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PubMed1996

Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells.

Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis.

Animals↗
PubMed1985

Hepatitis B virus integration in hepatocellular carcinoma DNA: duplication of cellular flanking sequences at the integration site.

The integrated form of hepatitis B virus (HBV) in the human hepatoma cell line huH2-2 and its cellular counterpart sequence have been cloned and analyzed. Blot hybridization analysis and nucleotide sequencing indicated that a single copy of the 1895-base-pair (bp) subgenomic region of HBV DNA, spanning from the middle of pre-S to the end of gene X, was integrated and flanked by the 12-bp directly repeating cellular sequences. A comparison of the sequencing data with that of the cellular counterpart DNA indicated the absence of deletion and rearrangement in the cellular flanking DNA following integration of the 1895-bp HBV DNA, except for generation of the 12-bp direct repeat at the virus-cell junction. A possible model for the mechanism of HBV integration is proposed.

Base Sequence↗
PubMed1990

Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

Base Sequence↗
PubMed1987

[Hepatitis B virus integration and oncogene activation].

In hepatitis B virus (HBV) related hepatoma samples there is a high frequency of HBV-DNA integration into the chromosome, but the frequency of integration in the early-stage of carcinogenesis is expected to be even higher. Structural an analysis of integrated HBV-DNA and chromosomal flanking DNAs disclosed that the marked rearrangement of chromosomal DNA is not directly linked to carcinogenesis. HBV-DNA integration and chromosomal DNA depletion occur even in chronic hepatitis tissues. Integration is, therefore, thought to trigger a series of reactions which lead to carcinogenesis. However, the oncogene is not detected within the HBV genome, and the HBV integration site in the chromosome is variable. As one approach to clarifying the cause and effect relationship between HBV-DNA integration and liver carcinogenesis DNA transfection experiments were conducted using mouse NIH3T3 cells to detect hepatoma-related oncogenes. As a result, the well-known N-ras gene and an unknown oncogene were independently isolated from different hepatoma tissues.

Carcinoma, Hepatocellular↗
PubMed1992

The open reading frames in the 3' long terminal repeats of several mouse mammary tumor virus integrants encode V beta 3-specific superantigens.

Mice expressing the minor lymphocyte stimulation antigens, Mls-1a, -2a, or -3a, singly on the B10.BR background have been generated. Mls phenotypes correlate with the integration of mouse mammary tumor viruses (MTV) in the mouse genome. The open reading frames within the 3' long terminal repeats of the integrated MTVs 1, 3, 6, and 13 encode V beta 3-specific superantigens. Sequence data for these viral superantigens is presented, indicating that it is the COOH-terminal portion of the viral superantigen that interacts with the T cell receptor V beta element.

Amino Acid Sequence↗
PubMed1998

Mapping of a mouse mammary tumor virus integration site by retroviral LTR--arbitrary polymerase chain reaction.

The de novo integration of retroviral genomes within the mammalian genome is believed to contribute to the tumorigenic process. Integration may result in the disruption or inappropriate transcription of key regulatory genes. We describe the application of an arbitrarily primed PCR method for the mapping and cloning of genomic integration sites of the mouse mammary tumor virus (MMTV). We have amplified DNA sequences between a selected retroviral MMTV-LTR and random sites in the 3' flanking DNA. Using this technique we were able to visualize several proviral integration sites present in a MMTV-induced mammary tumor derived cell line that were absent from the germ line. Cloning and sequencing of the PCR product corresponding to one site established its identification as an unique 3' flanking sequence.

Animals↗
PubMed1994

A multimegabase cluster of snRNA and tRNA genes on chromosome 1p36 harbours an adenovirus/SV40 hybrid virus integration site.

Adenovirus type 12 (Ad12) induces gaps at chromosomal bands 1p36, 1q21, 1q42-43 and 17q21 after infection of human embryonic kidney cells. Three of these bands harbour small nuclear RNA genes or pseudogenes, but the study of a possible relationship has been hampered by the lytic character of adenovirus infection. A non-lytic Ad5/SV40 hybrid virus preferentially integrates at 1p36 and the integration site has been cloned. Chromosomal band 1p36 encodes genes for small nuclear RNA U1 (RNU-1) and for the tRNAs of glutamic acid (TRE) and asparagine (TRN). Each of these genes is encoded by 15-30 copies. We studied the organization of these genes and of the viral integration site by pulsed field gel electrophoresis (PFGE) and analysis of yeast artificial chromosomes (YACs). We show that RNU-1, TRE and TRN genes are scattered over a region of probably more than 2 Mb with intergenic distances of up to 125 kb. The Ad5/SV40 integration site maps to identical chromosomal NotI fragments as RNU-1 and TRE. Fine mapping of a YAC shows that the integration site is within 40-70 kb of genes for RNU-1, TRN and TRE.

Adenoviruses, Human↗
PubMed1989

Comparison of non-ionic detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus integral membrane proteins.

The integral membrane proteins of Sendai virus haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with 2% of a non-ionic detergent, i.e., polyoxyethylene alkyl ethers varying by 8-14 hydrocarbon units in the alkyl chain and by 4-8 ethylene glycol units in the oxyethylene chain. Triton X-100 and octyl glucoside were included as reference detergents. The hydrophile-lipophile balance (HLB) and the critical micelle concentration (CMC) of the detergents were determined. A decrease in length of the oxyethylate by 8-5 ethylene glycol units and an increase in the alkylate by 8-12 hydrocarbon units resulted in higher yields of extracted proteins. The highest yields were obtained for C12E5 with an HLB of 11.7. Yields of extracted protein could be correlated with the HLB values of the polyoxyethylene alkyl ethers. The structural integrity of HN and F was not affected during extraction by either detergent as measured by their reactivity with monoclonal antibodies directed against native HN and F. Extracts were subjected to anion-exchange high-performance liquid chromatography (HPLC) on a Mono Q column in the presence of 0.1% of the detergent used for extraction. Eluate fractions were analysed by sodium dodecyl polyacrylamide gel electrophoresis and recoveries of HN and F protein were determined by size-exclusion HPLC. The immunological activity of HN and F was tested in an enzyme-linked immunosorbent assay. The highest recoveries of HN and F (80%) were obtained with C10E5 in the elution buffer. HN and F were partially purified and the immunological activity was well preserved.

Animals↗
PubMed1997

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Base Sequence↗
PubMed1990

Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma.

Hepatitis B virus (HBV) DNA frequently integrates into the genome of human primary liver cancer cells, but the significance of this integration in liver carcinogenesis is still unclear. Here we report the cloning of a single HBV integration site in a human hepatocellular carcinoma at an early stage of development, and of its germline counterpart. The normal locus was found to be transcribed into two polyadenylated messenger RNA species of 1.8 and 2.7 kilobases. We have isolated a complementary DNA clone from a normal adult human liver cDNA library which has an open reading frame with a coding capacity for a protein of 432 amino acids and relative molecular mass 48,536. The strong homology of the C-terminal half of the protein to the A-type cyclins of clam and Drosophila identifies it as a human cyclin A. The cyclin A gene has several exons, and the HBV integration occurs within an intron. As cyclins are important in the control of cell division, the disruption of a cyclin A gene by viral insertion might contribute to tumorigenesis.

Amino Acid Sequence↗
PubMed1992

Epstein-Barr virus integration in human lymphomas and lymphoid cell lines.

BACKGROUND: Epstein-Barr virus (EBV) is maintained as an episome in most infected cells. The presence of fused terminal restriction enzyme fragments distinguishes the circular DNA form from the linear virion form. METHODS: EBV genomic structure was analyzed in 8 lymphoid cell lines and 21 human lymphoma specimens by the Southern blot technique. RESULTS: Evidence of viral integration into host chromosomal DNA was identified in four cell lines. In the Namalwa and BL30-B95.8 cell lines, integration occurred through the terminal repeat (TR) sequences. In the BL41-P3HR1 and BL41-B95.8 cell lines, there was loss of left-end viral genomic sequences, including ori-P sequences required for episome maintenance, implying that integration was required for viral genome persistence. Integration was not detected in four other cell lines (Raji, Daudi, B95.8, and BL30-P3HR1). In 21 EBV-containing human lymphomas, including 18 immunodeficiency-related lymphomas, fused TR sequences were identified without evidence of viral genomic integration. CONCLUSIONS: These findings suggest that, although viral integration is common in Burkitt lymphoma cell lines infected in vitro, integration is not common in human lymphomas that develop in vivo in normal or immunodeficient people.

Adolescent↗
PubMed2006

Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis.

Chronic liver disease associated with long term hepatitis B virus (HBV) infection contributes importantly to the development of hepatocellular carcinoma (HCC). A salient feature of these chronic infections is the integration of subgenomic HBV DNA fragments into many different locations within the host DNA, suggesting that integration is random. Although this may promote genetic instability during liver regeneration which accompanies a bout of chronic liver disease, the actual role of integrated HBV DNA in hepatocarcinogenesis is uncertain. Importantly, most integration events retain the HBV open reading frame encoding the HBx antigen (HBxAg), which is the virus contribution to HCC. In addition, many integration events reported in the literature occur near or within fragile sites or other cancer associated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration potentially alters the expression of oncogenes, tumor suppressor genes, and microRNAs (miRNAs) that may contribute importantly to tumorigenesis. If so, then selected integration events may alter pathways that are rate limiting in hepatocarcinogenesis, thereby providing targets with diagnostic/prognostic potential and for therapeutic intervention.

Cell Transformation, Neoplastic↗
PubMed2003

Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey.

Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.

Animals↗
PubMed1999

Activation of the N-myc2 oncogene by woodchuck hepatitis virus integration in the linked downstream b3n locus in woodchuck hepatocellular carcinoma.

In the woodchuck hepatitis virus (WHV)/woodchuck model for hepatitis B virus-induced hepatocellular carcinoma, frequent activation of N-myc oncogenes by WHV integration has been firmly established. N-myc2, the most frequently affected gene, was reported to be activated by WHV insertion either in the proximity of the gene or in a distant uncoding locus, win. We previously reported that a WHV integration cloned from a liver tumor was located in a chromosomal locus already described by others as the site of WHV integration in another hepatocellular carcinoma. On this basis, the locus, named b3n, was defined as a recurrent site of WHV integration. A scaffold or matrix attachment region (S/MAR) element was subsequently shown to be located in this locus approximately 1 kb from the WHV insertion sites. S/MARs are genetic elements involved both in structural and functional organization of chromosomal DNA and in stimulation of gene expression. In the present work, we investigated the possibility that an N-myc gene might be affected by integration in b3n. Analysis of a liver tumor harboring WHV integration in this locus showed N-myc2 overexpression. By restriction analysis, the b3n locus was shown to be located downstream of N-myc2, so the known sites of viral insertion in b3n were approximately 11 kb downstream of the N-myc2 promoter. Although these data support that WHV insertion in b3n activates N-myc2, the mechanisms previously described to be involved in N-myc2 activation do not appear to properly account for activation in this subset of WHV integrations. Available data suggest that activation of N-myc2 by WHV integration in b3n might be mediated by the S/MAR located near the WHV insertion.

Animals↗
PubMed2006

Alternate polypurine tracts affect rous sarcoma virus integration in vivo.

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.

Avian Sarcoma Viruses↗
PubMed2000

Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3.

The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3. Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6. We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes. FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits. An int6 deletion (int6Delta) mutant was viable but grew slowly in minimal medium. This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein. These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation. Interestingly, haploid int6Delta cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6Delta cells formed abnormal spores. We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S. pombe.

Animals↗