Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells.

Some possible ways in which replication of plasmids containing the Epstein-Barr virus (EBV) plasmid maintenance origin, oriP, might be controlled were investigated. Virtually all plasmid molecules were found to replicate no more than once per cell cycle, whether replication was observed after stable introduction of the plasmids into cells by drug selection or during the first few cell divisions after introducing the DNA into cells. The presence in the cells of excess amounts of EBNA1, the only viral protein needed for oriP function, did not increase the number of oriP-replicated plasmids maintained by cells under selection. In the cell lines studied, EBNA1 and oriP seem to lack the capacity to override the cellular controls that limit DNA replication to one initiation event per DNA molecule per S phase. The multicopy status of EBV-derived, selectable plasmids appears to result from the initial uptake by cells of large numbers of plasmid molecules, the efficient maintenance of these plasmids, and the pressure of genetic selection against plasmid loss. Other unknown controls must be responsible for the amplification of EBV genomes soon after latent infection of cells.     

The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells

The genome of Epstein-Barr Virus (EBV) and plasmid derivatives of it are among the most efficient extrachromosomal replicons in mammalian cells. The latent origin of plasmid replication (oriP), when supplied with the viral Epstein-Barr Nuclear Antigen 1 (EBNA1) in trans, provides efficient duplication, partitioning and maintenance of plasmids bearing it. In this review, we detail what is known about the viral cis and trans elements required for plasmid replication. In addition, we describe how the cellular factors that EBV usurps are used to complement the functions of the viral constituents. Finally, we propose a model for the sequential assembly of an EBNA1-dependent origin of DNA synthesis into a pre-Replicative Complex (pre-RC), which functions by making use only of cellular enzymatic activities to carry out the replication of the viral plasmid.     

Control mechanisms of Epstein-Barr virus persistence

Epstein-Barr virus (EBV), a gammaherpesvirus widespread in all human populations, establishes an asymptomatic persistent infection in the majority of individuals. This review briefly summarizes what is known about the complex interactions between the virus and its host, with particular emphasis on the immune mechanisms that are involved in controlling persistent infection. EBV also persists in the malignant cells of a number of human tumors. The possible mechanisms by which these cells might avoid virus-specific immune responses and the potential for adapting these responses to clear the tumor are also discussed.     

Microcin plasmids: a group of extrachromosomal elements coding for low-molecular-weight antibiotics in Escherichia coli.

Microcins are low-molecular-weight compounds produced and excreted by Enterobacteriaceae. They inhibit the growth of a wide spectrum of microorganisms. Microcin-synthesizing transconjugants were obtained in seven out of eight experiments of conjugational transfer between wild-type microcinogenic strains of Escherichia coli and E. coli strain BM21. The physical analysis of one of the transconjugant strains that has acquired the ability to produce microcin 17 showed the presence of extrachromosomal DNA as a plasmid (pRYC17) of molecular weight 36 X 10(6) (18.3-micron length), which is absent in the "microcincured" derivative strain. pRYC17 was incompatible with plasmids of the IncFII group. Other suspected plasmids containing the information for the synthesis of microcins have not been clearly classified. Strains producing microcins 93, 136, and 140 show a partial incompatibility with IncFIII group of plasmids.     

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.     

Evidence for extrachromosomal elements in Lactobacillus.

Three strains of lactobacilli, Lactobacillus casei subsp. casei 64H, L. casei subsp. rhamnosus OC91, and L. coryniformis M34, were examined for the presence of plasmids. Plasmids of molecular weights of 23 x 10(6) and 16 x 10(6) were found in the first two strains respectively. This represents the first evidence for plasmids in lactobacilli; their function is not presently known.     

Strategies of immunoescape in Epstein-Barr virus persistence and pathogenesis

The establishment of persistent infections is a common feature of tumor-associated viruses. The capacity to maintain a long-term relationship with the host presupposes viral mechanisms for circumventing antiviral defenses. Recent findings have highlighted multiple strategies of immune evasion that allow the persistence of Epstein-Barr virus infected cells and promote the spread of the virus in immunocompetent hosts. These strategies are likely to play an important role in the pathogenesis of EBV-associated malignancies.     

Partial elimination of Epstein-Barr virus plasmids from Burkitt''s lymphoma cells by transfecting the BZLF1 gene.

Epstein-Barr virus (EBV) nonproducer Raji cells stably maintain approximately 45 copies of the EBV genome per cell, depending on the presence of the EBV-determined nuclear antigen 1 (EBNA-1) protein. We found that transfection of the EBV BZLF1 gene causes the disappearance of EBNA proteins on Western blots (immunoblots). On the basis of these results, we attempted to eliminate EBV plasmids in Raji cells by transfecting a BZLF1 plasmid. Among 33 clones that were cotransfected with a BZLF1 plasmid and a hygromycin B resistance plasmid and selected resistant for hygromycin B, 24 clones had decreased numbers of EBV plasmids, as revealed by the decrease in the intensity of the EBV band on Southern blots compared with that of nontransfected Raji cells.     

Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli

The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E. coli spheroplasts. With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved. The majority of DNA macromolecules were released in the form of single and connected rosettes. Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm). The mean length of the rosette loops was 1.06 +/- 0.4 micron. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops. Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. coli reflect one of the levels of organization of prokaryotic genetic material.     

Circular extrachromosomal copia-like transposable elements in Drosophila tissue culture cells

We find that in the circular extrachromosomal DNA from Drosophila tissue culture cells the transposable elements copia, 412, 297, and mdg 1 are present in variable amounts. There is no detectable circular DNA homologous to B104 . From the relationship between the intra- and extrachromosomal forms it appears that the amount of different circular elements is not related to the amount of the respective chromosomal elements.     

Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells.

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

EBNA1 partitions Epstein-Barr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in Saccharomyces cerevisiae and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in S. cerevisiae, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in S. cerevisiae can occur through chromosome attachment.     

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.