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At least 217 records · Page 12Linked to original sources
PubMed1987

Rous sarcoma virus is integrated but not expressed in chicken early embryonic cells.

We have developed a protocol that allows us to infect chicken early embryonic (CEE) cells with high efficiency. This was achieved by exposing the CEE cells to a semicontinuous dose of Rous sarcoma virus (RSV) for a period of 20 hr. Southern blot analysis indicated that an average of one proviral copy is integrated per embryonic cell. However, there was no production of infectious viral particles by the cells containing the proviral genome, although low levels of full-length genomic RNA could be detected by RNA transfer blot analysis. These low RNA levels contrast with the 100- to 1000-fold higher levels found in RSV-infected chicken embryo fibroblasts. We conclude that in cells derived from pregastrulating chicken embryos, RSV DNA is integrated into the cell genome but fails to be expressed in an efficient manner. These primary cells can therefore be used to identify factors involved in regulation of retroviral gene expression in normal cells. Such factors may also be instrumental in elucidating basic mechanisms involved in gene regulation during early development in higher vertebrates.

Animals↗
PubMed1980

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus.

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.

Animals↗
PubMed1993

Transactivation of cellular promoters by an integrated hepatitis B virus DNA.

A new Hepatitis B virus(HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma(HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3' truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6, and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Amino Acid Sequence↗
PubMed1987

Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.

Base Sequence↗
PubMed1997

The genome of Moloney murine leukemia virus can be integrated by the integrase of human immunodeficiency virus type 1 expressed alone in vivo.

An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.

Cell Line↗
PubMed1993

Trans-complementation among naturally occurring deletion mutants of hepatitis B virus and integrated viral DNA for the production of viral particles with mutant genomes in hepatoma cell lines.

Cultured hepatoma cells (HepG2) were cotransfected with two different plasmids carrying a head-to-tail dimer of recombinant hepatitis B virus (HBV) DNA cloned from deletion mutants isolated from the circulation of persistently infected hosts. They were tested for the secretion of viral particles with mutant genome encapsidation. A recombinant plasmid defective in the S gene and one defective in both the C and P genes complemented in trans for the production of viral particles. Mutant genomes from either of the recombinants were encapsidated. Similarly, a recombinant defective in the C gene and another defective in the P gene trans-complemented for the production of viral particles containing mutant genomes. A hepatoma cell line with integrated HBV DNA sequences defective in the C and P genes (PLC/PRF/5) when transfected with a recombinant defective in the S gene produced viral particles with the HBV genome from the transfecting recombinants. These results confirm the expected trans-complementation among the S, C and P genes of HBV, when either episomal or integrated into chromosomes, for the maintenance of defective HBV mutants in persistently infected hosts.

Base Sequence↗
PubMed1994

Integrated hepatitis B virus X and 3' truncated preS/S sequences derived from human hepatomas encode functionally active transactivators.

The hepatitis B virus (HBV) frequently integrates into hepatocellular genomic DNA during viral infection. Transcriptional transactivators encoded by integrated HBV X and 3' truncated preS/S sequences are known to stimulate gene expression from homologous and heterologous promoters. Here we demonstrate that 21 of 26 (81%) hepatocellular carcinoma tissues/cell lines contain coding sequences for at least one of the two known transactivators. Four integrated X and three preS/S transactivator sequences contained in five isolates from three hepatoma primary tissues or cell lines were used as examples to prove functionality of the encoded transactivators. In one case, where both X and preS/S sequences were present, dissection of X and preS/S transactivator sequences showed independent functionality. The investigation of X- and preS/S-specific RNA and protein expression revealed the existence of carboxyterminally truncated viral-cellular fusion proteins that were able to stimulate gene expression from the c-fos proto-oncogene promoter five- to ten-fold. These results demonstrate that structurally intact HBV transactivator sequences are integrated in the majority of HBV-associated HCCs/hepatoma cell lines. In all tested examples integrated DNAs had retained functionality as transactivators. This data thereby support indirectly the hypothesis of a possible involvement of HBV transactivators in liver cell proliferation and hepatocarcinogenesis.

Base Sequence↗
PubMed2005

High-resolution genome-wide mapping of transposon integration in mammals.

The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon- and virus-based integrating vectors.

Animals↗
PubMed1987

Analysis of retinoblastoma for human adenovirus and human JC virus genome integration.

Human adenovirus groups A and B have an oncogenic potential in newborn rodents. Especially, adenovirus type 12 is known to induce retinoblastoma-like tumor in baboons and transform human embryo retinoblast cells in vitro. Human JC virus is also known to produce a variety of tumors in newborn rodents, including retinoblastoma-like tumor. In this experiment, cell DNAs of human retinoblastomas were assayed for each of the transforming gene sequences of adenovirus groups A, B, C, D and E and for JC virus gene sequences, by using Southern blot hybridization. None of these viral gene sequences were detected at the level of 0.1-0.5 copy per diploid cell DNA in all of 11 retinoblastomas, including 6 retinoblastomas previously examined for adenovirus 12-transforming gene sequences. This led to a conclusion that most, if not all, adenoviruses and JC virus play no essential role in the etiology of human retinoblastoma, although there were experimental models of retinoblastoma induced by these viruses.

Adenoviruses, Human↗
PubMed1990

Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein.

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.

Avian Myeloblastosis Virus↗
PubMed1993

Efficient pathogen-derived resistance induced by integrated potato virus Y coat protein gene in tobacco.

The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines. Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations. The transcription and expression of the integrated CP gene was detected by Northern and Western blots. Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H. The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection. Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation. Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection. This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.

Agrobacterium tumefaciens↗
PubMed1983

Association of the lethal yellow (Ay) coat color mutation with an ecotropic murine leukemia virus genome.

The dilute (d) coat color mutation on chromosome 9 is closely associated with an ecotropic murine leukemia virus (MuLV) genome [Jenkins, N.A., Copeland, N.G., Taylor, B.A. & Lee, B.K. (1981) Nature 293, 370-374]. DBA/2J mice homozygous for the reverse mutation to wild type at the dilute locus (d+2J) lack ecotropic virus-specific sequences, suggesting that the dilute mutation was caused by virus integration. In the experiments described here, we analyzed the ecotropic MuLV DNA content of mice that collectively carry 10 different alleles at the agouti coat color locus (of chromosome 2) to determine whether any of these alleles also are associated with ecotropic virus sequences. Of the 10 alleles analyzed, one allele, lethal yellow (Ay), which is carried congeneically and heterozygously on C57BL/6J, 129/Sv, and LT/Sv mice, was closely associated with an ecotropic MuLV provirus. The close association of this provirus with the Ay allele suggests that this mutation also may be caused by virus integration. Furthermore, this association may be useful for molecular cloning and characterizing the many alleles at this locus.

Animals↗
PubMed1983

Approaches to the treatment of chronic hepatitis B viral infection.

In HBeAg positive patients with a high level of viral replication, antiviral therapy is the treatment of choice. The most promising agents at the moment are ARA-AMP and Interferon and both are being assessed in controlled clinical trials. In the anti-HBe positive patients in whom continued HBs antigenaemia is due to the presence of clones of cells containing integrated virus, some form of immune manipulation may be necessary. In rare cases in whom continuing inflammatory activity is related to an autoimmune reaction, low dose prednisolone may be beneficial. Treatment for delta infection has yet to be evaluated but antiviral agents such as interferon which inhibit both DNA and RNA viruses may prove effective. Although these forms of therapy are currently experimental, some are now entering phase III clinical trials. It seems probable that the ultimate regimen will include antiviral drugs and immune manipulation to adequately eliminate hepatocytes containing replicating and integrated virus. The latter is essential if we are to deal with the problem of neoplasia as well as infectivity and inflammatory liver disease.

Acyclovir↗
PubMed1984

Dilute-coat-color locus of mice: nucleotide sequence analysis of the d+2J and d+Ha revertant alleles.

The unstable dilute-coat-color mutation (d) of DBA/2J mice has been shown to be the result of integration of an ecotropic murine leukemia virus within the mouse genome. Molecular cloning and restriction enzyme analysis of the dilute allele and the viral preintegration site (+ allele), as well as two independent dilute revertants (d+2J and d+Ha), suggested that reversion is due to virus excision occurring by homologous recombination involving the viral long terminal repeats. The DNA sequence has now been determined for the cell-virus junctions of the provirus associated with the d mutation, for the viral preintegration site, and for the two revertant sites. These data (i) indicate that the d mutation was caused by a normal virus integration, (ii) confirm that virus excision occurs by precise homologous recombination, as exactly one long terminal repeat is present in each revertant site, and (iii) suggest that the virus induced the d mutation by integration into a noncoding sequence.

Alleles↗
PubMed2002

Spontaneous mobilization of integrated recombinant adenoassociated virus in a cell culture model of virus latency.

A cell line containing integrated recombinant adenoassociated virus (AAV) was investigated for spontaneous mobilization of vector sequence. Detection of these rare events was facilitated by using a vector design that allowed the circular rescue product (cAAV) to be individually scored by bacterial transformation. Restriction and sequence analysis of captured clones revealed five highly ordered classes of cAAV, each of which contained a defined segment of the integrated vector locus. A common feature of all cAAV classes was the presence of a modified inverted terminal repeat that joined the ends of the liberated sequence. Assembly of extrachromosomal vector genomes was accompanied by deletions in the integration locus that could be mapped to one of the five cAAV classes, suggesting an excision-type mechanism. We propose that the spontaneous deletion and mobilization of vector sequence from the recombinant adenoassociated virus (rAAV) integration locus is mediated by a recombination event between the inverted terminal repeats that define the boundaries of the individual genome subunits.

Cell Line↗
PubMed2006

DNA copy number amplification profiling of human neoplasms.

DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-of-origin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.

DNA Damage↗
PubMed1984

Cloning and analysis of integrated hepatitis virus sequences from a human hepatoma cell line.

We report here the isolation by molecular cloning and the analysis by heteroduplex and restriction enzyme mapping of seven distinct DNA fragments containing hepatitis B virus (HBV) sequences from genomic DNA of the PLC/PRF/5 human liver carcinoma cell line (the Alexander cell). No intact full-length HBV genomes were present. Three different patterns of organization of HBV fragments were detected. These included two linear fragments without detectable rearrangement, three other HBV fragments with internal deletions, and two HBV fragments containing long inverted duplications. HBsAg sequences are preferentially included in the integrated fragment, whereas the core gene is preferentially eliminated. Several of the integrated HBV fragments might act as templates for the synthesis of functional HBsAg mRNA, whereas only one clone could produce a full core antigen transcript.

Base Sequence↗