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PubMed1978

Replication of herpes simplex virus and cytomegalovirus in human leukocytes.

Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.

Antibodies, Viral↗
PubMed1990

Isolation of a human melanoma adapted Newcastle disease virus mutant with highly selective replication patterns.

The apathogenic Newcastle disease virus (NDV) strain Ulster has been used successfully as an adjuvant component for active specific immunotherapy of malignant mouse lymphoma, and in nude mice it was shown to be able to lead to retardation of the s.c. growth of xenotransplanted human melanoma cells. In order to improve in vivo effectiveness of virotherapy of human tumors without significantly increasing the risk of unspecific viral replication in host cells, we adapted the virus for growth in a human melanoma line (MeWo M). For this purpose NDV Ulster was mutagenized and a variant was selected which could replicate and reinfect the tumor line. The mutant (NDV 1E 10) performed late lysis on the melanoma line. Replication was found to be at least 100 times more efficient in MeWo M than in 6 of 8 other human tumor cell lines of different tissue origin. In 10 of 11 murine cell lines, NDV 1E 10 did not replicate via multicycles. Chick embryonic fibroblasts were permissive for nonlytic replication. Neither the virulent wild-type NDV Italian nor the avirulent strain NDV Ulster shared these specific replication properties with the new variant. We also established MeWo melanoma sublines with different metastatic capacities and tested them as targets for NDV 1E 10 infection. The MeWo subpopulations exhibited comparatively small differences in permissivity for multicyclic replication, but the more metastatic MeWo Met, like allogeneic melanoma lines, was more resistant to lysis. NDV Italian, in contrast, showed no differences in replication and lysis on any of the tested melanoma lines. Trypsin-activation experiments suggested an incomplete cleavage of mutant envelope glycoprotein F by the permissive cell line and, thus, mechanisms of specific infection and replication not requiring fully activated envelope glycoproteins.

Animals↗
PubMed1988

Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT.

The role of the primosome assembly and protein n' recognition site in replication of pBR322 plasmid was examined. The following evidence indicates that the primosome is involved in lagging-strand synthesis of pBR322 plasmid replication in vitro. Early replicative intermediates with newly synthesized leading strand, approximately 1 kilobase pair long, immediately downstream of the replication origin accumulate in products synthesized in extracts from a dnaT strain that lacks primosomal protein i or in wild-type extracts supplemented with anti-protein i antibody. These intermediates are converted efficiently into full-length DNA by addition of purified protein i. Consistent with the previously proposed role of the primosome (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73), an n' site on the lagging strand, but not on the leading strand, is required for efficient replication of the plasmid in vitro. Plasmids lacking an n' site on the lagging strand replicate only to a limited extent in vitro and early replicative intermediates carrying nascent leading strands are accumulated, although a portion of the intermediates complete replication to yield full-length DNA. The latter reaction is completely inhibited by addition of anti-protein i antibody. Insertion of the n' site of phage phi X174 into pBR322 plasmids lacking lagging-strand n' sites restores the replicative ability of the mutant plasmid comparable to that of the wild-type plasmid. These results indicate that protein i is essential for lagging-strand synthesis of pBR322 plasmid in vitro and that it may play an important role in the priming events as a part of either an n' site-dependent primosome or an n' site-independent, as yet unidentified, priming complex.

Bacterial Proteins↗
PubMed1988

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway.

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.

Bacteriophage phi X 174↗
PubMed1985

Human natural killer cells limit replication of herpes simplex virus type 1 in vitro.

Studies were undertaken to determine whether natural killer (NK) cells could inhibit the replication of herpes simplex virus type 1 (HSV-1) in culture. In the absence of effector cells, HSV-1 was found to replicate in fibroblasts with up to a 100-fold increase in virus titer from 4 to 16 hr after incubation at 37 degrees C. Human peripheral blood mononuclear cells were found to limit virus replication in a dose-dependent manner, with the greatest inhibition being observed at the highest concentration evaluated: i.e., an effector:target ratio of 800:1. The antiviral effect was not observed when nonactivated or virus-activated mononuclear cells were added to the virus preparations at the end (instead of the beginning) of the assay period, indicating that the observed effect was not due to a nonspecific toxicity of soluble factors released from freeze-thawed effectors. Neither was inhibition of HSV-1 replication due to the generation of interferon (IFN) during the NK assay, because the addition of anti-IFN did not abrogate the antiviral effect. Thus, the inhibition of viral replication was most likely due to a cytotoxic effector rather than to release of soluble factors. The effector cells responsible for limiting HSV-1 replication were shown to be NK cells by a number of criteria. Mononuclear cells from both HSV-1 seropositive and seronegative donors limited virus replication; their activity could be boosted by pretreatment of effector cells with IFN; the effector cells which limited virus replication were found in Percoll gradient fractions enriched for large granular lymphocytes; and the effector cells shared the cell surface phenotype of NK cells--they were enriched in populations depleted of T cells by panning with Leu-4 and were depleted of activity by treatment with the anti-NK antibody Leu-11b plus complement. We conclude that human NK cells are capable of recognizing and lysing HSV-1-infected target cells before infectious virus progeny are generated. These results suggest that NK cells, acting early in the course of an infection, might serve to limit HSV-1 replication and therefore reduce the virus load in the host before the development of the adaptive immune response and clearance of the infection.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1986

Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro.

The reconstituted pBR322 DNA replication system has been used to identify a mechanism for the processing and segregation of daughter DNA molecules by Escherichia coli topoisomerase I (Topo I) during the terminal stages of DNA replication. At low concentrations of Topo I (sufficient to confer specificity to the replication system for DNA templates containing a ColE1-type origin of DNA replication), the major products of the replication reaction were: multigenome-length, linear, double-stranded DNA molecules (an aberrant product); multiply interlinked, catenated, supercoiled DNA dimers; and a last Cairns-type replication intermediate. Thirty- to fifty-fold higher concentrations of Topo I led to the appearance of form II and form I pBR322 DNA as the only synthetic products. A model was developed in which Topo I, bound to a single-stranded gap on the parental H strand DNA just upstream of the origin of DNA replication, catalyzed the decatenation of the intermolecular linkages between the two daughter DNA molecules that were generated by primosome-catalyzed unwinding of the residual nonreplicated parental duplex DNA in the last Cairns-type intermediate. At low concentrations of Topo I, however, the intermolecular linkages persisted and, within the context of this replication system, were not removed by DNA gyrase. In support of this model it was demonstrated that: there was a single-stranded gap between the nonreplicated parental duplex region and the 5' end of the nascent leading-strand DNA; the number of intermolecular linkages in the catenated supercoiled DNA dimers was inversely related to the concentration of Topo I; the supercoiled DNA dimers did not serve as a precursor of the final form I DNA product; and maturation of the last Cairns-type replication intermediate to form I DNA was not affected by the presence of coumermycin, a potent inhibitor of the activities of DNA gyrase.

DNA Replication↗
PubMed1978

[Chronology of the replication of sex chromosome bands in lymphocytes of normal subjects and patients].

The replication sequence of the bands carried by chromosomes X and Y has been studied in normal individuals and in patients with structural abnormalities of the X. By comparing the segment with that of the autosomal bands (which had been previously studied), it was shown that the normal early X replicates in early X-phase for its R-bands and in late S-phase for its Q bands. The late X replicates entirely in late S-phase, and the sequence of band replication is not as stringent as for the early X and the autosomes. The study of fourteen cases of anomalies of chromosome X in females showed the following: in balanced reciprocal X-autosome translocations the rearranged X most often replicates early and the normal X late. Both show a normal replication sequence of their bands. In non-balanced X-autosome translocations, inactivation of the autosome fragment attached to the AUTOSOME FRAGMENT ATTACHED TO THE X may take place. In Xq- or in ter rea (X;X) (pter;pter), band p22 has a delayed replication. In iso-Xor Xp-, the long-arm-band sequence of replication shows a variation comparable to that of the late X in fibroblasts. These replication modifications are likely to induce partial inactivations or changes in activity which correspond to the so-called position effect in Drosophila.

DNA Replication↗
PubMed1982

[Analysis of chromosomal and extrachromosomal functions involved in the replication of bacterial plasmids].

A variety of functions codified by both the host chromosome and the plasmid genome are necessary for plasmid replication. We have found that dna B and dna F are essential for replication of both types of plasmids: R144 and Sa which have a restricted control of replication, and R6K with a relaxed control; on the other hand plasmids belonging to the first group absolutely depend on dna A and dna E gene products. Plasmid Sa, which shares with the 1st group a restricted control of replication but appears on a number of intermediate copies, shows a partial requirement to the dna A gene product. Location studies of plasmid-coded genes, involved in the replications of plasmids, were carried out in plasmid pSC102, a derivative of the resistance plasmic R6-5. All the genes required for replication are contained into a DNA segment which consists of two Pst I fragments: P-4 and P-6. The origin of replication On V is not itself sufficient for autonomous replication but needs in addition the presence of the Rep A gene. Genes responsible for copy control and incompatibility Cop/Inc are located on fragment P-6, i.e. they are physically distinct from Ori V. This indicates that the origin of replication is not responsible for, although may participate in, the incompatibility phenomenon.

Chromosomes, Bacterial↗
PubMed1984

Replication of herpes simplex virus in human T lymphocytes: characterization of the viral target cell.

Herpes simplex virus (HSV) replicated in mitogen-stimulated human T cells. Virus replication was obtained in highly enriched mitogen-stimulated T cells of the OKT 3+, OKT 4+, or OKT 8+ subtype, in stimulated B cells, and in macrophages precultured for 7 days. In contrast, no virus replication was obtained in unstimulated T or B cells, in macrophages grown in culture for 1 day, in Null/NK cells, or in granulocytes. Infectious center assays revealed that below 1% of the infected T cell subpopulations supported virus replication, whereas up to 42% of infected B cells and 80% of macrophages cultured for 1 wk were able to replicate HSV. By indirect double immunofluorescence studies, complement-mediated mass cytolysis, and positive selection experiments, it was shown that only T cells expressing Ia antigen actively replicated the virus. T cells activated in the mixed lymphocyte culture and with UV-inactivated HSV were also susceptible to HSV infection. Several human leukocyte cell lines were tested for their ability to support virus replication and were tested for a correlation with the expression of Ia antigen. Only cell lines expressing Ia antigen on more than about 5% of the total population produced new progeny virus. Ia-expressing T cells that spontaneously replicated HSV without any mitogenic prestimulation were found to occur in variable numbers in human cord blood. It is suggested, that such T cells, permissive for HSV replication, might contribute to an outspread of viral infection in vivo.

Antibodies, Viral↗
PubMed1982

Relationship between aberrant DNA replication and loss of cell viability in Chinese hamster ovary CHO-K1 cells.

We have previously presented evidence that a transient block to DNA replication induces an aberrant form of DNA synthesis. The most feasible explanation for this data is that the block to DNA replication results in some segments of the chromosomal DNA being replicated more than once in a single cell cycle. This form of aberrant DNA synthesis was demonstrated to occur following direct inhibition of DNA replication by 1-beta-D-arabinofuranosylcytosine or 9-beta-D-arabinofuranosyladenine or after indirect inhibition with cycloheximide. We have proposed mechanisms whereby this phenomenon could induce chromosome damage and cell death. In this paper, we present data on the relationship between this aberrant form of DNA replication and the loss of cell viability. Using Chinese hamster ovary CHO-K1 cells growing as monolayer cultures, we have simultaneously monitored the loss of cell viability as measured by colony formation and the relative extent of this aberrant DNA replication induced by 2-hr pulses of a series of concentrations of inhibitors of DNA replication. We have found that, with either direct inhibition of DNA replication with 1 beta-D-arabinofuranosylcytosine or with indirect inhibition, with cycloheximide, pulses of inhibitor administered to Chinese hamster ovary cells at increasing of this aberrant DNA replication which paralleled the increase in cell killing.

Animals↗
PubMed1994

Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.

Bacterial Proteins↗
PubMedDate not supplied

A comparative study of avian reovirus pathogenicity: virus spread and replication and induction of lesions.

This study examined the relationship of avian reovirus spread and replication to induction of lesions and the relevant role of the S1 segment encoding a virus-neutralizing antigen. One-day-old broiler chickens were infected via footpad or orally with two virus strains (883 and 176) that differ greatly in virulence and a reassortant (R44) that has the S1 segment from 176 and the remaining genome segments from 883. Virus replication and histological lesions in various tissues (heart, liver, spleen, kidney, bursa, hock joint, and bone marrow) were measured at 2-day intervals until day 8 postinoculation. The virulent strain 176 spread to and replicated efficiently in all tissues examined and caused extensive and severe lesions, whereas the mild strain 883 was detected only in tissues near inoculation sites and caused only minimal lesions. The appearance of lesions correlated with the presence of viral replication in each tissue tested. Together, these results indicate that induction of lesions, or pathogenicity, is directly related to virus spread and replication. Reassortant R44 behaved like strain 176 in chicken embryo fibroblasts (CEFs), i.e., both replicated much faster and produced larger plaques than strain 883. In broiler chickens, however, R44 behaved like strain 883, replicating and inducing lesions to an extent that was fat lower than that of strain 176. These results suggest that the S1 segment alone is capable of determining viral replication and plaque formation in cultured CEFs but is not sufficient to determine the virus spread and replication and the pathological change in broiler chickens.

Animals↗
PubMed1997

Replicative characteristics of primary isolates of the human immunodeficiency virus type 1 in peripheral blood mononuclear cells, primary macrophages and CD4+ transformed T-cell lines.

Macrophage-tropic strains of HIV-1 are selectively transmitted and play a significant role in HIV-1 persistence, dissemination and disease progression. Most primary HIV-1 isolates productively infect primary macrophages but fail to infect or induce syncytium (non-syncytium-inducing or NSI) in CD4+ transformed T-cell lines. Therefore, NSI isolates are considered to be macrophage-tropic. In order to select truly macrophage-tropic primary HIV-1 isolates for pathogenesis studies the replicative characteristics of a panel of eight primary (predominantly NSI) HIV-1 isolates were examined in primary macrophage cultures. This was not previously done. Stocks of these isolates were prepared in peripheral blood mononuclear cells (PBMC) and their replication in peripheral blood-derived macrophages and in CD4+ transformed T-cell lines examined based on the p24 antigen level in the cultures. Seven of the eight isolates did not replicate or induce syncytia (non-syncytium-inducing or NSI) in MT-2 cells, but one isolate replicated efficiently and induced large syncytia (syncytium-inducing or SI) in both the MT-2 and another transformed T-cell line (CEMx174). When replication of the isolates in peripheral blood-derived macrophages was examined, six of the seven NSI isolates replicated indicating that not all NSI isolates were capable of replicating in primary macrophages. Further, one of the six NSI isolates replicated (but did not induce syncytia) in the CEMx174 cell line, illustrating that characterization of primary isolates based on replication in one cell system does not allow predictions for other cell systems

CD4-Positive T-Lymphocytes↗
PubMed1998

DNA replication machinery of the mammalian cell.

The process of DNA replication in mammalian cells is highly complex and has several unique features that distinguish it from simpler prokaryotic systems. The study of mammalian DNA replication lagged behind that of prokaryotes for many years. This was because of the lack of a reliable and efficient mammalian cell-based in vitro DNA replication system. In 1984, the first mammalian-based DNA replication system that initiated DNA synthesis successfully in vitro was developed. The employment of the mammalian in vitro DNA replication system has led to the identification of several DNA replication proteins. This article describes the current knowledge regarding the proteins mediating mammalian DNA replication, as well as how they are proposed to function during DNA synthesis. There is also a discussion of the role the mammalian cell nuclear architecture plays in DNA replication. The evidence for the existence of an organized DNA replication machine in mammalian cells is also presented.

Animals↗
PubMed1999

Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.

Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.

Cell Line, Transformed↗
PubMed1996

Activation of oriLyt, the lytic origin of DNA replication of Epstein-Barr virus, by BZLF1.

oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is activated by the viral transactivator BZLF1 which belongs to the extended bZIP class of transcription factors. Seven binding sites for BZLF1, so-called ZRE sites, are located within oriLyt. By mutational analysis of individual ZRE sites, we found that lytic DNA replication is dependent on only four of these sites which colocate with the promoter of the BHLF1 gene. The remaining three ZRE sites distal to the BHLF1 promoter were dispensable for DNA replication and did not contribute to long-range transcriptional activation of this promoter by BZLF1. This finding indicated that a similar set of ZRE sites is involved in DNA replication and transcriptional activation. To determine the function of BZLF1 in DNA replication, BZLF1 mutants with successive deletions in the transactivation domain were analyzed in replication assays. Unexpectedly, most BZLF1 mutants which failed to support DNA replication were found to be equally defective in transcriptional activation. Therefore, similar trans-acting domains of BZLF1 are involved both in replication and in transcription.

Binding Sites↗
PubMed2001

Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin.

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.

Alleles↗
PubMed2006

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Caulobacter crescentus↗