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At least 199 records · Page 11Linked to original sources
PubMed1983

Integration of hepatitis B virus DNA: evidence for integration in the single-stranded gap.

The DNA of hepatitis B virus (HBV) can integrate into the genomes of infected human liver cells and may be related to the development of primary liver carcinoma (PLC). This report describes the analysis of three integrated HBV DNA sequences cloned from DNA of the human PLC cell line PLC/PRF/5. In all these sequences, integration of the viral DNA is at a site that is single-stranded (gap) in the unintegrated HBV DNA. Contrary to some speculations, there are no large terminal redundancies flanking the inserts as found in retroviral proviruses. The viral sequences show no obvious rearrangements in these clones, but integration can be associated with deletions in the viral DNA and also apparently in the host DNA.

Base Sequence↗
PubMed2000

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family.

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.

Amino Acid Sequence↗
PubMed1983

Integration of polyoma virus DNA into chromosomal DNA in transformed rat cells causes deletion of flanking cell sequences.

In order to find out whether polyoma virus (Py) integration into chromosomes causes rearrangements in the cell DNA flanking the integration site, we have mapped the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts, and then compared the two maps. To carry out this study we have cloned a segment including Py DNA and flanking sequences in the bacteriophage vector lambda gt WES and subcloned the flanking cell DNA in a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes and used the cloned flanking cell DNA and Py DNA as hybridization probes. Autoradiography of the LPT DNA blots revealed two sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the second set consists of fragments which contain no virus DNA sequences, and are identical to the fragments observed in the corresponding normal rat DNA digests. These data indicate that LPT cells are heterozygous with respect to the Py inserts. The same data were used to map the flanking sequences in the two types of cells. A comparison between the two maps revealed that a 3.0 kb cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, has been deleted from the LPT chromosome which carries Py DNA, but not from the LPT chromosome which does not carry the virus DNA. The implications for papovavirus integration are discussed.

Animals↗
PubMed1989

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

DNA, Recombinant↗
PubMed1984

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from a chronically infected liver.

We have isolated and determined the structure of a recombinant clone in lambda phage Charon 30 which contains woodchuck hepatitis virus sequences integrated in woodchuck genomic DNA sequences. This clone, in contrast to previously reported clones (Ogston et al., Cell 29:385-394, 1982), was isolated from a chronically infected liver which never developed hepatocellular carcinoma. Southern blot analysis of viral sequences in the clone in conjunction with electron microscope heteroduplex analysis showed that the integrated viral sequences did not contain internal rearrangements, as have those from hepatomas, but were colinear with the cloned viral genome except for the deletion of approximately 500 base pairs of viral sequences (between positions 1,000 and 1,550 on the viral map). Therefore, the integration was probably a defective genome incapable of supporting viral replication. However, the complete open reading frames coding for the viral X, core, presurface , and surface antigen genes were present, indicating that the viral sequences could code for viral antigens. Southern blot analysis of the normal cellular flanking sequences, using flanking sequence probes from the clone, showed that no detectable rearrangements of cellular DNA (less than 50 base pairs) had occurred at the site of viral integration.

Animals↗
PubMedDate not supplied

Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma.

Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.

Amino Acid Sequence↗
PubMed2000

Multiple integrations of human foamy virus in persistently infected human erythroleukemia cells.

Foamy viruses are complex retroviruses whose replication strategy resembles that of conventional retroviruses. However, foamy virus replication also resembles that of hepadnaviruses in many respects. Because hepadnaviruses replicate in an integrase-independent manner, we were interested in investigating the characteristics of human foamy virus (HFV) integration. We have shown that HFV requires a functional integrase protein for infectivity. Our analyses have revealed that in single-cell clones derived from HFV-infected erythroleukemia-derived cells (H92), there were up to 20 proviral copies per host cell genome as determined by Southern blot and fluorescent in situ hybridization analysis. Use of specific probes has also shown that a majority of the proviruses contain the complete tas gene, which encodes the viral transactivator, and are not derived from Deltatas cDNAs, which have been shown to arise rapidly in infected cells. To demonstrate that the multiple proviral sequences are due to integration instead of recombination, we have sequenced the junctions between the proviral sequences and the host genome and found that the proviruses have authentic long terminal repeat ends and that each integration is at a different chromosomal site. A virus lacking the Gag nuclear localization signal accumulates fewer proviruses, suggesting that nuclear translocation is important for high proviral load. Since persistently infected H92 clones are not resistant to superinfection, the relative importance of an intracellular versus extracellular mechanism in proviral acquisition has yet to be determined.

Animals↗
PubMed2002

The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

The genetic defects underlying the pathogenesis of acute myeloid leukemia (AML) are still largely unknown. Retroviral insertion mutagenesis in mice has become a powerful tool to identify candidate genes involved in the development of leukemia and lymphoma. We have used this strategy with the 1.4 strain of Graffi murine leukemia virus (MuLV), which predominantly causes myeloid leukemias. Here, we report that Graffi-1.4-induced AML frequently harbors virus integrations in the gene encoding the transcription factor Yin Yang 1 (YY1). These integrations occurred in both orientations, and all were located in the 5' promoter region of the gene, 0.5 to 1.5 kb upstream of the major transcriptional start site. Luciferase reporter assays showed that virus integration in this region increases promoter activity and renders it independent of a functional binding site for Sp1, a major transcriptional regulator of YY1. We used the murine 32D model to study the consequence of perturbed YY1 expression for myelopoiesis. YY1 protein levels were high in 32D parental cells maintained in interleukin-3-containing medium, but they dropped when the cells were induced to differentiate by granulocyte-colony-stimulating factor (G-CSF). Strikingly, G-CSF-induced neutrophilic differentiation was reduced in 32D cell transfectants ectopically expressing YY1. In similar experiments on primary bone marrow cells, enforced YY1 expression blocked the outgrowth of CFU-GM colonies. Increased YY1 expression was seen in some cases of human AML. Collectively, these data imply a possible role of perturbed expression of YY1 in the development of AML through interference with the myeloid differentiation program in the leukemic progenitor cells.

Animals↗
PubMed2002

Identify metastasis-associated genes in hepatocellular carcinoma through clonality delineation for multinodular tumor.

Disease recurrence and metastasis are frequently observed in many successfullytreated localized cancers, including hepatocellular carcinoma in which intrahepatic and extrahepatic recurrence (metastasis) are frequently observed after curative resection. The present study aimed at identifying metastasis-associated genes through delineation of the clonality for multinodular liver cancer. The clonal relationship of 22 tumor foci from six patients was investigated by the genome-wide expression profile via cDNA microarray consisting of 23,000 genes. Tumor molecular properties including p53 protein overexpression and gene mutation, hepatitis B virus integration pattern, and genetic alteration examined by comparative genomic hybridization were compared. Results indicated that gene expression patterns could serve as the molecular fingerprint for clonality identification. Together with the molecular data from p53, hepatitis B virus integration and comparative genomic hybridization profiles, tumor nodules from five patients were confirmed with clonal relationship, and the expression profiles of the primary nodules were compared with their corresponding intrahepatic metastatic nodules. A total of 90 clones were found to be correlated with intrahepatic metastasis by Student's t test (P < 0.05). With reference to the primary tumor, 63 clones (39 known genes and 24 express sequence tags) were down-regulated whereas 27 clones (14 known genes and 13 express sequence tags) were up-regulated in the metastatic nodules. These metastasis-associated genes may provide clues to reveal patients with increased risk of developing metastasis, and to identify novel therapeutic targets for the treatment of metastasis.

Adolescent↗
PubMed2000

Detection of integrated murine leukemia viruses in a mouse model of acute myeloid leukemia by fluorescence in situ hybridization combined with tyramide signal amplification.

This study was undertaken to develop a reliable method to enumerate and map somatically acquired, clonal, murine leukemia virus (MuLV) proviral insertions in acute myeloid leukemia (AML) cells from the BXH-2 mouse strain. This was achieved by using fluorescence in situ hybridization combined with tyramide signal amplification (FISH-TSA) and an 8.8 kilobase pair (kb) full-length ecotropic MuLV or 2.0 kb MuLV envelope (env) gene probe. Two-color FISH was utilized combining chromosome-specific probes for regions near the telomere and/or centromere and the MuLV probes. The technique reliably detected germline and somatically acquired, tumor-specific, MuLV proviruses in BXH-2 AML cell lines. It was possible to readily verify homozygous insertions at endogenous ecotropic MuLV loci, Emv1 (chromosome 5), Emv2 (chromosome 8) and a BXH-2 strain-specific locus (chromosome 11). This strategy also verified the presence of molecularly cloned proviral insertions within the mouse Nf1 gene and another locus on distal chromosome 11, as well as on chromosome 7 and chromosome 9 in BXH-2 AML cell line B117. The technique was also used to detect several new tumor-specific, proviral insertions in BXH-2 AML cell lines.

Acute Disease↗
PubMed2005

Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction.

In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.

Adult↗
PubMed1978

Another chromosomal assignment for a simian virus 40 integration site in human cells.

Somatic cell hybrids derived from fusion of GM637, a human cell line transformed by simian virus 40, and mouse B82 cells were examined for simian virus 40 T antigen, V antigen, and viral DNA. All hybrid cell lines that contained viral DNA were T-antigen positive. Cells that did not have viral DNA were T-antigen negative. We determined that there is a single viral insertion in these hybrid cells. Correlation of T-antigen expression and viral DNA with the partial complements of the human genome retained in the hybrids shwed that the inserted viral genome is in human chromosome 8. The integrated viral DNA is stable; free viral DNA found in GM637 does not insert at other potential sites in the human genome.

Antigens, Viral↗
PubMed2000

Integrated hepatitis B virus DNA preserves the binding sequence of transcription factor Yin and Yang 1 at the virus-cell junction.

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possible involvement of YY1 in the intramolecular recombination of linear replicative HBV DNA has been examined (Y. Hayashi et al., unpublished data). Data suggest that YY1 is involved in the joining reaction between HBV DNA and cellular DNA to form the virus-cell junction.

Amino Acid Sequence↗
PubMed1984

Virus genome integration as a function of the host cell genome replicative cycle.

Mouse 3T3 cells were grown and synchronized in monolayer with the double thymidine block. Their infection with SV40 took place continuously during the cellular cycle. However, integration of viral DNA into host cell DNA occurred preferentially during the S phase. Phase G1 appeared to be necessary for virus-cell DNA recombination in S phase. Phase G2 did not alter the stability of the integrated viral genome.

Animals↗
PubMed1985

Characterization of the influenza virus M2 integral membrane protein and expression at the infected-cell surface from cloned cDNA.

An investigation of properties of the influenza A virus M2 protein indicated that it is synthesized by 2 h postinfection together with other viral polypeptides and is transported to the infected-cell surface with a half-time of approximately 30 to 40 min. The available evidence suggests that M2 is not N-glycosylated even though it contains a potential glycosylation site, and the intracellular pattern of protein distribution includes localization to the Golgi apparatus. Proteolysis of intracellular microsome vesicles followed by immunoprecipitation with antiserum to a synthetic oligopeptide indicated that the M2 protein contains an extensive region of COOH-terminal amino acids exposed on the cytoplasmic side of the infected-cell membrane. A cDNA clone of the M2 mRNA was obtained and expressed in an SV40 recombinant vector. The M2 protein expressed by the vector became associated with the Golgi complex and was found on the surface of vector-infected cells. M2 is antigenically conserved among all strains of influenza virus both in regions exposed on the cell surface and intracellularly.

Cell Membrane↗
PubMed1985

Rat cells infected with anemia-inducing Friend leukemia virus contain integrated replication-competent but not defective proviral genomes.

The integrated proviral DNA in five murine cell lines transformed by the anemic strain of Friend leukemia virus (FLV-A) was examined by Southern hybridization to a cloned Friend virus (F-MuLV) probe. Kpn I fragments 9 kilobases (kb) and 5.7 kb long were observed for each cell line. However, the number of copies of each fragment in the cell genome varied according to the cell type. As compared to the adherent epithelioid cell lines, the anchorage-independent erythroleukemic cell lines contained more copies of the 5.7-kb fragment than of the 9-kb fragment, suggesting that the former may be biologically significant and perhaps related to the growth of erythroid cells. The presence of Kpn I fragments of the same sizes, albeit in fewer copies, in normal mouse spleen DNA made it difficult to distinguish exogenous virus from endogenous viral sequences. Therefore, rat 3Y1 cells, which contained no murine endogenous viruses, were infected with FLV-A stock virus prepared directly from the spleens of leukemic mice. Only the 9-kb Kpn I fragment, representing replication-competent Friend virus component, was detected in the infected rat cell DNA. No hybridization was observed to a 0.6-kb fragment of the spleen focus-forming virus env gene that is specific for xenotropic and dual-tropic mink cell focus-forming viruses. Since the virus synthesized by the infected rat cells was leukemogenic in adult mice, these data suggest that the wild-type FLV-A is replicative and fully pathogenic in the absence of other competent virus components.

Animals↗
PubMedDate not supplied

[The functional activity of the regulator region of virus SV40 integrated into recombinant plasmids].

We propose the system for transfection of eucaryotic SV40-based vectors. The method comprises simultaneous transfection of permissive monkey cells with plasmid DNA and the virus SV40. It is likely that the induction of replication of recombinant plasmids by the SV40 T-antigen is an indicator of functional activity of the SV40 regulatory region integrated into recombinant plasmids.

Animals↗
PubMed2004

Detection of infectious hepatitis A virus by integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction.

AIMS: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV). METHODS AND RESULTS: The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference. Water samples artificially contaminated with infectious or formalin-inactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively. The tagged RT-PCR had high specificity for HAV negative-strand RNA. By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV. The described method detected infectious HAV at inoculation level of 10(0) TCID50 per flask within 4 days. CONCLUSIONS: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples. This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines. It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water.

Cell Line↗