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At least 181 records · Page 10Linked to original sources
PubMed2004

Epstein-Barr virus is integrated between REL and BCL-11A in American Burkitt lymphoma cell line (NAB-2).

Epstein-Barr virus (EBV) initially isolated from the cultured Burkitt lymphoma (BL) cells, is one of the well-known oncogenic virus. The NAB-2 line, which was established from a North American Burkitt's tumor, was indicated to contain one copy of EBV DNA as the integrated form into chromosome 2p13 of the host genome. To demonstrate the integration site of EBV directly, and to clarify the relation between the integration sites and the oncogenes, fragments containing the nucleotide sequence of NAB-2 integration sites were cloned. EBV was integrated via the terminal repeats (TR), and integration sites located in the clone RP11-440P5 on chromosome 2, between two oncogenes, REL and BCL11A, which is apart from approximately 350 kbp from each other. Expression level of REL in NAB-2 was increased. The flanking region of chromosome 2 at the bilateral junction sites showed no homology to the junction sites of EBV. The integration site 2p13 overlaps with common fragile site, FRA2E. NAB-2 cells expressed almost all latent genes but LMP-2A that flanks the TR, indicating the type III of latent infection of EBV. Integration event in NAB-2 might alter the regulation of the oncogenes and provide advantage for continuous cell proliferation.

Base Sequence↗
PubMed1994

Purification of the integral membrane glycoproteins D of herpes simplex virus types 1 and 2, produced in the recombinant baculovirus expression system, by ion-exchange high-performance liquid chromatography.

Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Freijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr., 646 (1993) 37]. In the present study this novel approach was applied to the purification of the integral membrane glycoprotein D of Herpes simplex virus type 1 and 2. The glycoproteins D of types 1 (gD-1) and 2 (gD-2) were cloned into the baculovirus expression system and produced in protein-free cultured insect cells. Detergent extracts of recombinant baculovirus-infected insect cells containing gD-1 or gD-2 were prepared using pentaethyleneglycol monodecyl ether, for extraction (final concentration 2%, w/v). The same detergent was used as additive in the elution buffers for HPIEC on a Mono Q HR 5/5 column. At low (0.005%) detergent concentration, most of the proteins present in the extract including part of gD were eluted with the sodium chloride gradient whereas a subsequent blank run using the same gradient at higher detergent concentration (0.1%) resulted in selective elution of pure gD.

Animals↗
PubMed1988

Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.

Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).

Base Sequence↗
PubMed2006

Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites (+/-5kb), near CpG islands (+/-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

Cell Line↗
PubMed1977

Infectious DNA of spleen necrosis virus is integrated at a single site in the DNA of chronically infected chicken fibroblasts.

The infectious DNAs of a number of avian leukosis-sarcoma and reticuloendotheliosis viruses were digested with six nucleotide-specific restriction endonucleases, and the digests were tested for infectivity. All of the enzymes inactivated the viral infectivities except for EcoRI, which did not inactivate the infectivity of the DNA of two of the reticuloendotheliosis viruses, spleen necrosis and chick syncytial viruses. The infectious DNA of spleen necrosis virus after digestion with EcoRI had a buoyant density in CsCl solution greater than the density of the high-molecular-weight infectious viral DNA. The infectious EcoRI-digested spleen necrosis virus DNA from chronically infected chicken cells was uniform in size, 10 megadaltons, which indicated a single site of integration. The infectious EcoRI-digested spleen necrosis virus DNA from acutely infected cells was heterogeneous in size, ranging from 8-14 megadaltons, which indicated multiple sites of integration. These results are consistent with the hypothesis that cells that integrate infectious spleen necrosis virus DNA at a single site survive and multiply, whereas cells that integrate infectious viral DNA at additional sites either die or selectively lose or inactivate the DNA in the additional sites.

Acute Disease↗
PubMed1988

Herpes simplex virus-directed overreplication of chromosomal DNA physically linked to the simian virus 40 integration site of a transformed hamster cell line.

In the simian virus 40 (SV40)-transformed hamster cell line Elona herpes simplex virus (HSV) induces amplification of SV40 DNA sequences to high-molecular-weight head-to-tail concatemers indicating an extrachromosomal rolling circle replication. In order to enable investigations concerning intrachromosomal amplification of SV40 DNA sequences and flanking cellular sequences a genomic library of Elona DNA was constructed in phage lambda. Clones harboring cellular DNA adjacent to the SV40 integration site were isolated. Plasmid subclones devoid of SV40 DNA sequences were used as hybridization probes against total DNA from HSV-infected cells. Thus the amplification of both flanking cellular sequences was demonstrated, indicating a bidirectional replication mode.

Animals↗
PubMed2000

Lack of integrated TT virus (TTV) genomes in cellular DNA in infected human hematopoietic cells.

TT virus (TTV) isolated from the serum of a patient with posttransfusion hepatitis has been characterized as a member of the Circoviridae, a family of small DNA viruses with single-stranded circular genomes. TTV appeared to infect not only the serum and liver, but also the peripheral blood mononuclear cells (PBMC). We investigated the prevalence of TTV DNA in human hematopoietic cells, based on 84 mononuclear cell samples obtained from the bone marrow or lymph nodes of patients with hematopoietic malignancies including leukemia, malignant lymphoma and aplastic anemia. Forty-nine (58.3%) out of the 84 samples were positive for TTV DNA with polymerase chain reaction analysis, which was almost similar to the frequency found in the patients' serum. Southern blot analyses using a 3.2-kb fragment derived from the TTV DNA, however, showed no evidence supporting the fact that the TTV genomes are integrated into the human hematopoietic cell genomes, thus suggesting their existence as episomal forms.

Blotting, Southern↗
PubMedDate not supplied

[An analysis of the simian T-cell leukemia virus clonally integrated in baboon malignant lymphoma].

Proviral integration of a simian T-cell leukemia virus, S(H)TLV-I, highly homologous to human T-cell leukemia virus type 1 was examined in cellular DNAs isolated from lymphoid organs of lymphomatous and healthy baboons from the Sukhumi monkey colony. Most of the sick and some of healthy monkeys contained HTLV-I-related sequences in their chromosomal DNAs. Judging from the integration site of the provirus genome, S(H)TLV-I-infected cells had selective preferences and proliferated monoclonally.

Animals↗
PubMed1978

Morphological revertants of adenovirus type 12-transformed hamster cells.

Morphological revertants have been isolated from one line of adenovirus type 12-transformed hamster cells. This line, T637, is oncogenic in hamsters and contains multiple copies of the virus genome per cell. Different parts of the virus genome are represented in non-stoichiometric amounts and the virus DNA persists in the cells in an integrated form. The pattern of integrated virus genomes has been determined by the blotting technique. In the T637 line, morphological revertants arise spontaneously at relatively high frequency. Two of these revertants have been cloned. In contrast to the T637 line, the revertants F10 and G12 exhibit fibroblastic morphology. The patterns of integrated virus genomes in the revertants differs markedly from that of the T637 line; one of the revertant cell lines, F10, appears to have lost all virus DNA sequences. The morphological revertants continue to express the oncogenic phenotype, although the time required to produce tumours in animals appears to be prolonged compared to the parental BHK21 and the T637 cell lines. A number of biological parameters of the revertant lines have also been investigated.

Adenoviridae↗
PubMedDate not supplied

Integration site selection by retroviruses.

Integration into the host-cell genome is a critical step in the retrovirus life cycle. In particular, the choice of the integration site is crucial for retroviral replication, since integration at a site incompatible for high-level transcription may impair production of the progeny virus. Integration is not sequence specific, thus all chromosomal sites could potentially host integration events. However, this is not what is observed in vivo, where integrated viruses are preferentially detected in chromatin regions characterized by an open structure, a hallmark of actively transcribed genes. Target site selection might be influenced by several factors, including the function of cellular proteins that interact with integrase, the viral protein that catalyzes the integration reaction. Interestingly, a common functional feature that unifies these cellular co-factors is that, to a different extent, they are all involved in the regulation of chromatin structure or transcription. Inappropriate retroviral integration might lead to insertional mutagenesis and cellular transformation, as recently observed in a gene therapy clinical trial exploiting retroviral vectors for gene transfer into hematopoietic progenitors. Thus, the deeper understanding of the molecular mechanisms regulating integration site selection is also essential for the design of safer and more effective gene transfer vectors.

Chromatin↗
PubMed1991

[Hepatitis B virus and hepatocellular carcinoma].

Hepatocellular carcinomas in woodchuck were characterized for woodchuck hepatitis virus integration near c-myc oncogene. In one tumor, viral integration resulted in overexpression of a c-myc viral cotranscript. In a second tumor, viral insertion, 600 bp upstream of c-myc exon 1, was associated with increased levels of normal c-myc mRNA. These results demonstrate that integration of woodchuck hepatitis virus near a proto-oncogene can contribute to the genesis of liver tumors. From a comparison of a single hepatitis B virus (HBV) integration site in a human hepatoma with the corresponding unoccupied site have shown HBV DNA insertion in a putative cellular exon. This exon presented striking similarity to the DNA-binding domain of the thyroid/steroid hormones receptors. The corresponding cDNA has been isolated (hap gene) a shown to encode the retinoic acid receptor. It is most probable that consequent to HBV insertion, has became inappropriately expressed as an altered chimaeric gene retinoic acid receptor, thus contributing to the cell transformation. As for woodchuck these results strongly support the possibility that HBV may play a direct role in liver carcinogenesis by insertional mutagenesis.

Amino Acid Sequence↗
PubMed1993

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

3T3 Cells↗
PubMed1990

Mechanisms of thymic lymphomagenesis by the retrovirus SL3-3.

These studies report changes occurring in the thymus of AKR and NFS/N mice after infection with the lymphomagenic retrovirus SL3-3. In virus-infected AKR fetal thymus, the programmed cell death caused by treatment with antibody to CD3 was remarkably diminished. A method of establishing thymic stromal cultures from mice of 1 to 3 wk of age is described. Using this method, it was found that SL3-3 virus infection by neonatal inoculation allowed establishment of thymic stromal cultures from organs removed from AKR mice of 30 to 50 days of age and from lymphomas, whereas thymic stromal cultures could not be established from control mice after 30 days of age. Using NFS/N mice which have no endogenous virus, it was shown that infection of thymic stroma precedes infection of thymocytes and that thymocytes are permissive for infection with SL3-3 virus but not for the nononcogenic retrovirus, Akv, yet Akv virus replicates efficiently in thymic stroma. SL3-3 virus integrates randomly in each lymphoma induced by this virus. The lymphomas are clonal or oligoclonal. Pim-1 and c-myc genes commonly rearranged in other virus-induced thymic lymphoma showed rearrangement in only a few lymphomas. A theory is proposed, based on the work presented here and in recent studies, which states that SL3-3 virus infection of thymic stroma allows infection of thymocyte progenitors entering from the bone marrow. These cells are then altered so that their maturation is delayed and their intrathymic survival is prolonged. This permits virus integration and reintegration that results in the genetic changes which transform the cell.

Animals↗
PubMed1983

Characterization of a T-antigen-negative revertant isolated from a mouse cell line which undergoes rearrangement of integrated simian virus 40 DNA.

A transformation revertant has been isolated from an unusual line of simian virus 40 (SV40)-transformed BALB/c-3T3 cells in which rearrangements of integrated viral sequences are common. The revertant produces no SV40 T antigens, yields no virus on fusion with permissive cells, and can be retransformed by SV40 virions. SV40 DNA sequences are present within the cellular DNA, but interruption of the viral early transcription region by deletion and recombination with cellular sequences precludes the synthesis of T antigens. Analysis of this revertant lends further support to the notion that large T antigen plays an essential role in the maintenance of transformation in SV40-transformed BALB/c-3T3 cells. Examination of integration of SV40 DNA in this revertant, as well as in a temperature-sensitive A transformant, after retransformation by SV40 confirms that sequence homology plays little role in the insertion of SV40 DNA into cellular chromosomes.

Animals↗
PubMed2004

Human cell proteins and human immunodeficiency virus DNA integration.

Integration, catalyzed by the viral integrase (IN) protein, is a crucial step in the life cycle of all retroviruses including human immunodeficiency virus type 1 (HIV-1). Although purified HIV-1 IN protein is sufficient to catalyze the DNA breakage and joining steps of integration in the absence of any other protein factor, a number of studies indicate that cellular proteins participate in the integration process in cells. These host cell proteins have been proposed to act through binding the pre-integrated viral cDNA substrate, by directly interacting with the IN protein, and/or by repairing the single-stranded DNA gaps that occur at viral/chromosomal DNA junctions during integration. In this paper we summarize the identification and potential roles of specific cell factors in HIV-1 integration. We also present experimental results of human cell proteins that coimmunoprecipitated with HIV-1 IN following its expression in HeLa cells and discuss these results in light of the previously-identified integration cofactors.

Animals↗
PubMed1998

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence.

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

Amino Acid Sequence↗
PubMed1986

Analysis of six distinct integrated hepatitis B virus sequences cloned from the cellular DNA of a human hepatocellular carcinoma.

Six distinct hepatitis B virus (HBV) integrations and the flanking cellular sequences were cloned from a hepatoma DNA preparation. None of the cloned fragments retains the entire HBV sequences but the surface antigen (HBsAg) gene and the HBV enhancer are retained in three of the six clones. The other three clones carry only short and possibly highly rearranged HBV genomic sequences and seem to contain some GC-rich clusters. Members of the repetitive Alu family are also found in the vicinity of five of the six integration regions which may have contributed to genome instability. In these six clones, the preferred integration sites are shown to lie within the single-strand region of the HBV genome. None of the clones carries in the flanking cellular sequences any of the 17 oncogenes tested, although the possibility still exists that an oncogene may be found on the side of the genome which has not been cloned. This work thus paves the way for detailed sequence analysis of virus-host junctions, for transfection studies of the HBV integration events, and for a search of genes in the flanking cellular sequences which may have been activated by the retained HBV enhancer using the clones described.

Base Sequence↗