Adenoviral DNA integration and changes in DNA methylation patterns: a different view of insertional mutagenesis.
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Adenoviridae↗
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Selection of target sites for mobile DNA integration in the human genome.
DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn--thus, answering the question "What genomic features affect integration?" requires carefully specifying the length scale of interest.
Binding Sites↗
Immunogenicity of infectious bovine rhinotracheitis virus (BHV-1) proteins integrated into ISCOMs or liposomes.
Dynamics and persistence of antibody responses to immunization and reimmunization of rabbits with BHV-1 ISCOMs or BHV-1 liposomes were investigated for 20 weeks and compared with those induced by a commercial vaccine against infectious bovine rhinotracheitis (IBR) or free BHV-1 protein subunits. Comparable levels of antibodies to BHV-1, determined by ELISA, were found throughout the observation period in rabbits treated with BHV-1 ISCOMs or the commercial vaccine. The immunogenicity of BHV-1 liposomes was higher than that of the free virus proteins, but lower than those of BHV-1 ISCOMs or the commercial vaccine. Virus neutralization test confirmed the best immunostimulatory effect of BHV-1 ISCOMs as far as the levels and the persistence of antibodies were concerned. The reactivities of the specific virus protein subunits with anti-BHV-1 antibodies elicited in rabbits immunized with BHV-1 ISCOMs or the commercial vaccine were compared by Western blotting. The patterns of the bands were similar, but some differences were found in their numbers and intensities.
Animals↗
Integration of Epstein Barr virus near the breakpoint of a translocation 11;19 in a Burkitt's lymphoma cell line.
The physical state of Epstein Barr virus (EBV) DNA was analyzed in the Burkitt's lymphoma (BL) cell line BL60 and in the autologous EBV-immortalized lymphoblastoid cell line (LCL) IARC 277 by Gardella gel analysis, Southern blotting, and nonradioactive in situ hybridization to metaphase chromosomes. Although only episomal viral DNA was detected in the LCL, the 6-12 copies of EBV DNA in the BL cell line are integrated in one site of the host cell genome. The integration site is located near the breakpoint of a translocation 11;19 which is present in this cell line in addition to the BL-specific t(8;22).
Blotting, Southern↗
Activation of Fgf8 in S115 mouse mammary tumor cells is associated with genomic integration of mouse mammary tumor virus.
Fgf8 is an embryonally expressed mitogenic fibroblast growth factor which has transforming capacity. It is expressed in S115 mouse mammary tumor cells (S115 cells) and in parental tumors of DD/Sio mice as well as in some human breast and prostate cancer cell lines. In S115 cells androgens induce the expression of Fgf8 which seems to be associated with the androgen-maintained malignant phenotype of the cells. S115 cells also contain and express Mtv proviruses known to insertionally activate oncogenes in other tumor cells. Here we studied the possibility of insertional activation of Fgf8 in S115 cells by MMTV proviral integration. We demonstrate by Southern blotting that the genomic DNA from DD/Sio tumors and S115 cells contains Mtv-sequences (Mtv-6 and Mtv-17) which are not found in the DNA from spleen or liver of the DD/Sio mice. In addition, the newly integrated Mtv-6 was localized to the DNA fragment containing the Fgf8 gene. Furthermore, the expression of Fgf8 mRNA in DD/Sio tumors and S115 cells was not found in mammary gland or spleen and liver of DD/Sio mice. In S115 cells, Fgf8 mRNA expression was induced in parallel to MMTV mRNA by androgen and glucocorticoids which supports the possibility that Fgf8 is controlled by the steroid-regulated MMTV-LTR. In conclusion, our data provide evidence that the insertion of MMTV into the DD/Sio tumor DNA is associated with the transcriptional activation of Fgf8 in DD/Sio tumor and consequently in S115 mouse mammary tumor cells.
Animals↗
Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNA.
A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA synthesis (internally initiated DNA). Strand transfer of internally initiated DNA would result in "dead" viral DNA that lacks the att site needed for integration. Strand transfer of the internally initiated DNA could occur if DNA synthesis failed to initiate at the PPT or if the PPT-primed DNA was displaced before strand transfer. We sought to determine the efficiency of DNA synthesis initiating at the PPT and the proportions of PPT-primed DNA and internally initiated DNAs that are utilized for strand transfer. We constructed spleen necrosis virus-based retroviral vectors containing an internal PPT and an att site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were determined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and that internally initiated DNAs are rarely utilized for strand transfer. We hypothesize that efficient synthesis and strand transfer of PPT-primed DNA evolved to prevent lethal strand transfers of internally initiated DNAs.
Animals↗
Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA.
We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high M(r) DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.
Animals↗
Retroviral reverse transcription and integration: progress and problems.
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Animals↗
In vivo events of retroviral long terminal repeat integration into Marek's disease virus in commercial poultry: detection of chimeric molecules as a marker.
The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV. A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HS-cPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.
Animals↗
Regulation, replication, and integration functions of the Vibrio cholerae CTXphi are encoded by region RS2.
CTXphi is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae. CTXphi is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTXphi genome has two regions, the 'core' and RS2. Integrated CTXphi is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2. RS1 contains an additional ORF designated rstC. Functional analyses indicate that rstA2 is required for CTXphi replication and rstB2 is required for CTXphi integration. The amino terminus of RstR is similar to the amino termini of other phage-encoded repressors, and RstR represses the expression of rstA2. Although genes with related functions are clustered in the genome of CTXphi in a way similar to those for other filamentous phages, the CTXphi RS2-encoded gene products mediating replication, integration and repression appear to be novel.
Amino Acid Sequence↗
Evaluation of PCR-based methods for the quantitation of integrated HIV-1 DNA.
Integration of HIV-1 DNA is essential both for productive viral replication and for viral persistence in patients. Methods to measure specifically proviral HIV DNA are required for investigating the mechanisms of HIV integration, for screening novel integrase inhibitors in cell culture and for monitoring levels of persistent integrated viral DNA in patients. In this report, the linker primer polymerase chain reaction (LP-PCR) and Alu-PCR methods for the quantitation of integrated HIV-1 DNA have been modified and evaluated. Each of the two modified assays allowed the quantitative detection of 4 copies of integrated HIV DNA in presence of 2 x 10(5) cell-equivalents of human chromosomal DNA. The results show that proper DNA isolation procedures and the inclusion of appropriate controls in these assays are important for the accurate quantitation of integrated HIV DNA. With further improvements, it should be possible to use these methods as diagnostic tools to monitor closely the efficacy of antiretroviral therapy.
Base Sequence↗
Transcriptional activation of the integrated chromatin-associated human immunodeficiency virus type 1 promoter.
The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-kappaB sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-kappaB was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-kappaB failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-kappaB, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-kappaB. Taken together, these results suggest that Sp1 and NF-kappaB are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.
Chromatin↗
Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus.
A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.
DNA, Viral↗
Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.
Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.
Amino Acid Sequence↗
Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.
GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.
Alleles↗
A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein.
The mature flavivirus capsid protein (virion C) is commonly thought to be free in the cytoplasm of infected cells and to form a nucleocapsid-like complex with genomic RNA in mature virus particles. There is little sequence conservation among flavivirus virion C proteins, but they are similar in size (e.g., 99 amino acids [aa] for the dengue-4 [DEN4] C) and in bearing a net positive charge. In addition, we noted that C contained a conserved internal hydrophobic segment (spanning aa 45-65 in the DEN4 C). Results of in vivo expression and in vitro translation of wt and mutant forms of the DEN4 virion C demonstrated that the conserved internal hydrophobic segment in the DEN C functioned as a membrane anchor domain. Signal peptide function of this segment was also suggested by its requirement for the entry of C into membranes. Virion C was integrated in membranes in a "hairpin" conformation; positively charged segments amino- and carboxy-terminal to the hydrophobic signal-anchor segment were accessible to protease digestion in the "cytoplasm." The net positive charge in the amino-terminal extramembraneous portion of C (aa 1-44) was one determinant of the hairpin membrane orientation; a conserved positively charged residue within the hydrophobic segment (Arg-54 in the DEN4 C) was not.
Amino Acid Sequence↗
HIV-1 integration in the human genome favors active genes and local hotspots.
A defining feature of HIV replication is integration of the proviral cDNA into human DNA. The selection of chromosomal targets for integration is crucial for efficient viral replication, but the mechanism is poorly understood. Here we describe mapping of 524 sites of HIV cDNA integration on the human genome sequence. Genes were found to be strongly favored as integration acceptor sites. Global analysis of cellular transcription indicated that active genes were preferential integration targets, particularly genes that were activated in cells after infection by HIV-1. Regional hotspots for integration were also found, including a 2.4 kb region containing 1% of sites. These data document unexpectedly strong biases in integration site selection and suggest how selective targeting promotes aggressive HIV replication.
Base Sequence↗
Hepatocyte-based gene therapy.
Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells.
Adenoviridae↗