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1.
Sequence requirements of the Epstein-Barr virus latent origin of DNA replication. 总被引:6,自引:5,他引:6
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The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function. 相似文献
2.
Herpesvirus papio contains a plasmid origin of replication that acts in cis interspecies with an Epstein-Barr virus trans-acting function. 总被引:6,自引:6,他引:0
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Herpesvirus papio (HVP) and Epstein-Barr virus (EBV) are closely related biologically and biochemically; lymphoblastoid cells infected with either virus contain episomal viral DNA. The putative origin of replication for EBV plasmids (oriP) has been assigned to a 1,790-base-pair fragment (cis) in the short unique region of the genome which requires a viral function supplied in trans from elsewhere in the genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We report here the identification of the putative origin of replication (cis) in HVP; we assigned it to the HVP EcoRI K fragment. The results indicate that the HVP replication process requires both a cis and a trans-acting function, analogous to that found in EBV. 相似文献
3.
The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus. 相似文献
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5.
Structural requirements for simian virus 40 replication and virion maturation. 总被引:2,自引:1,他引:1
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The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis. 相似文献
6.
Architecture of replication compartments formed during Epstein-Barr virus lytic replication
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Daikoku T Kudoh A Fujita M Sugaya Y Isomura H Shirata N Tsurumi T 《Journal of virology》2005,79(6):3409-3418
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. 相似文献
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The Epstein-Barr virus glycoprotein 110 carboxy-terminal tail domain is essential for lytic virus replication. 总被引:2,自引:2,他引:0
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To investigate the importance of the Epstein-Barr virus (EBV) glycoprotein 110 (gp110) tail domain in the intracellular localization of gp110 and virus lytic replication, three carboxy-terminal truncation mutants of gp110 were constructed. Deletion of 16 amino acids from the carboxyl-terminal tail resulted in gp110 intracellular localization which was indistinguishable from that of wild-type gp110, whereas deletion of either 41 or 56 amino acids from the carboxyl-terminal tail of gp110 resulted in loss of retention of gp110 in the endoplasmic reticulum and nuclear membrane. None of the gp110 truncation mutants was able to complement EBV(gp110-)+ lymphoblastoid cell lines in transformation assays, indicating the importance of the gp110 tail domain in virus lytic replication. In electron microscopy analysis, no nucleocapsids or enveloped viruses were detected in EBV(gp110-)+ lymphoblastoid cell lines induced for lytic replication. 相似文献
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Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo. 总被引:7,自引:7,他引:0
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The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV. 相似文献
11.
The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the DeltaBMRF1 EBV phenotype. DeltaBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. DeltaBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome. 相似文献
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In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes. 相似文献
15.
The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed. 相似文献
16.
Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication. 总被引:14,自引:12,他引:2
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The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was approximately 5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol. 74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-kappaB responsive genes including IkappaBalpha and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication. 相似文献
19.
Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor 总被引:32,自引:0,他引:32
Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell. 相似文献
20.
Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. 总被引:31,自引:4,他引:27
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Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines. 相似文献