Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.     

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.     

Broad-host-range plasmid replication: an open question

Many factors can influence the ability of plasmids to colonize different hosts, efficient replication probably being the most critical one. Two major strategies seem to facilitate promiscuous plasmid replication: (i) initiation independent of host initiation factors; and (ii) versatile communication between plasmid and host initiation factors. Appropriate communication between a replicon and the different hosts, which becomes crucial at the initation of plasmid replication, plays a major role in plasmid promiscuity. Fused replicons or mechanisms that rescue collapsed replication forks may increase the efficiency of plasmid propagation. However, their contribution to plasmid promiscuous replication remains to be fully evaluated. Several examples of host-specific adaptation of promiscuous plasmids point to an enormous flexibility of these replicons.     

Initiation of DNA replication within oriP is dispensable for stable replication of the latent Epstein-Barr virus chromosome after infection of established cell lines

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.     

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ~750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.

The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

     

Epstein-Barr virus stimulates torque teno virus replication: a possible relationship to multiple sclerosis

Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis.     

Lymphoblastoid cell-induced suppression of human peripheral blood leukocyte mitogenic responses

Two lymphoblastoid tumor cell lines, the Burkitt lymphoma derived BJAB cell line which is free of Epstein-Barr virus (EBV) and B95-8 cells, which are marmoset lymphocytes transformed by EBV isolated from an infectious mononucleosis patient, were studied in regards to their effects on the blastogenic responsiveness of normal human peripheral blood leukocytes stimulated in vitro with mitogens. Mitomycin C treated tumor cell suspensions, when cocultured with normal human blood leukocytes, markedly depressed the expected blastogenic responses in vitro to concanavalin A, pokeweed mitogen, and phytohemagglutin. In addition, cell-free sonicates from the cell lines also depressed blastogenic responsiveness of the leukocytes in vitro. Heating the sonicates for 10 min at 100 degrees C markedly diminished the suppressive properties of the sonicates, as did ultraviolet light irradiation. The suppressive activity of the B95-8 sonicates was pelleted by high speed centrifugation as compared to the activity of sonicates derived from the BJAB cells. Further studies are warranted to determine the nature and mechanism of suppression of blastogenic responsiveness of normal human leukocytes by soluble components derived from such lymphoblastoid cell lines.     

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10–100 adjacent replicons (each ∼100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought – so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45–60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase. Received: 16 August 1999 / Accepted: 17 August 1999     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.     

Cis-acting negative control of DNA replication in eukaryotic cells

We show that in stable monkey cell lines, the replication of a chimeric SV40-BPV episomal replicon occurs once and only once per cell cycle. The copy number of this episome is stably maintained even when an excess of the limiting initiation factor T antigen is provided. These experiments therefore uncover a cis-acting negative control mechanism whereby replication control is not focused on limiting the activity of positive factors; rather, replication is permitted in unreplicated replicons but is actively prevented, in cis, in replicons that have already been duplicated. We also find that the initiation factor SV40 T antigen remains associated with its SV-BPV episomal template in a kinetically stable and potentially heritable state.     

Inhibition of mammalian cell DNA synthesis by ionizing radiation

A semi-log plot of the inhibitory effect of ionizing radiation on the rate of DNA synthesis in normal mammalian cells yields a two-component curve. The steep component, at low doses, has a D0 of about 5 Gy and is the result of blocks to initiation of DNA replicons. The shallow component, at high doses, has a D0 of greater than or equal to 100 Gy and is the result of blocks to DNA chain elongation. The target size for the inhibition of DNA replicon initiation is about 1000 kb, and the target size for inhibition of DNA chain elongation is about 50 kb. There is evidence that the target for both components is DNA alone. Therefore, the target size for inhibition of DNA chain elongation is consistent with the idea that an effective radiation-induced lesion in front of the DNA growing point somehow blocks its advance. The target size for inhibition of DNA replicon initiation is so large that it must include many replicons, which is consistent with the concept that a single lesion anywhere within a large group (cluster) of replicons is sufficient to block the initiation of replication of all replicons within that cluster. Studies with radiosensitive human cell mutants suggest that there is an intermediary factor whose normal function is necessary for radiation-induced lesions to cause the inhibition of replicon initiation in clusters and to block chain elongation; this factor is not related to poly(ADP-ribose) synthesis. Studies with radiosensitive Chinese hamster cell mutants suggest that double-strand breaks and their repair are important in regulating the duration of radiation-induced inhibition of replicon initiation but have little to do with effects on chain elongation. There is no simple correlation between inhibition of DNA synthesis and cell killing by ionizing radiation.     

Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.     

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.     

Size of the intracellular circular Epstein-Barr virus DNA molecules in infectious mononucleosis-derived human lymphoid cell lines.

The size of non-integrated circular Epstein-Barr virus (EBV) DNA molecules isolated from seven different human lymphoblastoid cell lines of infectious mononucleosis origin has been determined by sedimentation analysis and by direct contour length measurements on electron micrographs. Six lines had intracellular circular EBV genomes of the same size as linear virion DNA molecules. The seventh line, established with the B95-8 strain of EBV, was the only one found to have circular EBV DNA molecules significantly smaller than virion DNA. The data show that intracellular EBV DNA circles of reduced size do not generally occur in infectious mononucleosis-derived cell lines.     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

Chromosomal replication in Drosophila virilis

DNA fiber autoradiography was used to determine parameters underlying the DNA replication of the eukaryotic chromosome in Drosophila diploid brain cells in organ culture. The average rate of fork movement, estimated from 4 different labelling intervals, is 0.35 μm/min at 25 ° C. Of the tandem arrays 93% show patterns which are compatible with bidirectional replication, 7% show unidirectional replication. The unidirectional mode of replication is interpreted as being a consequence of the experimental schedule (using hot-cold pulse labelling) combined with the occurrence of termination signals. — Some autoradiograms showed the expected two grain tracks of different densities; others showed only a high density track. The latter were most prominent in arrays of short replicons (<10 μm) which correlate with replicating satellite sequences. — The majority of replicons fall into size classes < 100 μm. The frequency distribution is skewed towards larger replicon sizes; it spans 2–238 μm, has a mean of ˉx = 35.6 μm and a median of = 21.0 μm. If the distribution is corrected for supposed satellite replicons, the median increases to = 31.0 μm. — In experiments using warmhot pulse labelling, arrays were scored which must have been a consequence of fixed termination signals. Furthermore, grain tracks diverging from weak labelled centers often have different lengths, indicating that these replicons contain two diverging replicating sections of unequal length. Presented to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday with my best wishes