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PubMed2004

Switching the polarity of a bacteriophage integration system.

During lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage phiC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage lambda. We show that during phiC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that phiC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a phiC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB. Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.

Bacteriophages↗
PubMed2001

Computational analysis of retrovirus-induced scid cell death.

It was shown recently that retroviral infection induces integrase-dependent apoptosis (programmed cell death) in DNA-dependent protein kinase (DNA-PK)-deficient scid pre-B cell lines, and it has been proposed that retroviral DNA integration is perceived as DNA damage that is repairable by the DNA-PK-dependent nonhomologous end-joining pathway (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Very few infectious virions seem to be necessary to induce scid cell death. In this study, we used a modeling approach to estimate the number of integration events necessary to induce cell death of DNA-PK-deficient scid cells. Several models for integration-mediated cell killing were considered. Our analyses indicate that a single hit (integration event) is sufficient to kill a scid cell. Moreover, the closest fit between the experimental data and our computational simulations was achieved with a model in which the infected scid cell must pass through S phase to trigger apoptosis. This model is consistent with the findings that a single double-strand DNA break is sufficient to kill a cell deficient in DNA repair and illustrates the potential of a modeling approach to address quantitative aspects of virus-cell interactions.

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↗
PubMed2001

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC ) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

Animals↗
PubMed1997

Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5.

A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally hold view of D29 as a virulent phage must be reviewed.

Amino Acid Sequence↗
PubMed1993

Effect of gamma-interferon or cycloheximide treatment on viral and c-myc transcripts in bovine-papillomavirus-type-1-transformed primary mouse fibroblasts.

We have studied the effect of gamma-interferon (IFN-gamma) treatment on the transcription of viral and c-myc oncogenes in bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast cell lines. Upon IFN-gamma treatment, viral transcripts always decreased in cell lines containing episomal BPV-1 DNA, while the effect was variable in cell lines containing integrated BPV-1 DNA. Two series of tumour cell lines established by in vivo passage of B6B71 (episomal) and B6B31-J (integrated) cells via nude mice (NuTu A) into immunocompetent C57BL/6 mice (NuTuA B6Tul) also showed a decrease and an increase, respectively, in viral transcripts upon IFN-gamma treatment. IFN-gamma also reduced c-myc transcription in all cell lines derived from tumours, but increased it in the transformed cell cultures. There was selection of c-myc oncogene transcripts in all the tumour-derived cell lines as compared to their transformed cell cultures. Cycloheximide treatment increased both viral and c-myc gene transcripts 3- to 20-fold in all cell lines. These results imply that IFN-gamma produced locally by immunological mechanisms may influence the expression of Papillomavirus oncogenes, and the response to IFN-gamma treatment may change when the viral DNA is integrated into the host genome.

Animals↗
PubMed2000

[Effects of calcium on HPV16 gene transcription in cultured laryngeal epithelial cells].

Human papillomavirus (HPV) gene transcription is closely linked to the differentiation status of infected epithelial cells. A variety of physiological agents, including calcium, regulates the differentiation of cultured epithelial cells. The expression of cytokeratin No.13 (CK13) can be used as a marker for differentiation in cultured laryngeal epithelial cells (HLEC cells). The purpose of this study is to examine the effects of calcium on CK13 expression and HPV16 gene transcription in HLEC cells. We analyzed two types of HPV16-containing HLEC cells: HPV 16-immortalized HLEC cells (HLEC16 cells) and HPV16-positive (infected) cultured laryngeal papilloma cells (HLP16 cells). In the HLEC16 cells, the viral genes were integrated into the host cell chromosomes, while the HLP16 cells contained extra-chromosomal viral genes. The effects of increasing calcium concentrations on CK13 expression were then evaluated using immunocytochemistry. Both the HLP16 and the HLEC16 cells responded to an increased calcium concentration by inducing CK13 expression. In HLP16 and HLEC16 cells, the CK13 expression was undetectable at low calcium concentrations (0.1 mM) but became clearly detectable at high calcium concentrations (1.0 mM). The level of viral RNA was elevated in HLP16 cells with added calcium (1.0 mM) but was similar in HLEC16 cells grown in either low (0.1 mM) or high (1.0 mM) calcium concentrations. These results suggest that a calcium-induced differentiation results in the up-regulation of HPV16 gene transcription in HLP16 cells. The integration of viral gene into the host cell chromosomes may be an important determinant for the differentiation-independent transcription of HPV16 genes.

Calcium↗
PubMed1998

Recurrent integration of papillomavirus DNA within the human 12q14-15 uterine breakpoint region in genital carcinomas.

Genital carcinomas are associated with human papillomaviruses, and the viral DNA is frequently integrated in the host cell genome. Recurrent chromosomal alterations are genetic markers for specific tumor phenotypes. To demonstrate that papillomavirus DNA integration is indeed a recurrent chromosomal aberration, we mapped two independent papillomavirus integration sites in the human 12q14-15 region, one containing HPV16 DNA and the other HPV18 DNA. The two HPV integration sites map approximately 10 kbp from each other within the cosmid LLNL12NCO1-196E1 clone. The integration site corresponding to HPV16 DNA in SK-v cells is proximal to the 5' end of a DNA segment known to be rearranged by integration of HPV18 DNA in another cervical carcinoma cell line, SW756. Both integrations are located in the PAL2 locus within the uterine leiomyoma cluster region of translocation.

Base Sequence↗
PubMed2007

The prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of UK blood donors.

A lesser-recognized form of human herpesvirus 6 (HHV-6) persistence is integration of the viral genome in a host chromosome and high viral copy numbers in blood or sera are characteristic of this phenomenon. A cross-sectional study was performed to determine the frequency of high HHV-6 viral loads in whole blood (>6 log(10) copies/ml) in a population of blood donors in London, UK. Blood samples from 500 anonymized blood donors were collected from one donation center, DNA extracted, and quantitative realtime PCR used to measure viral load. Four samples (0.8%) were found to have high viral copy numbers of HHV-6 (median 6.7 log(10) copies/ml; range 6.5- 6.9 log(10) copies/ml). Cellular DNA was also quantitated using qRT-PCR for beta-globin. By comparing these two results, we calculated that there were between two and five copies of HHV-6 present per cell in these four donors. The median viral load detected in plasma from the four individuals was 3.8 log(10) copies/ml (range 3.5-4.0 log(10) copies/ml). All samples were HHV-6 variant B. In addition, a retrospective analysis of all diagnostic blood samples performed for HHV-6 in our center showed a prevalence of 2.9% of high viral loads characteristic of integration. In conclusion, high viral copy numbers of HHV-6, representing a population of viral integration, is detected in 0.8% of UK blood donors. The presence of high HHV-6 viral loads in healthy normal individuals reiterates the need to consider the confounding effect of HHV-6 viral integration in any laboratory diagnosis of HHV-6 infection.

Blood Donors↗
PubMed1996

Construction of an integrative food-grade cloning vector for Lactobacillus acidophilus.

An integrative cloning vector was constructed using a randomly cloned HindIII-digested chromosomal fragment from Lactobacillus acidophilus ADH inserted into an Escherichia coli vector, pBluescript II SK+. Southern hybridization studies demonstrated homology of the inserted fragment with one other L. acidophilus strain and one Bifidobacterium strain. Identification of a SauI site located near the middle of the 1.9-kb ADH chromosomal fragment made it possible to clone the Lactobacillus bulgaricus beta-galactosidase (EC 3.2.1.23) gene into this vector. The vector was unable to replicate in the homologous host, L. acidophilus ADH, following electroporation. The chromosomal fragment allowed the integration of the beta-galactosidase gene (beta gal) into the host chromosome via homologous recombination. The size of the two flanking L. acidophilus ADH chromosomal fragments, approximately 0.95 kb each, was sufficient to allow the double cross-over to take place. Southern hybridization demonstrated that only L. acidophilus and L. bulgaricus DNA had been integrated into the chromosome of the host strain. The beta-galactosidase activity of the transformant was increased approximately 200-fold when compared to the enzyme activity of the wild-type strain. The beta gal gene remained stable in the transformant strain after 30 transfers in growth media without selection pressure. This first-generation integrative cloning vector is constructed solely of DNA from organisms consumed by humans and could be considered a food-grade vector system.

Bacterial Proteins↗
PubMed2006

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells.

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, phiFC1, and phiRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of approximately 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, phiFC1, and phiRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.

Attachment Sites, Microbiological↗
PubMed2003

Development of a novel method of lytic phage delivery by use of a bacteriophage P22 site-specific recombination system.

Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. This lysogen was shown to be effective at decreasing the number of lambda-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars.

Bacteriophage P22↗
PubMed2004

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair.

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.

HeLa Cells↗
PubMed2003

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors.

Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.

Amino Acid Sequence↗
PubMed2001

Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors.

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.

Cell Line↗
PubMed2006

Use of multiple displacement amplification in the investigation of human papillomavirus physical status.

BACKGROUND AND AIMS: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities. METHODS: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation. RESULTS: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples. CONCLUSIONS: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.

Base Sequence↗
PubMed2004

DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri.

PURPOSE: Increasingly deregulated expression of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers. The expression of these genes in epithelial stem cells first results in chromosomal instability and induces chromosomal aneuploidy. It is speculated that this subsequently favors integration of HR-HPV genomes into cellular chromosomes. This in turn leads to expression of viral cellular fusion transcripts and further enhanced expression of the E6-E7 oncoproteins. Chromosomal instability and aneuploidization thus seems to precede and favor integration of HR-HPV genomes. EXPERIMENTAL DESIGN: To prove this sequential concept, we analyzed here the sequence of events of DNA aneuploidization and integration in a series of HPV-16-positive cervical dysplastic lesions and carcinomas. Eighty-five punch biopsies of HPV-16-positive cervical lesions (20 CIN1/2, 50 CIN3, and 15 CxCa) were analyzed for DNA ploidy by DNA flow cytometry and for integration of HPV E6/E7 oncogenes using the amplification of papillomavirus oncogene transcripts assay, a reverse transcription-PCR method to detect integrate-derived human papillomavirus oncogene transcripts. RESULTS: DNA aneuploidy and viral genome integration were both associated with increasing dysplasia (P < 0.001, chi(2) test for trend). In addition, DNA aneuploidy was associated with increased viral integration (P < 0.01, Fisher's exact test). Nineteen of 20 (95%) lesions with integrated viral genomes had aneuploid cell lines; however, only 19 of 32 (59%) lesions with aneuploid cell lines had integrated viral genomes. CONCLUSIONS: These data support the hypothesis that aneuploidization precedes integration of HR-HPV genomes in the progression of cervical dysplasia. Accordingly, deregulated viral oncogene expression appears to result first in chromosomal instability and aneuploidization and is subsequently followed by integration of HR-HPV genomes in the affected cell clones.

Adult↗