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PubMed2004

DNA-dependent PK inhibits adeno-associated virus DNA integration.

Recent studies have shown that recombinant adeno-associated virus (rAAV) can persist in episomal form; however, factors affecting rAAV persistence are poorly understood. DNA-dependent PK (DNA-PK) is a DNA repair enzyme, which we previously found played an important role in determining the molecular fate of the rAAV genome in mouse skeletal muscle. In the present study, we tested the effect of DNA-PK on AAV serotype 2 integration in vitro and in vivo in mouse liver. In an in vitro integration system, addition of DNA-PK decreased AAV integration, whereas antibody against DNA-PKcs increased integration. In vivo, matched doses of a recombinant AAV serotype 2 vector were injected into the portal vein of either C57BL/6 (DNA-PKcs(+/+)) or severe combined immunodeficient (DNA-PKcs(-/-)) mice. After partial hepatectomy to stimulate hepatocyte proliferation, retention of vector genomes and of transgene expression was substantially higher in severe combined immunodeficient mice, indicating that in the absence of DNA-PKcs, a greater proportion of genomes integrated into the cellular genome. In summary, we have provided evidence that DNA-PK inhibits AAV integration both in vitro and in vivo.

Animals↗

PubMed1997

Identification of a novel breast-cancer-anti-estrogen-resistance (BCAR2) locus by cell-fusion-mediated gene transfer in human breast-cancer cells.

Development of anti-estrogen resistance limits the benefit of endocrine therapy of breast cancer. The mechanistic basis for resistance to the anti-estrogen tamoxifen may involve (epi)genetic alterations within tumor cells. We have initiated a random search for genes allowing estrogen-dependent ZR-75-1 human breast-cancer cells to proliferate in the presence of tamoxifen. The strategy was based on insertion mutagenesis of ZR-75-1 cells using defective retrovirus and subsequent identification of common integration sites. As an alternative approach to identify integration loci involved in anti-estrogen resistance, we have applied cell fusion. Integration regions from lethally irradiated, tamoxifen-resistant cells were transferred to hygromycin B-resistant ZR-75-1 cells. Somatic cell hybrids were established by selection for resistance to G418 (encoded by the integrated virus) and hygromycin B. Individual integration loci were thus separated among different cell hybrids and tested for their role in anti-estrogen resistance. Analysis of a panel of 29 somatic-cell hybrids revealed that tamoxifen resistance co-segregated with only 1 of the 2 integration loci present in the tamoxifen-resistant donor cell line. This locus was further identified as a common integration site in our panel of tamoxifen-resistant cell clones. Our results designate this integration site as the second breast-cancer-anti-estrogen-resistance locus (BCAR2), which most likely contains a gene responsible for the anti-estrogen-resistant phenotype in close proximity to the integrated virus. Our data also imply that individual genes can alter the estrogen dependency of human breast-cancer cells in a dominant manner in vitro.

Breast Neoplasms↗

PubMed2006

Genome-wide mapping of foamy virus vector integrations into a human cell line.

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.

Cell Line↗

PubMed1978

A herpes simplex virus 1 integration site in the mouse genome defined by somatic cell genetic analysis.

Transfection experiments with HSV 1 in which one uses herpes simplex virus (HSV) thymidine kinase (TK) as a selectable prototrophic marker yield two classes of transformed cells: stable and unstable. In this report, we test the hypothesis that the stability phenotype can be explained by virus genome integration into a recipient cell chromosome. The method of analysis is by means of somatic cell genetics. We have isolated a series of microcell hybrids between a TK- Chinese hamster cell line and a transformed mouse cell line expressing the TK encoded by HSV 1. Several of the hybrid lines contain a single murine chromosome and express only the viral TK. Karyotypic analysis of these hybrids and of TK- derivatives generated by BrdUrd counterselection reveals that the TK+ phenotype is correlated with the presence of the terminal portion of the long arm of a specific murine chromosome. Results of extensive isozyme analyses of the hybrids and their TK- segregants fully corroborate the karyologic data. The results are consistent with the hypothesis that the viral tk gene is covalently integrated into this chromosomal region which itself does not appear to carry the endogenous murine tk locus. Other more complicated models are discussed. Our findings also show that somatic cell genetics can be used to localize viral integration sites in host chromosomes with high resolution.

Animals↗

PubMed2005

Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.

PURPOSE: Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. EXPERIMENTAL DESIGN: We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. RESULTS: The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. CONCLUSIONS: HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.

Adult↗

PubMed1982

Integrated shope virus DNA is present and transcribed in the transplantable rabbit tumour Vx-7.

To facilitate a molecular analysis of Shope papilloma virus-induced neoplastic cells, we have established a cell line from Vx-7, a transplantable tumour originally induced by the Shope virus. Single phase molecular hybridization and Southern transfer methods were employed to assess copy number and physical state of the DNA, and the extent of transcription. Both tumour and cell line were found to contain multiple copies of the virus genome and these were all integrated into the host cell DNA. Transcripts corresponding to a complexity of approx. 1% of the virus genome were detected at low abundance. These results are discussed relative to our earlier findings with tumours induced directly by virus, and to requirements for maintenance of the Vx-7 tumour over the 30 years that it has been in existence.

Animals↗

PubMed2005

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.

The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.

Chromosome Mapping↗

PubMed1991

[Hepatitis B virus and hepatocellular carcinoma].

Hepatocellular carcinoma in woodchuck were characterized for woodchuck hepatitis virus integration nea 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/steriod hormones receptors. The corresponding cDNA has been isolated (hap gene) as shown to encode the retinoic acid receptor. It is most probable that consequent to HBV insertion, hap gene 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↗

PubMed2000

Duck hepatitis B virus DNA integration induced by oxidative radicals and its transmission from parent cells to offspring ones.

OBJECTIVE: To investigate the role of oxidative radicals in duck hepatitis B virus DNA (DHBV DNA) integration and the transmission of integrated DHBV DNA from parent cells to their offspring cells. METHODS: TUNEL assay and Southern blot hybridization were used to detect the cellular DNA strand breaks and the incidence of new type of DHBV DNA integration into cellular DNA of LMHD(21-6) cell line after the cells treated by low dose of hydrogen peroxide (10.0 micromol/L) during its growth from one cell to 3 +/- 10(7) to 5 +/- 10(7) cells. RESULTS: The rate of cell death induced by low dose of H(2)O(2) (10.0 micromol/L) was 32.0%, but that induced by high dose (>10.0 micromol/L) was more than 50.0%. At the same time, the incidence of new DHBV DNA integration into cellular DNA induced by 10.0 micromol/L of H(2)O(2) was 50.0% (6/12) and that in control group was 8.33% (1/12). There was a significant difference between the two groups (P<0.05). Besides, the new integrated DHBV-DNA in the parent cells could be found in the DNA of their offspring cells with the same size of base pair. CONCLUSION: Oxidative radicals is one of the important inducing factors in DHBV-DNA integration into cellular DNA and the integrated DHBV-DNA is able to transmit from the parent cells to their offspring cells stably.

Animals↗

PubMed1990

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1988

The structure and function of the integrated polyoma virus DNA in 82-rat and 53-rat transformed cells.

Integrated viral sequences and adjacent cellular sequences from the polyoma virus (Py)-transformed 53-Rat and 82-Rat cell lines which contain two and three partial early regions respectively, each in a single viral insert, have been molecularly cloned. Each of the cloned partial early regions have been subcloned and assessed with regard to their transcription, translation products (T antigens, T Ags) and biological activity including their transforming ability. The 53-Rat 5.3 kb EcoRI fragment is an intact Py EcoRI linear genome (derived from within the tandem duplicated sequences) which transforms rat cells with high efficiency and produces infectious virus when circularized and transfected into mouse cells. The 82-Rat cell line expresses three novel T Ag species of 63K, 40K and 32K in addition to the Py middle and small T Ags. The 63K protein was found to be a truncated form of large T Ag produced as the result of an addition/deletion in early region B sequences unique to large T Ag. The 40K and 32K proteins are hybrid viral-cellular middle and large T Ags respectively, which are expressed from early region A that has been truncated by recombination with rat cellular DNA. Differences in the nuclear and cytoplasmic location of the different 82-Rat early region RNAs are due to RNA stability and/or transport from the nucleus to the cytoplasm most likely as a result of different cellular sequences at their 3' ends. Finally no common structural feature or sequence specificity was observed at the virus-host DNA joins of the two cell lines.

Animals↗

PubMed1996

Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells.

The vaccinia virus Western Reserve A38L protein is a hydrophobic integral membrane glycoprotein with amino acid similarity to mammalian integrin-associated protein. The protein has an N-terminal immunoglobulin superfamily domain, followed by five membrane-spanning domains and a short cytoplasmic tail. Deletion of the protein reduces virus plaque size but does not affect virus virulence (J. E. Parkinson, C. M. Sanderson, and G. L. Smith, Virology, in press). In this study, we have used a recombinant vaccinia virus in which the A38L gene may be inducibly overexpressed by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG), to demonstrate that overexpression of the vaccinia virus A38L gene produces drastic changes in the morphology, permeability, and adhesion of infected cells. In particular, A38L overexpression caused swelling of cells, marginalization of nuclear chromatin, and vacuolization of the endoplasmic reticulum, features characteristic of cell necrosis. By 18 h postinfection, cells become permeable and lytic as defined by the free entry of propidium iodide and loss of the cytoplasmic enzyme lactate dehydrogenase. Chelation of extracellular Ca2+ 22 h postinfection inhibited further release of lactate dehydrogenase, showing that Ca2+ influx was required for A38L-induced lysis. Direct measurement of 45Ca2+ influx showed that the rate of Ca2+ uptake was directly related to the period of A38L induction. The A38L protein, therefore, promotes the formation of pores within the plasma membrane of cells, and these pores facilitate Ca2+ entry and induce necrosis. Addition of rifampin inhibited virus assembly but not the ability of A38L to induce necrosis, indicating that pore formation is independent of viral morphogenesis. Finally, overexpression of the A38L protein resulted in a reduced plaque size and a threefold decrease in production of infective particles in vitro. The A38L protein represents the first example of a virus protein which directly or indirectly promotes the influx of extracellular Ca2+.

Calcium↗

PubMed1986

Primary hepatocellular carcinoma with hepatitis B virus-DNA integration in a 4-year-old boy.

The autopsy findings in a case of hepatocellular carcinoma with hepatitis B virus (HBV)-DNA integration in a Japanese boy, 4 years and 10 month of age, are reported. The boy was an HBV carrier born in a typical familial cluster of HBV infection in Japan. He had been asymptomatic until the sudden manifestation of the liver tumor. Histopathologic examination revealed a well-differentiated, adult-type hepatocellular carcinoma without hepatic cirrhosis. HBV-DNA sequences were detected in the tumor cell DNA by the Southern blot hybridization method. This is the youngest patient with hepatocellular carcinoma with HBV-DNA integration reported to date, suggesting that HBV may manifest its oncogenic properties after a shorter incubation period than generally believed.

Antigens, Viral↗

PubMed1997

Direct detection of hepatitis B virus gene integrated in the Alexander cell using fluorescence in situ polymerase chain reaction.

Prolonged infection of hepatitis B virus (HBV) is reported to cause hepatocellular carcinoma (HCC) via liver cirrhosis. However, it is still unknown whether the HCC is induced by the HBV DNA integration or by inflammatory stimulation during the phase of liver cirrhosis. The aim of this study is to determine the intracellular or intranuclear distribution of HBV DNA with a highly sensitive assay. Here we directly detected the integration of HBV DNA by fluorescence in situ polymerase chain reaction (FISPCR). Since FISPCR products directly incorporate rhodamine-4-dUTP, the nucleus of Alexander cells integrated with HBV gene reacted with the HBV primers emits obvious fluorescence. The fluorescence values which were measured with an imaging analyzer show a significant difference between Alexander cells as compared to the controls. In conclusion, the target sequences of HBV were specifically amplified as fluorescent DNA after the present FISPCR procedure. This method could provide a novel and simple strategy for determining the quantitative role of viral DNA integration in oncogenesis.

Carcinoma, Hepatocellular↗

PubMed1992

Friend murine leukaemia virus is integrated at a common site in most primary spleen tumours of erythroleukaemic animals.

A common proviral integration site was identified and characterized in erythroleukaemias induced by Friend murine leukaemia virus (F-MuLV). Using inverse polymerase chain reaction, we found a proviral integration site common to at least 90% of 20 primary tumours tested. This site was identical to Fli-1, a locus recently reported by others to be rearranged in 75% (9/12) of cell lines established from spleens of erythroleukaemic mice and to code for a member of the ets gene family. Our data suggest that about half of the F-MuLV-induced erythroleukaemias contained more than one cell clone with a proviral integration in Fli-1, with different individual integration sites within Fli-1 in each cell clone. All proviruses were found to be integrated in the same transcriptional orientation with respect to flanking cellular DNA. We discuss these findings in relation to multistage models of neoplasm.

Animals↗

PubMed1983

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome.

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.

Animals↗

PubMed2006

Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor.

The mechanisms controlling retroviral integration have been the topic of intense interest, in part because of adverse clinical events that occurred during retrovirus-mediated human gene therapy. Here we investigate the use of artificial tethering interactions to constrain retroviral integration site selection in an in vitro model. During normal infection, HIV DNA integration is favored in active cellular transcription units. One component of the targeting mechanism is the cellular LEDGF/p75 protein. LEDGF/p75 binds tightly to HIV integrase (IN) protein, and depletion of LEDGF/p75 from target cells results in reduced integration in transcription units, suggesting integration targeting by a tethering mechanism. We constructed and analyzed fusions of LEDGF/p75 or its IN-binding domain (IBD) to the DNA-binding domain of phage lambda repressor protein (lambdaR). In the presence of the lambdaR-LEDGF/p75 fusions, increased strand transfer by IN was seen in target DNA near lambdaR-binding sites in vitro . These data support the idea that a direct interaction between LEDGF/p75 and IN can mediate targeting via a tethering mechanism, and provide proof of concept for the idea that protein-protein interactions might be engineered to constrain integration site selection during human gene therapy.

Base Sequence↗

PubMed2002

Deletion of T cells bearing the V beta8.1 T-cell receptor following mouse mammary tumor virus 7 integration confers resistance to murine cerebral malaria.

Plasmodium berghei ANKA induces a fatal neurological syndrome known as cerebral malaria (CM) in susceptible mice. Host genetic elements are among the key factors determining susceptibility or resistance to CM. Analysis of mice of the same H-2 haplotype revealed that mouse mammary tumor virus 7 (MTV-7) integration into chromosome 1 is one of the key factors associated with resistance to neurological disease during P. berghei ANKA infection. We investigated this phenomenon by infecting a series of recombinant inbred mice (CXD2), derived from BALB/c (susceptible to CM) and DBA/2 (resistant to CM) mice, with P. berghei ANKA. We observed differences in susceptibility to CM induced by this Plasmodium strain. Mice with the MTV-7 sequence in their genome were resistant to CM, whereas those without integration of this gene were susceptible. Thus, an integrated proviral open reading frame or similar genomic sequences may confer protection against neuropathogenesis during malaria, at least in mice.

Animals↗