 1 can abolish the effect of the E12 inhibitory domain and results in the efficient DNA binding of the E12 homodimer. Competition experiments revealed that the inhibitory domain, in fact, blocks the dimerization of E12 rather than DNA binding. MyoD contains two glutamic residues in helix 2 that are required for efficient dimerization with E12. More importantly, these residues were not essential for dimerization with E12 mutants in which the dimerization inhibitory domain had been relaxed, or for dimerization with E47 which does not contain the inhibitory domain owing to the use of an alternative exon. The positions of these glutamic residues are conserved among the four myogenic factors. Thus, members of the MyoD family of gene regulatory proteins can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2. This result describes a novel mechanism facilitating the selective formation of the MyoD(MRF)-E12 heterodimer that enhances dimerization specificity and may apply to other members of the E-protein family.

Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues.

The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with E12. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with E12. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and E12 and determine the dimerization specificity of the bHLH proteins.

Phosphorylation inhibits the DNA-binding activity of MyoD homodimers but not MyoD-E12 heterodimers.

MyoD is a member of the basic helix-loop-helix (bHLH) family of muscle gene regulatory proteins that includes myogenin, myf-5, and MRF4. These proteins have been shown to heterodimerize with E2A bHLH proteins, E12/E47, and to bind to a consensus sequence known as an E-box, CANNTG, the target for transcriptional activation by these myogenic regulators. MyoD is also a phosphorylated nuclear protein that is present in muscle cells prior to the transcriptional activation of the muscle-specific genes, many of which contain E-box elements in their regulatory regions. Here we report that phosphorylated chicken MyoD, called CMD1, produced in sf9 cells using the baculovirus system, is qualitatively similar to CMD1 isolated by immunoaffinity purification from primary cultures of embryonic chick breast muscle. Functional analysis of phosphorylated and dephosphorylated CMD1 produced in sf9 cells indicates that, in the presence of magnesium, DNA binding of phosphorylated CMD1 is inhibited whereas binding in association with E12 is not affected. However, CMD1 binding alone is equally efficient when either EDTA is added in excess or dephosphorylated or bacterially expressed CMD1 is used in the assay. Our results suggest that cellular phosphorylation changes the CMD1 homodimer-heterodimer equilibrium which, in turn, modulates and/or eliminates binding site competition between CMD1 homodimers and CMD1/E-protein heterodimers in the cell.

Isolation and functional comparison of Dmyd, the Drosophila homologue of the vertebrate myogenic determination genes, with CMD1.

We have isolated a cDNA clone, called Dmyd for Drosophila myogenic determination gene, from a 0-16 hour Drosophila embryo library that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family (Paterson et al 1991). Dmyd encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix region and 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle specific gene activation. The gene is unique and maps to 95A/B on the right arm of the third chromosome. Low stringency hybridizations indicate Dmyd is not a member of a multigene family, similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear gene regulatory factor, and is proposed to be a transiently expressed marker for a unique subset of muscle founder cells. We have used an 8kb promoter fragment from the gene, which contains the first 55 amino acids of the Dmyd protein, joined to lac Z to follow myogenic precursor cells into muscle fibers using antibodies to beta-galactosidase and Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles so it cannot be followed continuously as a muscle marker. This is reflected in the loss of expression of Dmyd RNA in 12-24 hour embryos, a major period of early myogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle. Functional differences between Dmyd and CMD1 are described and explained in terms of a model which may give insight to the nature of homo and heterodimer formation in the bHLH family of proteins.

HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.

Giant intracranial aneurysms--magnetic resonance imaging follow-up and clinical symptoms.

Twenty-four intracranial aneurysms over 20 mm in diameter were studied with magnetic resonance (MR) imaging. MR imaging follow-up of eight cases revealed induced thrombus with homogeneous intensity and decreased size even after complete intraluminal thrombosis. Most cases demonstrated homogeneous intensity thrombus in contrast to the heterogeneous intensity of spontaneous thrombus. The clinical symptoms could not be explained retrospectively by the thrombus characteristics. Perianeurysmal high intensity, indicating cerebral edema, was detected in one case presenting with a rapid increase in size. MR imaging is useful for following these pathological intra- and perianeurysmal changes.

Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene.

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.