Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Virus Integration”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources
PubMed1994

Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509 were determined. A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.

Amino Acid Sequence↗
PubMed1995

The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence.

The de novo methylation of foreign DNA integrated into the mammalian genome is a fundamental process whose mechanism has not yet been elucidated. We have studied de novo methylation in adenovirus type 12 (Ad12) genomes inserted into the genomes of Ad12-induced hamster tumor cells. De novo methylation of Ad12 DNA, which is not methylated in the virion, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultivation of cloned Ad12-induced hamster tumor cell lines, the same segments in integrated Ad12 DNA in different cell lines become methylated or remain unmethylated, depending on their positions in the viral genome. (ii) When Ad12 DNA or Ad12 DNA fragments are transfected into hamster cells and permanent cell lines are established by selection for the cotransfected neomycin phosphotransferase gene, patterns of de novo methylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines. (iii) A detailed study on the topology of the integrated viral genomes in the Ad12-transformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of the integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearranged. An originally internally located Ad12 DNA segment has become transposed to the left-terminal sequences of the viral genome in several cell lines and tumors. In the complete Ad12 genomes, the internally located PstI-D fragment becomes extensively methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences. When this DNA segment has been juxtaposed to the left-terminal, hypomethylated fragment of Ad12 DNA in rearranged genomes, the PstI-D fragment remains unmethylated. We therefore reason that the initiation of de novo methylation in integrated Ad12 DNA cannot be directed exclusively by the nucleotide sequence. Other parameters, such as site of integration, conformation of integrates, mode of cell selection, or chromatin structure related to transcriptional activity, may play decisive roles.

Adenoviridae↗
PubMed2001

Sequence analysis of HIV-1 insertion sites in peripheral blood lymphocytes.

An essential component of the HIV-1 life cycle involves insertion in the genome of an infected cell. The site of HIV-1 integration has the potential to disrupt a gene and perturb a normal cellular function. To begin to address whether disease pathogenesis may correlate with the site of insertion, flanking cellular sequences at these HIV integrated regions were directly amplified from peripheral blood mononuclear cells DNA from a broad range of infected individuals using an inverse polymerase chain reaction strategy. Amplified flanking regions were sequenced and examined for similarity to the nucleic acid database. In this group of analyzed samples, the HIV-1 provirus was inserted within non-coding regions throughout the genome of the infected host, in which 7/14 sites were positioned in close proximity to different Alu repetitive elements while 2/14 sites were located within intron sequences. Insertions were also detected at sites without a specific gene designation but not within short tandem repetitive sequences, telomeres or centromeric repeat regions. Altogether, it is expected that this approach will yield new information on sites of integration by HIV-1 that may be associated with the pathogenic manifestations of disease progression.

Base Sequence↗
PubMed1980

Integration pattern of human JC virus sequences in two clones of a cell line established from a JC virus-induced hamster brain tumor.

The physical state of the JC virus (JCV) genome was studied in two clonal cell lines (clones 2 and 7) derived from a tissue culture cell line (HJC-15) established from a hamster brain tumor induced by JCV. Saturation-hybridization and reassociation kinetic analyses, using in vitro (32)P-labeled JCV DNA, indicated that clone 7 and 2 cells contain 9 to 10 and 4 to 5 copies per cell, respectively, of all or most of the viral genome. Both cell DNAs were analyzed by using the Southern blotting procedure with three restriction endonucleases: XhoI, which does not cleave JCV DNA; EcoRI, which cleaves once; and HindIII, which cleaves three times. With each DNA, a variety of JCV-specific DNA fragments were detected. The following conclusions are possible: (i) JCV DNA is integrated into cell DNA in both clonal lines; (ii) both clonal lines contain multiple copies of the viral genome integrated in a tandem head-to-tail orientation; (iii) neither clonal line contains detectable free-form I, II, or III JCV DNA; (iv) each clonal line contains multiple independent sites of JCV DNA integration; and (v) most or all of the sites of integration on the cellular or the viral genome, or both, are different in clone 7 DNA than in clone 2 DNA. Thus, although both clone 7 and clone 2 cells were established from the HJC-15 tumor cell line, they differ in the copy number and integration pattern of JCV DNA.

Animals↗
PubMed1992

Requirement for a conserved serine in both processing and joining activities of retroviral integrase.

Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.

Avian Sarcoma Viruses↗
PubMed1996

Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR.

Alleles↗
PubMed2006

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia.

INTRODUCTION: The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. METHODS: The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. RESULTS: The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. CONCLUSION: These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

Animals↗
PubMed2005

Physical status of HPV-16 in esophageal squamous cell carcinoma.

BACKGROUND: Infection with high-risk human papillomavirus (HPV) has been implicated as one of the risk factors of esophageal squamous cell carcinoma (ESCC). Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of the HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. OBJECTIVES AND STUDY DESIGN: To investigate the viral integration status of HPV-16 infection in ESCC, 35 HPV-positive ESCC specimens collected from Chinese patients were subject to real-time quantitative PCR for determination of physical status of HPV-16 by analyzing the ratios of E2 to E6 genes. RESULTS: Our results showed that only 8.6% (3/35) of the HPV-16 positive specimens harbored exclusively the episomal form (i.e. E2/E6 ratio > or = 1), whereas the remaining 91.4% contained either only the integrated form (5.7%, with E2/E6 ratio = 0) or a mixture of episomal and integrated forms of viral molecules (85.7%, with E2/E6 ratios > 0 but < 1). Amongst the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/integrated E6 ratios of less than 1, indicating a predominance of integrated form of viral genes in these lesions. CONCLUSION: Our finding of frequent integration of viral DNA in the host genome suggests that integration HPV-16 is common in ESCC from Chinese patients and implies that HPV infection may play a role in the pathogenesis of ESCC.

Carcinoma, Squamous Cell↗
PubMed2005

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study.

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.

Attachment Sites, Microbiological↗
PubMed1996

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1.

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3 By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.

Amino Acid Sequence↗
PubMed2006

Human herpesvirus 6 chromosomal integration in immunocompetent patients results in high levels of viral DNA in blood, sera, and hair follicles.

Six immunocompetent patients with human herpesvirus 6 (HHV-6) chromosomal integration had HHV-6 and beta-globin DNA quantified in various samples by PCR. The mean HHV-6 DNA concentration (log(10) copies/milliliter) in blood was 7.0 (>/=1 HHV-6 DNA copies/leukocyte), and in serum it was 5.3 (>/=1 HHV-6 DNA copies/lysed cell). The mean HHV-6 DNA load (log(10) copies)/hair follicle was 4.2 (>/=1 copies/hair follicle cell), demonstrating that viral integration is not confined to blood cells. The characteristically high HHV-6 DNA levels in chromosomal integration may confound laboratory diagnosis of HHV-6 infection and should be given due consideration.

Adult↗
PubMed1993

Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method.

We describe a two-step polymerase chain reaction method that can be used for the amplification of cellular DNA sequences adjacent to an integrated retroviral provirus. The technique involves a partly degenerate, arbitrary primer that will hybridize in the provirus-flanking cellular DNA. By using this primer in combination with a biotinylated provirus-specific primer, a provirus-cellular DNA junction fragment can be isolated from the nonspecific amplification products by using streptavidin-coated magnetic beads. A second amplification employing a nested provirus-specific primer and a biotinylated nondegenerate primer derived from the partly degenerate primer followed by purification with streptavidin-coated beads enhances the specificity and the efficiency of recovery of a fragment(s) containing the unknown flanking sequences. In addition to being relevant in studies of viral integration sites, the method should be generally useful to analyze DNA sequences either upstream or downstream from a known sequence.

Animals↗
PubMed2001

Intact EAV-HP endogenous retrovirus in Sonnerat's jungle fowl.

The EAV-HP group of chicken endogenous retrovirus elements was previously shown to be defective, with large deletions of the pol gene. In this report, we demonstrate that genomes of other Gallus species also maintain EAV-HP elements with similar deletions. The chicken EAV-HP1 locus was detected in both red (Gallus gallus gallus) and Sonnerat's (Gallus sonneratii) jungle fowl with identical integration sites, indicating that these elements had integrated before separation of the Gallus species. Furthermore, we demonstrate for the first time that the G. sonneratii genome carries EAV-HP elements with intact pol regions.

Animals↗
PubMed1994

High rate of morphological reversion in tumor cell line H-19 associated with permanent transcriptional suppression of the LTR, v-src, LTR provirus.

The highly malignant line of morphologically transformed H-19 hamster tumor cells that harbor a single LTR, v-src, LTR provirus segregates morphologically flat revertants at the rate of 1.4 to 2.4 x 10(-3)/cell/cycle. Revertants behave like almost nonmalignant cells; they keep the provirus within an unaltered junction DNA fragment. However, the provirus is methylated, permanently transcriptionally silent, and not rescuable. Using the polymerase chain reaction, we have synthesized the whole proviral structure from two revertants and established that the left-hand long terminal repeats assuring transcription remained structurally intact. Moreover, the cloned proviral DNAs from three revertants were shown to produce tumors in chickens. The unusually high reversion rate together with the finding of structural integrity of proviral transcriptional signals in revertants indicate strongly that the reversion has been mediated by cell-regulatory mechanisms.

Animals↗
PubMed2006

[Natural history of hepatitis B infection].

Hepatitis B virus (HBV) is transmitted by parenteral, sexual and perinatal routes. While fulminant hepatitis may occur in 1% of cases of symptomatic acute hepatitis, the principal problem of HBV infection is that it may become chronic, classically defined by carriage of HB surface antigens (HBsAg) for more than 6 months. This occurs in only 0.5 to 3% of immunocompetent adults but more frequently in children (up to 90%) and in immune-compromised patients (30 to 100%). The course of chronic HBV infection is characterized by variations in viral replication with spontaneous reactivation or discontinuation, and potential exacerbations observed clinically or by laboratory testing. The pathogenesis of HBV infection is mainly immune-mediated, resulting from host-virus interactions but also from the complexity of the virus itself (integration, mutation, occult replication). These factors explain the variety of presentations of chronic HBV infection, which range from immune tolerance to inactive carriage of HBsAg, passing through a stage of immune clearance, where chronic active hepatitis which may lead to cirrhosis (yearly incidence of 1.3 to 5.9%). Cirrhosis may be complicated by portal hypertension, liver failure, or hepatocellular carcinoma, which together explain 80% of the morbidity and mortality associated with HBV. The 5-year survival rate for HBV-related cirrhosis ranges from 52 to 82%. Immunosuppression, hepatitis D virus superinfection, and chronic alcohol consumption are the principal factors that modify this natural history. Chronic HBV infection is a major public health problem, particularly in developing countries, and it requires that efforts to make HBV vaccination universal be intensified.

Acute Disease↗
PubMed2007

Human papillomavirus-related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture.

BACKGROUND: Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real-time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples. METHODS: Human papillomavirus (HPV) 16 and 18 E7 mRNA expression was measured using quantitative RT-PCR. DNA index and chromatin measures were taken from image cytology samples. The chromatin features, through discriminant analysis, were aggregated into a score, and both measurements were related to mRNA expression. RESULTS: mRNA level and DNA index show an increasing trend over increasing histological grades. However, DNA index and chromatin score were not correlated to mRNA levels in these samples. Chromatin score differed by mRNA type found with HPV 18 infected samples having a higher score than those with HPV 16. Samples infected with HPV 16 and HPV 18 had even higher chromatin scores. CONCLUSIONS: DNA index and chromatin score were not directly correlated with mRNA levels. However both mRNA and DNA index were related to histological grade, and chromatin score was associated with HPV type. Therefore, DNA index and mRNA levels could be independent predictors of cervical dysplasia, and chromatin score could be related to the viral integration process in cells infected with HPV 18 or dual infections.

Adult↗
PubMed2000

Insertional mutagenesis as a route to identifying genes involved in self renewal of haemopoietic stem cells.

The genes controlling self renewal in the haemopoietic system are still unknown. Using retroviral insertional mutagenesis we have established multipotent haemopoietic stem cell lines (FDCP-mix) that possess an increased self renewal capacity in vitro. To identify genes involved in the regulation of self renewal, proviral integration sites were cloned from FDCP-mix cells and used as probes to screen independently isolated FDCP-mix cell lines for a common proviral insertion site. So far, two common integration sites have been identified, A25 and M4. A25 is rearranged in 50% of the FDCP-mix cell lines and M4 in 10%. Genes located at or near these sites are likely candidates for the control of self renewal of haemopoietic stem cells.

Cell Differentiation↗
PubMed2002

Disruption of HIV-1 integrase-DNA complexes by short 6-oxocytosine-containing oligonucleotides.

We recently found that oligonucleotides containing the 6-oxocytosine heterocyclic base are efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al. (2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we demonstrate that the inhibition arises from a noncompetitive mechanism in which the modified oligonucleotide attacks the integrase-DNA complex, leading to its active disruption. This conclusion is based on the following results. First, despite the fact that the respective affinities of a 6-oxocytosine-containing oligonucleotide and of its nonmodified counterpart for integrase were identical, only the modified compound inhibited the enzyme activities. Second, DNA binding and UV cross-linking assays indicated that the 6-oxocytosine-containing oligonucleotide prevented the formation of a stable integrase-DNA complex. Third, the kinetics of the dissociation of the integrase-DNA complex were dramatically accelerated in the presence of the modified ODN, whereas the nonmodified counterpart did not influence the dissociation. This mechanism was supported by the ability of the 6-oxocytosine-containing oligonucleotide to inhibit the strand transfer activity of HIV-1 preintegration complexes in vitro. Disruption of integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides constitutes an original mechanism of integration inhibition, therefore suggesting a strategy for searching for inhibitors of the HIV-1 preintegration complexes.

Binding, Competitive↗