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PubMed2002

[Detection and quantification of HIV-1 provirus provides a prognostic marker for the therapy-monitoring].

By the effective application of highly active antiretroviral therapy (HAART), the plasma HIV-1 RNA level can be dramatically suppressed to below the detectable level in a short period. As the amount of total HIV-1 DNA in peripheral CD4+ cells sharply decline in the first phase of the treatment in accordance with the decline of HIV-1 RNA, total HIV-1 DNA, mainly containing the unintegrated form, can be a useful marker for therapy-monitoring just as HIV-1 RNA. In the next phase, after the transcription and reverse transcription of HIV-1 are profoundly inhibited by continuing HAART, the mass of the latent HIV-1 reservoir can be quantified by measuring the amount of total HIV-1 DNA which mainly reflects the integrated HIV-1 provirus in this phase.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Clinical indications of multiple integrations of human T-cell lymphotropic virus type I proviral DNA in adult T-cell leukemia/lymphoma.

In this review, we discuss the possible relationship between the clinical characteristics and the multiple integration of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA in patients with adult T-cell leukemia/lymphoma (ATL). Some patients with ATL show multiple HTLV-I integrations and exhibit clinical characteristics unlike those of ATL patients who show the typical integration of a single provirus. Multiple HTLV-I integrations can be detected by Southern blotting as multiple bands having varied intensities. These multiple integration conditions can arise from one tumor cell clone carrying multiple copies of the provirus, or from multiple cell clones, each carrying one copy of the provirus. The former patients manifest an extremely aggressive clinical course with the infiltration of unusual organs such as the retina and uvea. The latter patients show an indolent clinical course with skin lesions. These findings suggest that the clinical implications for multiple HTLV-I integrations exist in ATL. This may be one of the explanations for the heterogeneous findings in the disease. Such observations may provide information linking viral integration with clinical manifestations, and improve our understanding of the pathogenesis of ATL.

DNA, Viral↗
PubMed2000

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system.

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.

Adenoviridae↗
PubMed1980

Integration, expression, and infectivity of exogenously acquired proviruses of Rous-associated virus-O.

We investigated the integration sites, infectivities, and expression of Rous-associated virus-0 (Rav-0) DNAs in exogenously infected turkey and chicken cells. Restriction endonuclease analyses of the DNAs of RAV-0-infected cells indicated that unique integration sites of RAV-0 DNA were detectable in clones of RAV-0-infected cells but not in mass-infected cell cultures. In addition, the sites of integration of RAV-0 DNA differed in each of the seven clones of RAV-0-infected cells examined. Thus, endogenous RAV-0 proviruses appeared to integrate at multiple sites in cellular DNA, which were distinct from the sites of integration of endogenous RAV-0 genomes. Since exogenous RAV-0 proviruses are expressed at 10(3)- to 10(4)-fold-higher levels and are 10(3)- to 10(4)-fold more infectious in transfection assays than the endogenous RAV-0 genome of uninfected V+ chicken cells, these results are consistent with the hypothesis that transcription of the endogenous RAV-0 genome is regulated by flanking cellular DNA sequences. Although all RAV-0-infected cells contained infectious RAV-0 DNA and produced high titers of RAV-0 compared with uninfected V+ cells, different clones of RAV-0 infected chicken cells differed by as much as 30-fold in their levels of virus production. The infectivity of the DNA of each clone of RAV-0-infected cells correlated with the amount of virus produced by that clone. However, these differences did not appear to be correlated either with the number of exogenous RAV-0 proviruses in different clones or with the infectivity of RAV-0 produced by different clones, indicating that differences either in modification of RAV-0 DNAs or in the cellular sequences flanking exogenous RAV-0 DNAs were responsible for the observed differences in expression and infectivity.

Animals↗
PubMed1996

Target-sequence preferences of HIV-1 integration complexes in vitro.

Integration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes.

Binding Sites↗
PubMed1995

A role for bacteriophages in the evolution and transfer of bacterial virulence determinants.

A virulence-associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and phi R73, together with open reading frames (vapB, C and D) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola. Similar to P4 and phi R73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage-related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.

Attachment Sites, Microbiological↗
PubMed1979

The integration sites of endogenous and exogenous Moloney murine leukemia virus.

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.

AKR murine leukemia virus↗
PubMed2006

Porcine endogenous retrovirus integration sites in the human genome: features in common with those of murine leukemia virus.

Porcine endogenous retroviruses (PERV) are a major concern when porcine tissues and organs are used for xenotransplantation. PERV has been shown to infect human cells in vitro, highlighting a potential zoonotic risk. No pathology is associated with PERV in its natural host, but the pathogenic potential might differ in the case of cross-species transmission and can only be inferred from knowledge of related gammaretroviruses. We therefore investigated the integration features of the PERV DNA in the human genome in vitro in order to further characterize the risk associated with PERV transmission. In this study, we characterized 189 PERV integration site sequences from human HEK-293 cells. Data showed that PERV integration was strongly enhanced at transcriptional start sites and CpG islands and that the frequencies of integration events increased with the expression levels of the genes, except for the genes with the highest levels of expression, which were disfavored for integration. Finally, we extracted genomic sequences directly flanking the integration sites and found an original 8-base statistical palindromic consensus sequence [TG(int)GTACCAGC]. All these results show similarities between PERV and murine leukemia virus integration site selection, suggesting that gammaretroviruses have a common pattern of integration and that the mechanisms of target site selection within a retrovirus genus might be similar.

3' Flanking Region↗
PubMed1985

Molecular cloning of integrated caprine arthritis-encephalitis virus.

A full-length DNA clone of the exogenous retrovirus, caprine arthritis-encephalitis virus (CAEV), was isolated from high molecular weight DNA of CAEV-infected Himalayan tahr ovary cells. Although other restriction maps of CAEV have been published, this is the first time that the proviral DNA has been cloned. The restriction enzyme map of the clone was determined and found to be identical to that of unintegrated linear CAEV DNA except for the presence of cellular flanking sequences. These findings establish that lentiviruses are able to integrate within the infected host cellular genome. The cloned CAEV genome was shown to contain terminal repeats of approximately 450 base pairs in length, and its restriction enzyme map was oriented with respect to the direction of viral RNA transcription. When the cloned CAEV DNA was used as a molecular probe, it failed to detect related proviral sequences in the genomes of a variety of vertebrate species, including the goat, sheep, horse, mouse, and man. When CAEV DNA was hybridized under relaxed conditions to a variety of cloned DNAs, representing different oncoviral genera, homology to mouse mammary tumor virus (MMTV) was observed, while no homology to avian type C or mammalian type A, C, and D retroviruses was detected. This homology was localized to a region in MMTV corresponding to the 3' end of the gag gene and the 5' end of the pol gene.

Animals↗
PubMed1996

A new concept in (adenoviral) oncogenesis: integration of foreign DNA and its consequences.

A new concept for viral oncogenesis is presented which is based on experimental work on the chromosomal integration of adenovirus DNA into mammalian genomes. The mechanism of adenovirus DNA integration is akin to non-sequence-specific insertional recombination in which patch homologies between the recombination partners are frequently observed. This reaction has been imitated in a cell-free system by using nuclear extracts from hamster cells and partly purified fractions derived from them. As a consequence of foreign DNA insertion into the mammalian genome, the foreign DNA is extensively de novo methylated in specific patterns, presumably as part of a mammalian host cell defense mechanism against inserted foreign DNA which can be permanently silenced in this way. A further corollary of foreign (adenovirus or bacteriophage lambda) DNA integration is seen in extensive changes in cellular DNA methylation patterns at sites far remote from the locus of insertional recombination. Repetitive cellular, retrotransposon-like sequences are particularly, but not exclusively, prone to these increases in DNA methylation. It is conceivable that these changes in DNA methylation are a reflection of a profound overall reorganization process in the affected genomes. Could these alterations significantly contribute to the transformation events during viral or other types of oncogenesis? These sequelae of foreign DNA integration into established mammalian genomes will have to be critically considered when interpreting results obtained with transgenic, knock-out, and knock-in animals and when devising schemes for human somatic gene therapy. The interpretation of de novo methylation as a cellular defense mechanism has prompted investigations on the fate of food-ingested foreign DNA. The gastrointestinal (GI) tract provides a large surface for the entry of foreign DNA into any organism. As a tracer molecule, bacteriophage M13 DNA has been fed to mice. Fragments of this DNA can be found in small amounts (about 1% of the administered DNA) in all parts of the intestinal tract and in the feces. Furthermore, M13 DNA can be traced in the columnar epithelia of the intestine, in Peyer's plaque leukocytes, in peripheral white blood cells, in spleen, and liver. Authentic M13 DNA has been recloned from total spleen DNA. If integrated, this DNA might elicit some of the described consequences of foreign DNA insertion into the mammalian genome. Food-ingested DNA will likely infiltrate the organism more frequently than viral DNA.

Adenoviridae↗
PubMed2005

The single-stranded genome of phage CTX is the form used for integration into the genome of Vibrio cholerae.

A major determinant of Vibrio cholerae pathogenicity, the cholera enterotoxin, is encoded in the genome of an integrated phage, CTXvarphi. CTXvarphi integration depends on two host-encoded tyrosine recombinases, XerC and XerD. It occurs at dif1, a 28 bp site on V. cholerae chromosome 1 normally used by XerCD for chromosome dimer resolution. The replicative form of the phage contains two pairs of binding sites for XerC and XerD in inverted orientations. Here we show that in the single-stranded genome of the phage, these sites fold into a hairpin structure, which creates a recombination target for XerCD. In the presence of XerD, XerC can catalyze a single pair of strand exchanges between this target and dif1, resulting in integration of the phage. This integration strategy explains why the rules that normally apply to tyrosine recombinase reactions seemed not to apply to CTXvarphi integration and, in particular, why integration is irreversible.

Base Sequence↗
PubMed2004

Limited lentiviral transgene expression with increasing copy number in an MGMT selection model: lack of copy number selection by drug treatment.

Retroviral vector integration into the human genome carries increased risk of oncogenesis with increasing integrations. To boost transgene expression for gene therapy, multiple integrations are often sought. We studied the relationship between the number of vector integrations and transgene expression and the effect that drug selection in an MGMT-selection model would have on vector copy number. K562 cells were transduced using a lentiviral vector and a library of clones was generated. Median proviral copy number was 4 and a positive correlation with transgene expression was observed. Transgene expression increased at a linear rate between 1 and 4 vector copies/cell, but was unpredictable at >4 integrations/cell. When lentivirus MGMT(P140K)-transduced K562 cells were treated with O(6)-benzylguanine (BG)/BCNU, there was no selection for increased median copy number in colony-forming units, despite strong selection pressure and an increase in transgene expression and activity. These data show a direct and linear correlation between MGMT(P140K) transgene expression and vector copy number. Strong BG/BCNU selective pressure does not result in preferential survival of high-copy-number clones but does select for strong transgene expression. Thus drug selection would not be expected to increase the risk of oncogenesis due to exaggerated selection in favor of high-copy-number vector integration.

Alkyl and Aryl Transferases↗
PubMed2002

Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies.

Most classical integrases of prokaryotic genetic elements specify integration into tRNA or tmRNA genes. Sequences shared between element and host integration sites suggest that crossover can occur at any of three sublocations within a tRNA gene, two with flanking symmetry (anticodon-loop and T-loop tDNA) and the third at the asymmetric 3' end of the gene. Integrase phylogeny matches this classification: integrase subfamilies use exclusively either the symmetric sublocations or the asymmetric sublocation, although tRNA genes of several different aminoacylation identities may be used within any subfamily. These two familial sublocation preferences imply two modes by which new integration site usage evolves. The tmRNA gene has been adopted as an integration site in both modes, and its distinctive structure imposes some constraints on proposed evolutionary mechanisms.

Base Sequence↗
PubMed2006

High efficiency site-specific genetic engineering of the mosquito genome.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Aedes↗
PubMed1996

Sequence tags of provirus integration sites in DNAs of tumors induced by the murine retrovirus SL3-3.

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. The proviral integration site sequences were surveyed in tumor DNAs by a simple two-step PCR method. From 20 SL3-3-induced tumors a total of 39 provirus-host junctions were amplified and sequenced. Seven showed homology to known sequences. These included the known common integration site c-myc as well as genes not previously identified as targets of provirus integration, namely N-ras and the genes coding for major histocompatibility complex class 11 E-beta, protein kinase C-eta, and T-cell receptor beta-chain. Among these genes, the integrations in c-myc as well as the one in N-ras were found to be clonal. One of the remaining 32 proviral integration site sequences that show no similarities to known sequences may represent a common integration site, as 2 of the 20 tumors demonstrated clonal provirus insertion into this region.

Animals↗
PubMed1994

Clinical importance of extraordinary integration patterns of human T-cell lymphotropic virus type I proviral DNA in adult T-cell leukemia/lymphoma.

The proviral DNA of human T-cell lymphotrophic virus type I (HTLV-I) is known to be integrated monoclonally in the malignant cells of adult T-cell leukemia/lymphoma (ATL), which is a peripheral T-cell malignancy caused by this virus. We studied the relationship between the integration patterns of HTLV-I and clinical characteristics in 89 patients with ATL. The proviral DNA of HTLV-I was examined by the standard Southern blot analysis using the endonucleases EcoRI and Pst I. One clear band of greater than 9 kb was detected in most of the patients (83 case) when cellular DNA was digested with EcoRI. On the other hand, extraordinary integration patterns of HTLV-I proviral DNA were detected in 6 patients; 3 of them showed two bands, while the other 3 showed one band smaller than 9 kb. When cellular DNA was digested with PstI, the band patterns of these patients were quite different from those of typical patients. The patients with the extraordinary integration patterns had clinical characteristics dissimilar to those of the other 83 patients with the ordinary integration pattern. The patients with two bands by EcoRI digestion always had severe hypoxemia with extremely high levels of serum lactate dehydrogenase at first presentation and showed peculiar organ infiltrations, such as retina and muscle, which were less frequent in the other ordinary 83 patients. They all died within 8 months after the onset. In contrast, the patients with one smaller band by EcoRI digestion always had small and mature T lymphocytes with bilobulated nuclei without lymphadenopathy and showed a favorable clinical course, which was uncommon in the ordinary cases. They were alive 20 to 38 months after diagnosis. Rearranged bands of the T-cell receptor gene were detected in all patients with unusual integration. These findings indicate that the integration patterns of HTLV-I proviral DNA have a clinical implication and may be one of the explanations for heterogeneity in the behavior of this disease.

DNA, Viral↗
PubMed1994

Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach.

A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.

Chromosome Mapping↗