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PubMed2004

Integration target site selection for retroviruses and transposable elements.

When a retrovirus infects a cell, its RNA genome is reverse transcribed into a double-stranded DNA, which is then permanently integrated into the host chromosome. Integration is one of the essential steps in the retroviral life cycle. Many transposable elements also move around and integrate into the host genome as part of their life cycle, some through RNA intermediates and some through 'cut and paste' mechanisms. Integration of retroviruses and transposable elements into 'sensitive areas' of the genome can cause irreparable damage. On the other hand, because of their ability to integrate permanently, and the relatively efficient rates of transgenesis, retroviruses and transposable elements are widely used as gene delivery tools in basic research and gene therapy trials. Recent events in gene therapy treatments for X-linked severe combined immunity deficiencies (X-SCID) have highlighted both the promise and some of the risks involved with utilizing retroviruses. Nine of 11 children were successfully treated for X-SCID using a retrovirus carrying the gene mutated in this disease. However, later two of these children developed leukemias because of retroviral integrations in the putative oncogene LMO2 [1]. A third child has also been demonstrated to have an integration in LMO2, but is as of yet nonsymptomatic [2]. It is a bit difficult to explain the high frequency of integrations into the same gene using a random model of retroviral integration, and there has been evidence for decades that retroviral integrations may not be random. But the data were somewhat limited in their power to determine the precise nature of the integration biases. The completion of the human genome sequence coupled with sensitive polymerase chain reaction techniques and an ever-decreasing cost of sequencing has given a powerful new tool to the study of integration site selection. In this review, we describe the findings from several recent global surveys of target site selection by retroviruses and transposable elements, and discuss the possible ramifications of these findings to both mechanisms of action and to the use of these elements as gene therapy vectors.

Animals↗
PubMed2004

Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.

Animals↗
PubMed2003

Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. Greater than 99% of all cervical tumors contain HPV DNA. Integration of high-risk HPV has been temporally associated with the acquisition of a malignant phenotype. Recent work from our lab has shown that HPV16, the most common high-risk HPV associated with cervical carcinoma, preferentially integrates at loci containing human common fragile sites (CFSs). CFSs are regions of genomic instability that have also been associated with deletions, translocations, and gene amplification during cancer development. The current work shows that HPV18, the second most prevalent high-risk HPV type found in cervical tumors, preferentially targets the CFSs. We identified 27 unique HPV18 integrations in cervical tumors, of which 63% (P<0.001) occur in CFSs. However, the distribution of HPV18 integrations found were profoundly different from those found for HPV16. Specifically, 30% of all HPV18 integrations occurred within the chromosomal band 8q24 near the c-myc proto-oncogene. None of the HPV16 integrations occurred in this region. Previous low-resolution mapping suggested that c-myc may be a target of HPV integration. Our data at nucleotide resolution confirm that in HPV18-positive cervical tumors, the region surrounding c-myc is indeed a hot spot of viral integration. These results demonstrate that CFSs are preferred sites of integration for HPV18 in cervical tumors. In addition, we have identified multiple cellular genes that have been disrupted by HPV18 integration in cervical tumors. Our results suggest that the sites of HPV18 integration are nonrandom and may play an important role in the development of cervical tumors.

Chromosome Mapping↗
PubMed2002

Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors.

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of approximately 10(-4) per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA(Arg) gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.

Animals↗
PubMed2004

Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Animals↗
PubMed1994

Integration of human papillomavirus type 6a DNA in a tonsillar carcinoma: chromosomal localization and nucleotide sequence of the genomic target region.

Human papillomavirus type 6a (HPV 6a) DNA was detected in a tonsillar carcinoma both as integrated and episomal molecules, and one viral-cellular junction was molecularly cloned (Bercovich et al., J. Gen Virol., 72: 2569-2572, 1991). The cellular sequence was used as a probe for the isolation of a cosmid from a normal human genomic DNA library. A 2.7-kilobase subclone including the integration site was sequenced. It was shown to contain sequences with similarities to the E2 and L2 regions of human papillomaviruses, a 5' truncated long interspersed repeated DNA element type 1 retrotransposon, and a fragment of an O-repeat element. The chromosomal localization of the integration site was determined to be at region 24 of the long arm of chromosome 10 (10q24). This is the region where the fragile site is located in which HPV 18 DNA is integrated in the cell line FEP18-5. In addition it contains the site of breakpoints affecting protooncogenes Hox11 and Lyt10. Other genes related to cell division and DNA repair have also been mapped to this chromosomal band. Analysis of genomic DNA of cell lines and patients using 10q24-derived probes is presented. The integration of human papillomavirus type 6 DNA into chromosome 10q24 may have disrupted a cellular gene critical for normal cell growth, which further analysis should help to identify.

Base Sequence↗
PubMed1994

HTLV-II non-integrated malignant lymphoma induction in Japanese white rabbits following intravenous inoculation of HTLV-II-infected simian leukocyte cell line (Si-IIA).

Lymphoma induction in rabbits by an unknown factor derived from an HTLV-II-producing simian (Cynomolgus) leukocyte cell line (Si-IIA) is reported. Thirteen of 17 male Japanese white rabbits (76%) inoculated intravenously with Si-IIA cells developed malignant lymphoma including Hodgkin-like lymphoma between 62 and 167 days after inoculation. Histologically, there was extensive diffuse or nodular infiltration of either large cell type or mixed type lymphoma cells in many organs, frequently involving the spleen, liver, lymph nodes and kidneys, and less frequently the thymus, bone marrow, lungs, heart, skin and gastrointestinal tract. Hodgkin-like lymphoma was also observed in two rabbits. Chromosomal analysis of five cell lines established from tumor-bearing rabbits revealed the male rabbit karyotype. The immunophenotype of these tumor cells was usually T-cell (CD5+ or -, RT1+, RT2+ or -, CD45+, CD4-, RABELA- and MHC class II-DQ+) except for Hodgkin-like lymphoma cells which expressed only CD45. However, integration of the HTLV-II provirus genome could not be demonstrated in the tumor tissues or any of the rabbit cell lines by polymerase chain reaction or Southern blot analysis. Moreover, no lymphoma was induced by inoculation of HTLV-IIC, MOT (other HTLV-II-producing human cell lines) or TALL-1 (control). Two of four rabbits injected with cell-free pellets from Si-IIA cultures died of malignant lymphoma (15-20 days). Five irradiated rabbit cell lines were inoculated but only one (Ra-SLN) induced lymphoma in 1 of 3 rabbits at 27 days. Neither Herpesvirus saimiri nor Herpesvirus ateles (simian oncogenic viruses) was detected in Si-IIA cells by immunofluorescence testing. These data suggest that the high rate of lymphoma induction in rabbits may be caused not by only HTLV-II or well known simian oncogenic viruses, but rather by an unknown passenger agent derived from Si-IIA or HTLV-IIA, with which Si-IIA was established.

Animals↗
PubMed2006

Extensive phage dynamics in Staphylococcus aureus contributes to adaptation to the human host during infection.

Bacteriophages serve as a driving force in microbial evolution, adaptation to new environments and the pathogenesis of human bacterial infections. In Staphylococcus aureus phages encoding immune evasion molecules (SAK, SCIN, CHIPS), which integrate specifically into the beta-haemolysin (Hlb) gene, are widely distributed. When comparing S. aureus strain collections from infectious and colonizing situations we could detect a translocation of sak-encoding phages to atypical genomic integration sites in the bacterium only in the disease-related isolates. Additionally, significantly more Hlb producing strains were detected in the infectious strain collection. Extensive phage dynamics (intragenomic translocation, duplication, transfer between hosts, recombination events) during infection was shown by analysing cocolonizing and consecutive isolates of patients. This activity leads to the splitting of the strain population into various subfractions exhibiting different virulence potentials (Hlb-production and/or production of immune evasion molecules). Thus, phage-inducing conditions and strong selection for survival of the bacterial host after phage movement are typical for the infectious situation. Further in vitro characterization of phages revealed that: (i) SAK is encoded not only on serogroup F phages showing a conserved tropism for hlb but also on serogroup B phages which always integrate in a distinct intergenic region, (ii) the level of sak transcription correlates to phage inducibility but is independent of the phage localization in the chromosome, and (iii) phages can be stabilized extra-chromosomally during their life cycle.

Attachment Sites, Microbiological↗
PubMed1993

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Adenoviruses, Human↗
PubMed1993

Nucleotide sequence of attP and cos sites of phage CTX and expression of cytotoxin in Pseudomonas aeruginosa PA158.

The gene for Pseudomonas aeruginosa cytotoxin (CTX) has been found to be part of a temperate phage with a total size of 35.5 kb. We have investigated several DNA fragments of this phage for CTX production. For phage integration, the phage genome cohesive (cos) ends covalently associate with host DNA of strain PA158. The cos ends and the CTX gene are found on a 3.4 kb EcoRI fragment B and are included in the 11 kb HindIII fragment A and the 8.5 kb BamHI fragment B of the phage DNA. The cos ends are 20 nucleotides long and are located at 338-357 nucleotides upstream of the CTX transcriptional initiation site. The phage attachment (attP) site is also present on the 3.4 kb EcoRI fragment B. The attP site consists of 34 bp and is located at 974-1007 nucleotides upstream of the CTX gene start site. Replication of the vegetative form of the phage is increased at 37 degrees C compared to that at 30 degrees C, while cytotoxin production in infected cells is similar at 30 degrees C and 37 degrees C. It can be concluded, therefore, that the integrated form of the CTX gene is responsible for CTX production. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed ten proteins in purified phage preparations; however, CTX could not be detected on Western blots using an enzyme-linked immunofluorescence assay.

Base Sequence↗
PubMed1995

Human papillomavirus and disease-free survival in FIGO stage Ib cervical cancer.

To determine if human papillomavirus (HPV) in the primary tumor was associated with disease-free survival of stage Ib cervical cancer patients, archival tissues from 47 patients were analyzed for HPV DNA by in situ hybridization (ISH) and polymerase chain reaction. HPV integration was determined by ISH signal pattern. Laboratory data were correlated with clinical parameters and disease-free survival. Kaplan-Meier estimates of 4-year disease-free survival were 56% in women with HPV detected in the primary tumor by ISH and 100% for women in whom HPV was not detected (P = .02). Four-year disease-free survival was 39% for patients with integrated HPV in the primary tumor (P = .005 vs. HPV-negative tumors and .05 vs. HPV episomal or episomal/integrated). HPV detection and integration state was not associated with any other clinical variable. Detection of integrated HPV DNA in the primary tumor was strongly associated with treatment failure.

Adult↗
PubMed1993

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.

Bacteriophage lambda↗
PubMed2004

Diversity in the lysis-integration region of oenophage genomes and evidence for multiple tRNA loci, as targets for prophage integration in Oenococcus oeni.

The central genomic regions of Oenococcus oeni phages fOg30 and fOgPSU1 have been compared with the equivalent regions of oenophages fOg44 and phi 10MC. In all cases, an almost identical endolysin gene was followed by one of two orfs, encoding putative holins (orf117 and orf163). The fOg44 endolysin was established as a secretory protein when expressed in Lactococcus lactis. Orf117 (from fOg44) promoted lysis of Escherichia coli cultures upon induction of a defective lambda Sam7 prophage, but Orf163 (from fOg30) failed to elicit a lysis response in this system. fOg44 and fOgPSU1 were shown to integrate at the 3' end of a tRNA(Glu) and a tRNA(Lys), respectively. Searching the available sequence of the O. oeni MCW genome for attP-like elements, two other tRNA targets could be proposed for prophage establishment. Between the lysis and integration elements, a diverse cluster of genes (absent in phi 10MC) was observed. One common gene in this "lysogenic conversion cluster" was experimentally confirmed as a transcriptional repressor, affecting the expression of a putative permease gene.

Amino Acid Sequence↗
PubMed2006

Only those genes of the KIAA1245 gene subfamily that contain HERV(K) LTRs in their introns are transcriptionally active.

Insertion of LTRs into some genome locations might seriously affect regulation of the neighboring genes expression. This hypothesis is widely accepted but, however, not confirmed directly. Earlier, we have identified a family of closely related genes highly similar to the KIAA1245 mRNA counterpart. This family included a subfamily of genes some of which contained and the others lacked an LTR in their structure. We compared transcription of several closely related genes of the subfamily differing in the presence or absence of LTRs. Only LTR-containing genes were transcribed in transformed cell lines, tumorous and embryonic human tissues, whereas LTR-lacking genes remained silent. Since the genes were in the same intracellular microenvironment, we suggested that this effect was most probably due to intrinsic cis-characteristics of integrated LTRs and confirmed this by demonstrating high enhancer activity of KIAA1245 LTRs. The expression of the LTR-containing genes in embryonic tissues might suggest their involvement in evolutionary events during primate speciation.

Brain↗
PubMed2003

Retrovirally transduced muscle-derived cells contribute to hematopoiesis at very low levels in the nonhuman primate model.

Recent studies have suggested a remarkable potential of adult stem cells from a variety of organs to give rise to cells of disparate organs, but evidence of such potential at a clonal level is lacking in most if not all studies to date. To assess directly the hematopoietic potential of muscle-derived cells in a relevant large animal, we initiated retroviral-tagging studies in the rhesus macaque to allow tracking at the clonal level by integration site analysis. Four rhesus macaques underwent transplantation with transduced muscle-derived cells after lethal irradiation followed by delayed infusion of an autologous hematopoietic graft. The first animal showed no evidence of hematopoietic recovery and, despite infusion of the backup hematopoietic graft, succumbed due to complications of prolonged cytopenias. In the remaining three animals, the overall contribution of retrovirally tagged muscle-derived cells toward hematopoiesis was exceedingly low. Retroviral integration site analysis among clonally derived muscle cells and bone marrow cells in vivo in one animal suggests a common source. These results demonstrate that harvesting disparate organs for cellular therapy is currently highly inefficient at best.

Animals↗
PubMed2000

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene.

The integrase gene (int) on the genome of &phi;FSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The &phi;FSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.

Amino Acid Sequence↗
PubMed2006

Transmission of integrated human herpesvirus 6 through stem cell transplantation: implications for laboratory diagnosis.

We identified a stem cell donor with chromosomally integrated human herpesvirus (HHV)-6 and monitored the recipient for HHV-6 after transplantation. The appearance and subsequent increase in HHV-6 load paralleled engraftment and an increase in white blood cell count. Fluorescent in situ hybridization analysis showed integrated HHV-6 on chromosome band 17p13.3 in the donor and in the recipient after transplantation but not in the recipient before transplantation. The increase in viral load due to the genetic transmission of integrated HHV-6 could have been misinterpreted as substantial active infection and, thus, led to the administration of toxic antiviral therapy. We suggest that the confounding influence of integration be considered in laboratory investigations associating HHV-6 with disease.

Adult↗
PubMed1997

Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability.

BACKGROUND: Several lymphomas are associated with Epstein-Barr virus (EBV) infection. However EBV is not detectable in 100% of cases using standard staining techniques. It still remains an open question whether in these EBV-negative cases EBV has never infected the cell, whether it has infected the cell and escapes conventional screening methods, or whether it has been lost again after initial infection. MATERIALS AND METHODS: The physical status of EBV in the Burkitt's lymphoma cell line BL60-P7 as well as in three somatic cell hybrids between BL60-P7 and its autologous EBV-immortalized lymphoblastoid cell line IARC 277 was analyzed using conventional cytogenetics, Southern blotting, and fluorescence in situ hybridization (FISH). RESULTS: Integration of EBV into the host genome of the lymphoma cell line BL60-P7 leads to an achromatic gap which causes a 'vulnerable site'. In hybrid cells, loss of integrated EBV, together with an adjacent chromosomal fragment, occurs during long-term cultivation. The integrated EBV genome, including genes encoding for LMP and EBER, is partly deleted. CONCLUSION: We assume that integration of EBV into the host cell genome could be a more common event in lymphoma cells. Partially deleted EBV might escape standard detection assays. The integration might constitute a chromosomal region prone to break events akin to the phenomenon of fragile sites, leading to the loss of viral DNA as well as chromosomal DNA. This observation makes it tempting to speculate that under certain conditions EBV can act in lymphomagenesis by a so-called hit-and-run mechanism.

Blotting, Southern↗