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PubMed1993

Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.

Animals↗

PubMed1998

Retrotransposal integration of mobile genetic elements in human diseases.

Approximately one-third of the mammalian genome is composed of highly repeated DNA sequences, of which the two major families, the long and short inter-spersed nucleotide elements (LINEs and SINEs), are represented in humans by L1 and Alu elements respectively. Both 'types of element are considered to be retrotransposable and to play significant roles in genomic function and evolution. The majority of inserted elements are truncated and often rearranged relative to full-length elements; usually, such retrotransposed sequences are flanked by target-site duplications of various lengths and contain 3' polyA tracts, common characteristics of retrotransposal integration. Retrotransposal integrations of Alu and L1 sequences into biologically important genes appear to play significant roles in some human diseases. Most of the inserted sequences that cause human diseases seem to belong to one or a few subsets of each type of retrotransposon, suggesting that only a few active elements can function as templates for retrotransposition. Integrations observed in oncogenes and in tumor suppressor genes may participate in carcinogenesis by altering the activity of the affected genes. The exact mechanism of these events is unclear; however, retrotransposal integration may be a general mechanism of mutation in humans.

Animals↗

PubMed1995

Unusual long target duplication by insertion of intracisternal A-particle element in radiation-induced acute myeloid leukemia cells in mouse.

Retrotransmission into the IL-3/GM-CSF gene locus by the retrotransposon intracisternal A-particle (IAP) had been observed in distinct tumor cell lines. We analyzed the locus in genomes from 7 different myeloid leukemia cell strains which were originally generated by whole-body X-irradiation of the inbred C3H/He mice at a dose of 3 Gy and maintained by in vivo passage. In one leukemia cell strain out of 7 such cases, RFLP of an allele of the interleukin-3 gene was found. Sequence analysis after cloning from the genomic library showed that a type I delta 2 IAP element was inserted in the region upstream of the IL-3 gene in the head-to-head orientation. This suggests that the locus in myeloid cells is sensitive for integration of IAP elements. Additionally, an unusual long target duplication of 82 bp, 14-fold larger than normal one, was found at the junction of the element. This suggests the possibility of a radiation-induced integration mechanism which is distinct from normal retrotransmission.

Animals↗

PubMed2006

Sec61alpha and TRAM are sequentially adjacent to a nascent viral membrane protein during its ER integration.

Co-translational integration of a nascent viral membrane protein into the endoplasmic reticulum membrane takes place via the translocon. We have been studying the early stages of the integration of a double-spanning plant viral movement protein to gain insights into how viral membrane proteins are transferred from the hydrophilic interior of the translocon into the hydrophobic environment of the bilayer, where the transmembrane (TM) segments of the viral proteins can diffuse freely. Photocrosslinking experiments reveal that this integration involves the sequential passage of the TM segments past Sec61alpha and translocating chain-associating membrane protein (TRAM). Each TM segment is first adjacent to Sec61alpha and subsequently is adjacent to TRAM. TRAM crosslinking extends for a long period during nascent chain biogenesis. In addition, the replacement of the first viral TM segment with a non-viral TM sequence still yields nascent chain photo-adducts with TRAM. TRAM therefore appears to be involved in viral membrane protein integration, and nascent chain recognition by TRAM does not appear to rely solely on the TM domains.

Animals↗

PubMed2006

HIV integration site selection: targeting in macrophages and the effects of different routes of viral entry.

We have studied the selection of HIV DNA integration sites in primary macrophages to investigate two questions. First, mature macrophages do not divide, allowing us to investigate whether HIV integration targeting differs between dividing cells and nondividing cells. We sequenced and analyzed 754 unique integration sites and found that integration in macrophages is favored in active transcription units (TUs), as was observed previously for other cell types. However, HIV integration in genes was slightly less favored in macrophages than in dividing PBMC or T cell lines. Second, we compared integration targeting by HIV-vector particles bearing either of two different envelope proteins (HIV R5 Env or VSV-G) to determine whether the mechanism of entry influenced subsequent integration targeting. Integration sites generated by HIV R5- or VSV-G-bearing particles showed no significant differences in their distributions in the human genome. Analysis of additional published integration site sequences also indicated that the route of entry did not affect integration site selection for other viral envelopes as well.

Base Sequence↗

PubMed2006

Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response.

Integration of high-risk human papillomavirus (HRHPV) into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and acquisition of a selective growth advantage for cells containing integrants. Overexpression of the viral transcriptional regulator E2 from heterologous promoters has an inhibitory effect on transcription from integrated HRHPV. Therefore, we hypothesized that loss of E2-expressing episomes from cells in which integration had previously occurred would be required for such cells to gain a growth advantage. Using the unique W12 model of cervical squamous carcinogenesis, we show that cells containing integrated HPV16 reproducibly emerged during long-term culture when there had been a rapid fall in episome numbers. During the period of emergence, it is possible to isolate single-cell clones containing an intracellular mixture of the integrant being selected and episomes at reduced load. The lower level of E2 expression seen in such cells is associated with partial inhibition of transcription from the HPV16 integrant. Full deregulation is not observed until complete loss of E2-expressing episomes occurs. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by the type I IFN pathway. Taken together, our results indicate that episome loss, associated with induction of antiviral response genes, is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. We conclude that cervical carcinogenesis requires not only HRHPV integration, but also loss of inhibitory episomes.

Cell Line↗

PubMed2000

CTXphi contains a hybrid genome derived from tandemly integrated elements.

CTXphi is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5' prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3' element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXphi, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXphi DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXphi-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXphi does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXphi to new hosts.

Bacteriophages↗

PubMed2000

Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours.

We previously described two genital carcinomas (IC2, IC4) containing human papillomavirus type 16 (HPV-16)- or HPV-18-related sequences integrated in chromosomal bands containing the c-myc (8q24) or N-myc (2p24) gene, respectively. The c-myc gene was rearranged and amplified in IC2 cells without evidence of overexpression. The N-myc gene was amplified and highly transcribed in IC4 cells. Here, the sequence of an 8039 bp IC4 DNA fragment containing the integrated viral sequences and the cellular junctions is reported. A 3948 bp segment of the genome of HPV-45 encompassing the upstream regulatory region and the E6 and E7 ORFs was integrated into the untranslated part of N-myc exon 3, upstream of the N-myc polyadenylation signal. Both N-myc and HPV-45 sequences were amplified 10- to 20-fold. The 3' ends of the major N-myc transcript were mapped upstream of the 5' junction. A minor N-myc/HPV-45 fusion transcript was also identified, as well as two abundant transcripts from the HPV-45 E6-E7 region. Large amounts of N-myc protein were detected in IC4 cells. A major alteration of c-myc sequences in IC2 cells involved the insertion of a non-coding sequence into the second intron and their co-amplification with the third exon, without any evidence for the integration of HPV-16 sequences within or close to the gene. Different patterns of myc gene alterations may thus be associated with integration of HPV DNA in genital tumours, including the activation of the protooncogene via a mechanism of insertional mutagenesis and/or gene amplification.

Amino Acid Sequence↗

PubMed2001

Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia.

Juvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1 gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137-143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of the Myb gene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1 cooperate with loss of Nf1 to cause AML.

Acute Disease↗

PubMed2002

Overexpression of cyclin A in hepatocellular carcinoma and its relationship with HBx gene integration.

OBJECTIVE: To investigate the frequency of cyclin A overexpression in hepatocellular carcinoma (HCC) and its relationship with clinical significance and HBx gene integration. METHODS: PCR, RT-PCR and Western blot were used to detect the gene, mRNA and protein level of cyclin A in the tumor and nontumorous tissue. PCR and Southern blot were used to detect the integration of HBx gene in HCC. RESULTS: Amplification of cyclin A gene was found in 1 of 35 patients; overexpression of cyclin A mRNA and protein was found in 16 of 35, 21 of 35 patients, respectively. Overexpression of cyclin A protein was correlated with patient's age, tumor size and HBx integration. CONCLUSION: Overexpression of cyclin A occurs in the early stage of HCC carcinogenesis. It may be one of the important approaches by which HBV affects the normal cell cycle of hepatocyte.

Adult↗

PubMed1993

[The use of Miniphage D3112 deltaHcts as an integrative vectorin Pseudomonas aeruginosa cells].

Catechol 2,3-dioxygenase gene (xylE) was cloned into the Pseudomonas aeruginosa miniphage D3112 delta Hcts. Miniphage provides an effective transduction of gene xylE and its transposition into different replicons. It can be used as an integrative vector in the P. aeruginosa cells. Besides, gene xylE is rather convenient genetic marker for identification of miniphage both in Excherichia coli and Pseudomonas species.

Bacteriophages↗

PubMed1998

Somatic mutations that contribute to breast cancer.

Cytogenetic and molecular analyses of primary sporadic human breast carcinomas have documented at least 12 different chromosome arms affected by loss of heterozygosity (LOH). This has been taken as evidence for the presence of putative tumour suppressor genes in the remaining allele within the affected regions. We have previously identified three regions on chromosome 17q that are affected by LOH in primary human breast tumours. A physical map of one of these regions (17q21) has been prepared. The putative target gene appears to be located between the D17S846 and D17S746 loci. We are currently determining whether either of two genes located in this region is the target for LOH. The mouse mammary tumour model system provides an approach for identifying genes which, when activated or inactivated by mouse mammary tumour virus (MMTV) integration, contribute to specific stages of mammary tumorigenesis. Using this approach we have identified two genes, designated NOTCH4/INT3 and INT6 respectively. Interruption of NOTCH4/INT3 by MMTV represents a gain-of-function mutation that has profound consequences for mammary gland development and tumorigenesis. INT6 was found to be interrupted by an integrated MMTV genome in a mammary hyperplastic outgrowth line and two independent mammary tumours. In each case the transcriptional orientation of the viral genome was opposite to that of INT6. The rearranged allele was expressed as a truncated chimaeric RNA species composed of INT6 coding sequences, intron sequences and MMTV sequences. Since the non-rearranged allele contained no mutations, we conclude that MMTV integration into INT6 causes a dominant-negative mutation or biologically activates its function. The nucleotide sequence of INT6 is unrelated to any of the known genes in the GenBank database, but is evolutionarily highly conserved.

Animals↗

PubMed1996

Mouse mammary tumor virus production by thymic epithelial cells in vivo.

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.

Animals↗

PubMed2003

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.

Alpharetrovirus↗

PubMed1992

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flow-sorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.

Animals↗

PubMed2000

Identification and characterization of mycobacteriophage L5 excisionase.

The well-characterized mycobacteriophage L5 forms stable lysogens in Mycobacterium smegmatis. Establishment of lysogeny involves integration of the phage genome into the chromosome of its mycobacterial hosts through an integrase-mediated site-specific recombination event. As L5 lysogens spontaneously generate free phage particles, prophage excision must also occur, although an L5 excisionase gene had not been identified. We show here that L5 gene 36 encodes the phage excisionase and is a small, heat-stable 56-amino-acid protein that strongly stimulates excisive recombination both in vivo and in vitro. The ability to manipulate the highly directional phage integration and excision reactions will provide powerful tools for the introduction, curing and recovery of foreign genes in recombinant mycobacterial strains.

Amino Acid Sequence↗

PubMed1995

Deletion in human chromosome region 12q13-15 by integration of human papillomavirus DNA in a cervical carcinoma cell line.

In human cervical carcinomas papillomavirus DNA is frequently integrated in the cell genome. We have cloned the integration site of human papillomavirus-18 DNA in human chromosome region 12q13-15 present in the SW756 cervical carcinoma cell line. Viral DNA is broken from nucleotides 2643 to 3418 in the E1 and E2 open reading frames, resulting in a deletion of 775 bases of viral DNA. Cloning and sequence analysis of the rearranged and germline alleles shows that there is no homology between the target cellular and viral DNA, suggesting it is a nonhomologous recombination. The target cellular region is called papillomavirus associated locus 2 (PAL2). The 5'- and 3'-flanking probes derived from the hybrid viral-cellular clone detect completely different germline restriction fragments in DNA from cells with normal chromosome 12. There is no overlap between the restriction maps of the target germline clones obtained with 5'- and 3'-flanking probes. Probes from these germline clones beyond the breakpoint position do not detect any DNA rearrangement in SW756 cells DNA. These data prove that there is a deletion of cellular DNA as consequence of the integration, with an estimated minimum size of 14 kilobases. Both cellular flanking probes are outside the amplicon of this chromosome region identified in the OSA and RMS13 sarcoma cell lines, comprising SAS-CHOP-CDK4-MDM2 genes and where translocation breakpoints are located in liposarcomas. The integration at 12q13-15 might have been selected by its contribution to the tumor phenotype.

Alleles↗

PubMed1995

Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages phi 13 and phi 42.

Functions required for site-specific integration and excision of the Staphylococcus aureus serotype F virulence-converting phages phi 13 and phi 42 were localized and characterized. Like other temperate phages, integration of phi 13 and phi 42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and phi 11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5' to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of phi 13 and phi 42 are quite distinct from each other, and from the corresponding regions of phi 11 and L54a which encode the Xis proteins that are required with Int to mediate site-specific excision of the latter phages. Surprisingly, phi 13 and phi 42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site-specific excision of integrated phage DNA sequences.

Amino Acid Sequence↗