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At least 865 records · Page 48Linked to original sources
PubMed1996

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Amino Acid Sequence↗
PubMed2004

Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen.

The integration of hepatitis B virus (HBV) DNA in the liver of chronic HBV carriers has been documented extensively. However, the status of the viral genome during acute infection has not been assessed conclusively. While HBV DNA sequences are detected often in serum, liver, and peripheral blood mononuclear cells (PBMCs) after the clearance of serum the hepatitis B virus surface antigen (HBsAg), the precise status of the viral genome, and in particular the possible persistence of integrated genomes in PBMCs, has not been established. A highly sensitive PCR-derived assay (Alu-PCR) was employed to re-examine liver and PBMC specimens obtained from patients with acute (n = 19) and chronic (n = 22) hepatitis in whom serum HBsAg was present (n = 12) (HBV-related chronic active hepatitis) or absent with anti-HCV (n = 10) (HCV-related chronic active hepatitis). Viral integration was demonstrated in 3 out of 19 liver specimens from patients with acute hepatitis and 12 out of 12 specimens from patients with chronic hepatitis. Viral integration was also observed in 4 out of 7 PBMC samples from HBV-related chronic active hepatitis patients and 2 out of 10 liver and PBMC samples from HCV-related chronic active hepatitis patients. In one liver specimen from an acute hepatitis patient, HBV DNA was found integrated in the intronic sequence of the tumour necrosis factor (TNF)-induced protein gene; viral integration into cellular sequences was also found in the PBMCs of four HBV-related chronic active hepatitis and two HCV-related chronic active hepatitis. The results demonstrate the early integration of HBV genome during acute viral infections and the persistence of the viral genome in an integrated form in PBMCs.

Acute Disease↗
PubMed1994

The nature and expression of int-5, a novel MMTV integration locus gene in carcinogen-induced mammary tumors.

Our previous studies have resulted in the identification and cloning of int-5, a novel site of mouse mammary tumor virus (MMTV) integration, from BALB/c D2 precancerous hyperplastic alveolar nodules (HAN). This paper presents a detailed characterization of the int-5 locus from both D2 HAN and normal genome and expression of the unique gene from the MMTV integration site. Our results show that the cellular gene at the MMTV integration site in the int-5 locus is identical to the gene encoding aromatase (CYP19), a member of the cytochrome P-450 gene superfamily. MMTV is integrated within exon 10 in the 3' untranslated region of the aromatase gene. This gene (int-5/aromatase) is expressed in normal mammary gland and overexpressed in mammary tumors. These results suggest that the overexpression of this gene may be responsible for mammary tumorigenesis. This is the first demonstration of integration of MMTV in a cellular gene that plays a role in hormone-dependent breast cancers.

Animals↗
PubMed1978

The integrated genome of murine leukemia virus.

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection. The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.

Animals↗
PubMed1987

Provirus integration of bovine leukemia virus into DNA of infected human myeloma cells.

Sixty clones of bovine leukemia virus-infected B-human myeloma ARH77 cells were isolated. The DNAs of all clones were examined for BLV provirus integration by Southern blotting analysis. Proviral sequences were found in DNAs of two clones. One of them (clone I B3) contained one proviral copy with a deletion of approximately 5.5 kb; the other one (clone I F9) carried three integrated proviruses. Viral proteins of 70,000, 42,000 and 35,000 M. W. were found in extracts of clone I F9. In clone I B3 only the 70,000 M.W. protein was detected.

Cell Line↗
PubMed1979

Integration of bovine leukemia virus DNA in the bovine genome.

DNA preparations from circulating leukocytes, lymph node tumors, and spleens of three bovine leukemia virus-infected cattle were fractionated by Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient centrifugation. Bovine leukemia virus proviral sequences were found in large GC-rich fragments having a buoyant density in CsCl close to 1.708 g/cm3. Provirus integration, therefore, does not take place at random locations in the host genome, but in a specific class of DNA segments. Hybridization of cDNA synthesized on viral RNA to EcoRI and Xba I restriction fragments of the DNA from infected cells showed that: (i) only one copy of proviral DNA is integrated per haploid genome; (ii) different restriction patterns were found in the proviral DNAs present in the genomes of different animals, providing evidence for the existence of several strains or mutants; and (iii) different integration sites for the proviral DNA were found in the genome of different animals and of different infected cells in the same animal. The latter finding strongly suggests a polyclonal origin of bovine leukemia virus-infected cells.

Animals↗
PubMed2006

Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts.

The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though approximately 10(14) molecules are inoculated in the studies in rabbits, by day 8 or 9 ( approximately 1 week postinoculation), already all but on the order of 10(4)-10(6) molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.

Acquired Immunodeficiency Syndrome↗
PubMed2006

The integrase interactor 1 (INI1) proteins facilitate Tat-mediated human immunodeficiency virus type 1 transcription.

Integration of human immunodeficiency virus type 1 (HIV-1) into the host genome is catalyzed by the viral integrase (IN) and preferentially occurs within transcriptionally active genes. During the early phase of HIV-1 infection, the incoming viral preintegration complex (PIC) recruits the integrase interactor 1 (INI1)/hSNF5, a chromatin remodeling factor which directly binds to HIV-1 IN. The impact of this event on viral replication is so far unknown, although it has been hypothesized that it could tether the preintegration complex to transcriptionally active genes, thus contributing to the bias of HIV integration for these regions of the genome. Here, we demonstrate that while INI1 is dispensable for HIV-1 transduction, it can facilitate HIV-1 transcription by enhancing Tat function. INI1 bound to Tat and both the repeat (Rpt) 1 and Rpt 2 domains of INI1 were required for efficient activation of Tat-mediated transcription. These results suggest that the incoming PICs might recruit INI1 to facilitate proviral transcription.

Chromosomal Proteins, Non-Histone↗
PubMed2025

The common murine retroviral integration site activating Hhex marks a distal regulatory enhancer co-opted in human early T-cell precursor leukemia.

The Hhex gene encodes a transcription factor that is important for both embryonic and post-natal development, especially of hematopoietic tissues. Hhex is one of the most common sites of retroviral integration in mouse models. We found the most common integrations in AKXD (recombinant inbred strains) T-ALLs occur 57-61kb 3' of Hhex and activate Hhex gene expression. The genomic region of murine leukemia virus (MLV) integrations has features of a developmental stage-specific cis regulatory element (CRE), as evidenced by ATAC-seq in murine progenitor cells and high H3K27 acetylation at the syntenic CRE in human hematopoietic cell lines. With ChIP-exonuclease, we describe occupancy of LIM domain binding protein 1 (LDB1), the constitutive partner of the LIM Only-2 (LMO2), GATA1, and TAL1 transcription factors at GATA sites and at a composite GATA-E box within the CRE. With virtual 4C analysis, we observed looping between this +65kb CRE and the proximal intron one enhancer of HHEX in primary human ETP-ALLs and in normal progenitor cells. Our results show that retroviral integrations at intergenic sites can mark and take advantage of CREs. Specifically, in the case of HHEX activation, this newly described +65kb CRE is co-opted in the pathogenesis of ETP-ALL by the LMO2/LDB1 complex.

Humans↗
PubMed2004

Hepatocellular carcinoma: role of hepatitis B and hepatitis C viruses proteins in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer, being a common cancer type worldwide. Many aetiological factors have been related with HCC development, such as cirrhosis, hepatitis viruses and alcohol. Chronic infection with hepatitis B (HBV) and C viruses (HCV) often results in cirrhosis and enhances the probability of developing HCC. The underlying mechanisms that lead to malignant transformation of infected cells, however, remain unclear. HBV is a DNA virus that integrates into the host genome, and this integration is believed, in part, to be carcinogenic. Besides, the virus encodes a 17 kDa protein, HBx, which is known to be a causative agent in the formation of HCC. On the contrary, HCV is a RNA virus that does not integrate into the host genome but likely induces HCC through host protein interactions or via the inflammatory response to the virus. Products encoded in the HCV genome interfere with and disturb intracellular signal transduction. Some HCV proteins, such as the core protein, NS3 and NS5A, have seen to have a regulatory effect on cellular promoters, to interact with a number of cellular proteins, and to be involved in programmed-cell death modulation under certain conditions. The identification of these proteins functions in HCC development and the subsequent development of strategies to inhibit protein-protein interactions may be the first step towards reducing the chronicity and/or of the carcinogenicity of these two viruses.

Carcinoma, Hepatocellular↗
PubMed2004

Integration of adeno-associated virus (AAV) and recombinant AAV vectors.

The driving interest in adeno-associated virus (AAV) has been its potential as a gene delivery vector. The early observation that AAV can establish a latent infection by integrating into the host chromosome has been central to this interest. However, chromosomal integration is a two-edged sword, imparting on one hand the ability to maintain the therapeutic gene in progeny cells, and on the other hand, the risk of mutations that are deleterious to the host. A clearer understanding of the mechanism and efficiency of AAV integration, in terms of contributing viral and host-cell factors and circumstances, will provide a context in which to evaluate these potential benefits and risks. Research to date suggests that AAV integration in any context is inefficient, and that the persistence of AAV gene delivery vectors in tissues is largely attributable to episomal genomes.

Cell Line↗
PubMed1987

Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

Carcinoma, Hepatocellular↗
PubMed2004

Sequences of flavivirus-related RNA viruses persist in DNA form integrated in the genome of Aedes spp. mosquitoes.

Flavivirus-related sequences have been discovered in the dsDNA genome of Aedes albopictus and Aedes aegypti mosquitoes, demonstrating for the first time an integration into a eukaryotic genome of a multigenic sequence from an RNA virus that replicates without a recognized DNA intermediate. In the Aedes albopictus C6/36 cell line, an open reading frame (ORF) of 1557 aa with protease/helicase and polyprotein processing domains characteristic of flaviviruses was identified. It is closely related to NS1-NS4A genes of the Cell Fusing Agent and Kamiti River virus and the corresponding mRNAs were detected. Integrated sequences homologous to the envelope, NS4B and polymerase genes of flaviviruses were identified. Overall, approximately two-thirds of a flavivirus-like genome were characterized. In the Aedes aegypti A20 cell line, a 492 aa ORF related to the polymerase of the Cell Fusing Agent and Kamiti River virus was identified. These flavivirus-related integrated DNA sequences were detected in laboratory-bred and wild Aedes albopictus and Aedes aegypti mosquitoes, demonstrating that their discovery is not an artefact resulting from the manipulation of mosquito cell lines, since they exist under natural conditions. This finding has major implications regarding evolution, as it represents an entirely different mechanism by which genetic diversity may be generated in eukaryotic cells distinct from accepted processes.

Aedes↗
PubMed1981

Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepatocellular carcinoma. Studies in percutaneous liver biopsies and post-mortem tissue specimens.

We used recombinant-DNA technology and gel electrophoresis to find hepatitis B virus DNA (HBV-DNA) in liver and tumor tissue from patients with hepatocellular carcinoma and chronic liver disease, and to study the integration of HBV-DNA into the genome of these tissues' cells. In 12 patients with hepatocellular carcinoma who had hepatitis B surface antigen (HBsAg) in their serum, integrated HBV-DNA was identified in the tumors; it was also found in tumors from three of eight patients who were seronegative for HBsAg but positive for antibody to HBsAg. In some cases, integrated HBV-DNA was also present in nontumorous liver tissue that had the same hybridization pattern or one different from that of the tumor. In five carriers of HBsAg who had evidence of the carrier state and chronic liver disease for less than two years, HBV-DNA was present but not integrated in liver tissue. In the two patients who had carried HBsAg for more than eight years, HBV-DNA was integrated into the host genome. These data suggest that integration of HBV-DNA into hepatocytes occurs during the course of persistent HBV infection and precedes development of gross neoplasm.

Biopsy↗
PubMed2002

Subcellular localization and integration activities of rous sarcoma virus reverse transcriptase.

Reverse transcriptases (RTs) alphabeta and beta from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT alphabeta and the beta subunit, when expressed independently, can be detected in the nucleus, whereas the separate alpha subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of beta, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT alphabeta with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT alphabeta is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT alphabeta could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).

3T3 Cells↗
PubMed1971

The G-IX system. A cell surface allo-antigen associated with murine leukemia virus; implications regarding chromosomal integration of the viral genome.

This report concerns a cell surface antigen (G(IX); G = Gross) which exhibits mendelian inheritance but which also appears de novo in cells that become productively infected with MuLV (Gross), the wild-type leukemia virus of the mouse. In normal mice, G(IX) is a cell surface allo-antigen confined to lymphoid cells and found in highest amount on thymocytes. Four categories of inbred mouse strains can be distinguished according to how much G(IX) antigen is expressed on their thymocytes. G(IX) (-) strains have none; in the three G(IX) (+) categories, G(IX) (3), G(IX) (2), and G(IX) (1), the amounts of G(IX) antigen present (per thymocyte) are approximately in the ratios 3:2:1. A study of segregating populations derived mainly from strain 129 (the prototype G(IX) (3) strain) and C57BL/6 (the prototype G(IX) (-) strain) revealed that two unlinked chromosomal genes are required for expression of G(IX) on normal lymphoid cells. The phenotype G(IX) (+) is expressed only when both genes are present, as in 129 mice. C57BL/6 carries neither of them. At one locus, expression of G(IX) is fully dominant over nonexpression (G(IX) fully expressed in heterozygotes). At the second locus, which is linked with H-2 (at a distance of 36.4 +/- 2.7 units) in group IX (locus symbol G(IX)), expression is semidominant (50% expression of G(IX) in heterozygotes); gene order T:H-2:Tla:G(IX). As a rule, when cells of G(IX) (-) mice or rats become overtly infected with MuLV (Gross), an event which occurs spontaneously in older mice of certain strains and which also commonly accompanies malignant transformation, their phenotype is converted to G(IX) (+). This invites comparison with the emergence of TL(+) leukemia cells in TL(-) mouse strains which has been observed in previous studies and which implies that TL(-) --> TL(+) conversion has accompanied leukemic transformation of such cells. So far the only example of G(IX) (-) --> G(IX) (+) conversion taking place without overt MuLV infection is represented by the occurrence of GCSA(-):G(IX) (+) myelomas in BALB/c (GCSA:G(IX) (-)) mice. Unlike the other Gross cell surface antigen described earlier, GCSA, which is invariably associated with MuLV (Gross) infection and never occurs in its absence, G(IX) antigen sometimes occurs independently of productive MuLV infection; for example, thymocytes and some leukemias of 129 mice are GCSA(-):G(IX) (+), and MuLV-producing sarcomas may be GCSA(+):G(IX) (-). The frequent emergence of cells of G(IX) (+) phenotype in all mouse strains implies that the structural gene coding for G(IX) antigen is common to all mice. There is precedent for this in the TL system, in which two of the Tla genes in linkage group IX appear to be ubiquitous among mice, but are normally expressed only in strains of mice carrying a second (expression) gene. It is not yet certain whether either of the two segregating genes belongs to the MuLV genome rather than to the cellular genome. This leaves the question whether MuLV may have a chromosomal integration site still debatable. But there is a good prospect that further genetic analysis will provide the answer and so elucidate the special relationship of leukemia viruses to the cells of their natural hosts.

AKR murine leukemia virus↗
PubMed2000

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus.

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.

Attachment Sites, Microbiological↗