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PubMed2003

Scaffold attachment region located in a locus targeted by hepadnavirus integration in hepatocellular carcinomas.

The role of viral integration in HBV induced hepatocellular carcinoma (HCC) is still controversial. In the WHV/woodchuck animal model, WHV integration was found to activate the N-myc2 oncogene either by enhancer insertion in proximity of the gene, or by integration in a distantly located uncoding locus, win. In addition, we have reported that N-myc2 activation also results from WHV integration in the b3n locus, located several kilobases downstream of N-myc2. In this work we report the search for function(s) of the b3n locus that might be possibly affected by WHV integration and indirectly activate N-myc2. A 0.5 kb region of the sequence of this locus exhibited unusual features, typical of scaffold/matrix attachment regions (S/MAR). Standard in vitro binding assays are commonly used to assess if a DNA fragment is a S/MAR. DNA fragments cloned from the b3n locus were tested for in vitro binding affinity for both heterologous and autologous nuclear scaffold preparations. Only the fragment spanning the region rich of S/MAR motifs was found to bind specifically nuclear scaffolds, thus demonstrating that a S/MAR element is present in the b3n locus. Based on these findings, we speculate that WHV integration might deregulate the S/MAR element and indirectly affect the expression of the N-myc2 gene located upstream of the S/MAR. Our findings also suggest that the role of HBV integration should be reconsidered, because a similar mechanism has not been investigated to date in human HCC.

Animals↗
PubMed1997

A highly efficient and stable system for site-specific integration of genes and plasmids into the phage phiLC3 attachment site (attB) of the Lactococcus lactis chromosome.

An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed. A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication. Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L. lactis. The above results, the observation that the phiLC3 attB site appear to be conserved in L. lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L. lactis.

Bacteriophages↗
PubMed2003

A process for specifying integration for multi-tier applications in healthcare.

Integration of the heterogeneous application systems in healthcare is needed to support the clinical and administrative work, patient-centric care, regional interoperability and utilisation of both valuable legacy systems and new technologies. The integration activity is complicated, and must be supported by flexible processes and methods. The integration methods should support the evaluation and specification of different integration approaches, standards and technologies on many interoperability levels. We introduce a process for specifying many integration decisions for a given integration situation. The process is part of an integration method, which is validated in PlugIT project in Finland.

Decision Making, Computer-Assisted↗
PubMed2003

Quantitative analysis of human immunodeficiency virus type 1 DNA dynamics by real-time PCR: integration efficiency in stimulated and unstimulated peripheral blood mononuclear cells.

We established a set of real-time PCR assay to accurately quantify human immunodeficiency virus type 1 (HIV-1) DNA in infected cells. Using this assay we were able to measure the strong-stop, full-length/ 1-LTR circle, 2-LTR circle, and integrated forms of viral DNA, and the data provided was quite consistent with the characteristics of mutant viruses in early phase of infection. Since our assay is particularly applicable to quantify the integrated DNA in small scale of samples, we measured the level of integrated DNA in wild-type virus (WT)- or Vpr-defective virus (deltaVpr)-infected peripheral blood mononuclear cells (PBMC), and examined whether quiescent condition of the PBMC influences integration step of HIV-1. Under stimulating condition approximately 25% of total viral DNA was in integrated form in either WT- or DeltaVpr-infected cells. In contrast, under unstimulated condition the level of integration efficiency was not significantly reduced in WT-infected cells, while this efficiency was severely impaired in the absence of vpr gene. This result clearly demonstrated a crucial role of the Vpr for nuclear localization and subsequent integration of viral DNA in nondividing cells. Therefore, our assay is useful for analyzing the events in early phase of HIV-1 infection under various conditions.

Cell Culture Techniques↗
PubMed2004

Evidence of chromosomal integration of AAV DNA in human testis tissue.

Persistent infection with adeno-associated virus (AAV) has been demonstrated in human tissues, most frequently in the female and male genital tract. The clinical significance of latent AAV infection remains, however, uncertain to date. The mode of latency of AAV is not known, i.e., it is unclear whether the viral genome is integrated in the cellular genome, and if integration occurs site-specifically in chromosome 19 as has been observed in cell culture. Therefore we investigated if viral DNA in AAV DNA-positive human testis samples from two patients, is integrated in the cellular genome. Using two different molecular approaches, uni-directional PCR and Walking Primer PCR, we could demonstrate that AAV DNA is present in an integrated form in testis tissue. Virus-cell DNA junction fragments were cloned and sequenced. A detailed analysis revealed integration within sequences of the so-called AAVS1 region on chromosome 19. These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases.

Base Sequence↗
PubMed2002

Filamentous phage integration requires the host recombinases XerC and XerD.

Many bacteriophages and animal viruses integrate their genomes into the chromosomal DNA of their hosts as a method of promoting vertical transmission. Phages that integrate in a site-specific fashion encode an integrase enzyme that catalyses recombination between the phage and host genomes. CTX phi is a filamentous bacteriophage that contains the genes encoding cholera toxin, the principal virulence factor of the diarrhoea-causing Gram-negative bacterium Vibrio cholerae. CTX phi integrates into the V. cholerae genome in a site-specific manner; however, the approximately 6.9-kilobase (kb) CTX phi genome does not encode any protein with significant homology to known recombinases. Here we report that XerC and XerD, two chromosome-encoded recombinases that ordinarily function to resolve chromosome dimers at the dif recombination site, are essential for CTX phi integration into the V. cholerae genome. The CTX phi integration site was found to overlap with the dif site of the larger of the two V. cholerae chromosomes. Examination of sequences of the integration sites of other filamentous phages indicates that the XerCD recombinases also mediate the integration of these phage genomes at dif-like sites in various bacterial species.

Attachment Sites, Microbiological↗
PubMed1997

Recurring proviral integration suggests a role for proto-oncogene activation in thymomas induced with Mo-MuLV-rescued BCR/ABL virus.

Intrathymic injection of Moloney murine leukemia virus (Mo-MuLV)-pseudotyped bcr-abl retrovirus (bcr-abl/M) causes thymic lymphoma but only after a prolonged latent period similar to that seen after intrathymic injection of Mo-MuLV alone. Since thymomas induced by Mo-MuLV show recurring proviral integration near certain cellular proto-oncogenes, it was reasoned that if the pathogenesis of bcr-abl/M thymomas is affected by viral integration, then it may be possible to detect proviral insertion near common Mo-MuLV integration sites in bcr-abl-induced thymomas. A panel of thymomas induced by intrathymic injection of Mo-MuLV, Abelson murine leukemia virus (A-MuLV), or the bcr-abl/M virus was analyzed for proviral integration near c-myc, N-myc, Pim-1, and Mlvi-1 loci that are frequently occupied by provirus in Mo-MuLV-induced T cell lymphomas, and for integration near Ahi-1 that is often occupied in A-MuLV/M-induced pre-B cell lymphoma. As expected, thymomas induced with Mo-MuLV showed frequent rearrangements in these loci while thymomas induced with A-MuLV/M (which does not require Mo-MuLV) did not. The bcr-abl/M-induced tumors also showed recurring proviral integration near c-myc, Pim-1 and Mlvi-1, albeit at a lower frequency than seen in the Mo-MuLV tumors. Unexpectedly, four independent thymomas that were clearly of T cell origin demonstrated proviral integration within the Ahi-1 region which was previously thought to only occur in A-MuLV/M induced pre-B cell lymphoma. These observations suggest that recurring proviral insertion in c-myc, Pim-1, Mlvi-1, and Ahi-1 may provide a selective advantage for bcr-abl/M transformed T lymphoid cells. This model may provide a tool for identifying cellular genes that can cooperate with bcr-abl in lymphoid transformation.

Fusion Proteins, bcr-abl↗
PubMed1999

Mrvi1, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1.

Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5' end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is down-regulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene.

Alternative Splicing↗
PubMed2002

Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites.

The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes.

Animals↗
PubMed2003

Common fragile sites are preferential targets for HPV16 integrations in cervical tumors.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at both the cytogenetic and molecular levels. To further explore this relationship at the molecular level, we used RS-PCR to rapidly isolate cellular sequences flanking the sites of HPV16 integration in 26 primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on aphidicolin-stimulated metaphases. Our data demonstrate that 11/23 HPV16 integrations in cervical tumors occurred within CFSs (P&<0.001). In addition, we show that deletions and complex rearrangements frequently occur in the cellular sequences targeted by the integrations and that integrations cluster in FRA13C (13q22), FRA3B (3p14.2), and FRA17B (17q23). Finally, our data suggest that cellular genes, such as Notch 1, are disrupted by the HPV16 integrations, which may contribute to the malignant phenotype.

Carcinoma, Squamous Cell↗
PubMed1994

Human immunodeficiency virus type 1 may preferentially integrate into chromatin occupied by L1Hs repetitive elements.

Human DNA flanking sites of eight human immunodeficiency virus type 1 (HIV-1) proviral integrations have been analyzed in isolates derived both from integrations in an infected individual and from tissue culture. Sequence analysis encompassing 80-3000 bp of human DNA on one or both sides of the site of integration revealed that seven of the eight HIV-1 proviruses had integrated directly into or within one nucleosome's distance from an L1Hs or Alu repetitive element. To compare this with the frequency at which human L1 or Alu elements sharing > or = 70% identity with L1Hs and Alu consensus sequences would be encountered at random, > 200 bp from each of 82 individual anonymously cloned segments of human DNA were sequenced: L1Hs elements were encountered in 8.5% of the 82 clones and Alu elements were encountered in 13.4+ by using these homology windows. From these data it appears that HIV-1 integrates into or near L1Hs elements with an approximately 6-fold higher frequency than would be expected if HIV-1 integration events were distributed uniformly throughout the genome. A cumulative binomial probability test shows that there is a 0.26% chance that one would arrive at these figures by chance and puts the data well within a 99% confidence interval. We propose that sites of L1Hs and Alu insertions originally occurred in regions of chromatin that were more easily accessible to the retroposon machinery and that these regions are now acting as preferred integration sites for HIV-1.

Base Sequence↗
PubMed2002

Changes in the mechanism of DNA integration in vitro induced by base substitutions in the HIV-1 U5 and U3 terminal sequences.

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994-3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.

Base Pairing↗
PubMed2004

Literature on integrated HIV care: a review.

With a significant proportion of HIV-infected patients now presenting with co-occurring substance abuse disorders and mental disorders, interest in integrated HIV care is growing. However, no review of integrated HIV care has been conducted. Using relevant key word searches of the Medline and Psychlit databases, the authors identified about 450 publications. The few evaluations of integrated models tended to focus on measurements of engagement and retention in medical care, and their findings indicated an association between integrated HIV care and increased service utilization. No random assignment controlled studies were identified, except in the peripheral area of integrated care (without HIV primary care) for persons with co-occurring substance abuse disorders and mental disorders. The majority of reviewed articles described integrated models operating in the field and various aspects of implementation and sustainability. Overall, they supported use of a wide range of primary and ancillary services delivered by a multidisciplinary team that employs a 'biopsychosocial' approach. Despite the lack of scientific knowledge regarding the effects of integrated HIV care, those wanting to optimize treatment for patients with multiple interacting disorders can gain useful and practical knowledge from this literature.

Delivery of Health Care, Integrated↗
PubMed2002

Novel integrating adenoviral/retroviral hybrid vector for gene therapy.

A hybrid adenoviral vector system was designed to incorporate an excisable retroviral cassette that can be stably integrated into the host cell genome. The vector contains the terminal sequences of two Moloney murine leukemia virus retroviral long terminal repeats (LTRs), fused to form a junction fragment, and is flanked by two loxP recognition sequences. Cre recombinase-directed excision liberates a circular, double-stranded DNA molecule containing the LTR junction fragment. Despite the natural intermediate for retroviral integrase being a linear DNA molecule, we show that, in the presence of Cre and retroviral Gag and Pol, the excised circle can be integrated into the target cell genome through both specific integrase (Int)-directed mechanisms and by a random integration process. The loxP cassette, carrying in addition a selectable marker gene, was incorporated into the E1-deleted region of an adenoviral vector. Infection of cells expressing Cre, Gag, and Pol generated clones that survived long term in drug selection (>3 months). Int-mediated integration was demonstrated in seven of nine clones by sequencing of the integration sites. In addition, the introduction of the loxP cassette into 293 cells coexpressing Cre and Int alone in the absence of other Gag and Pol proteins was sufficient to catalyze the integration mechanism. These experiments demonstrate that it is possible to generate high-titer adenovirus-mediated delivery of a C-type retroviral provirus that can subsequently undergo retroviral Int-mediated integration into dividing and nondividing cells.

Adenoviruses, Human↗
PubMed2004

Transgene expression after rep-mediated site-specific integration into chromosome 19.

We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks).

Chromosomes, Human, Pair 19↗
PubMed2006

Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration.

Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.

Animals↗
PubMed2002

Single copy heterozygote integration of HPV 33 in chromosomal band 5p14 is found in an epithelial cell clone with selective growth advantage.

Infection with human papillomavirus (HPV) of specific high-risk type triggers a series of events in target cells, which will eventually lead to development of genital neoplasia. The integration of high-risk HPV DNA into the cell genome has been regarded as a crucial event in tumor progression. With respect to different HPV types, the knowledge of HPV integrated loci is still limited. We have now determined the genomic variation and chromosomal location of HPV 33 DNA in the cell line UT-DEC-1, established from a vaginal mild dysplasia lesion. The viral sequence of the cell line was determined, and a variant of the prototype HPV 33 strain was identified, showing nucleotide substitutions resulting in amino acid changes in the E2, L2 and E4 open reading frames. In late passage UT-DEC-1 cells, a deletion of more than half of the 3' part of E1 and major parts of the E2 and E4 genes provided evidence for integration. The flanking sequences of the integration site were completely homologous to published sequences from chromosomal band 5p14, and remained unchanged in all subclones established from late passage cells. There were no chromosomal deletions or gross rearrangements at the integration site, and only a single heterozygotic copy of HPV 33 was detected. The karyotype of late passage cells showed only minor changes compared with early passage cells. During passaging of the cell line, there were progressive changes towards a malignant phenotype, and in parallel to this, the cells carrying episomal HPV 33 of the early passages was completely superseded by cells containing the integrated virus. Thus, our results show that this single copy heterozygote integration of HPV 33 into chromosome band 5p14 appears to be associated with emergence of cells escaping senescence, and with growth advantage compared with cells carrying episomal virus.

Base Sequence↗
PubMed1997

A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration.

We have probed the nucleoprotein organization of Moloney murine leukemia virus (MLV) pre-integration complexes using a novel footprinting technique that utilizes a simplified in vitro phage Mu transposition system. We find that several hundred base pairs at each end of the viral DNA are organized in a large nucleoprotein complex, which we call the intasome. This structure is not formed when pre-integration complexes are made by infecting cells with integrase-minus virus, demonstrating a requirement for integrase. In contrast, footprinting of internal regions of the viral DNA did not reveal significant differences between pre-integration complexes with and without integrase. Treatment with high salt disrupts the intasome in parallel with loss of intermolecular integration activity. We show that a cellular factor is required for reconstitution of the intasome. Finally, we demonstrate that DNA-protein interactions involving extensive regions at the ends of the viral DNA are functionally important for retroviral DNA integration activity. Current in vitro integration systems utilizing purified integrase lack the full fidelity of the in vivo reaction. Our results indicate that both host factors and long viral DNA substrates may be required to reconstitute an in vitro system with all the hallmarks of DNA integration in vivo.

Animals↗