scholarly journals Genome-wide mapping of foamy virus vector integrations into a human cell line

2006 ◽  
Vol 87 (5) ◽  
pp. 1339-1347 ◽  
Author(s):  
Ali Nowrouzi ◽  
Marcus Dittrich ◽  
Chuck Klanke ◽  
Martin Heinkelein ◽  
Matthias Rammling ◽  
...  
Keyword(s):  
Human Genome ◽  
Integration Site ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Consensus Sequence ◽  
Cpg Islands ◽  
Retroviral Vectors ◽  
Inverse Pcr ◽  
Integration Pattern ◽  
Integration Sites

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.

scholarly journals Integration Site Choice of a Feline Immunodeficiency Virus Vector

2006 ◽  
Vol 80 (17) ◽  
pp. 8820-8823 ◽  
Author(s):  
Yubin Kang ◽  
Christopher J. Moressi ◽  
Todd E. Scheetz ◽  
Litao Xie ◽  
Diane Thi Tran ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Integration Site ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Moloney Murine Leukemia Virus ◽  
Human Hepatoma Cells ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Primate Lentiviruses ◽  
Transcriptional Units

ABSTRACT We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a “bendable” structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus.

Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 289-289 ◽  
Author(s):  
Akihiro Konno ◽  
G. Jayashree Jagadeesh ◽  
Daniele Moratto ◽  
Marita Bosticardo ◽  
Ingeborg Holt ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Peripheral Blood Lymphocytes ◽  
Inverse Pcr ◽  
Blood Lymphocytes ◽  
Integration Sites ◽  
Insertion Sites

Abstract Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking. We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001). These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.

scholarly journals VISDB: a manually curated database of viral integration sites in the human genome

2019 ◽  
Vol 48 (D1) ◽  
pp. D633-D641 ◽  
Author(s):  
Deyou Tang ◽  
Bingrui Li ◽  
Tianyi Xu ◽  
Ruifeng Hu ◽  
Daqiang Tan ◽  
...  
Keyword(s):  
Human Genome ◽  
Motif Discovery ◽  
Target Genes ◽  
Integration Site ◽  
Cpg Islands ◽  
Fragile Sites ◽  
Viral Integration ◽  
Sequence Motif ◽  
Host Interactions ◽  
Integration Sites

Abstract Virus integration into the human genome occurs frequently and represents a key driving event in human disease. Many studies have reported viral integration sites (VISs) proximal to structural or functional regions of the human genome. Here, we systematically collected and manually curated all VISs reported in the literature and publicly available data resources to construct the Viral Integration Site DataBase (VISDB, https://bioinfo.uth.edu/VISDB). Genomic information including target genes, nearby genes, nearest transcription start site, chromosome fragile sites, CpG islands, viral sequences and target sequences were integrated to annotate VISs. We further curated VIS-involved oncogenes and tumor suppressor genes, virus–host interactions involved in non-coding RNA (ncRNA), target gene and microRNA expression in five cancers, among others. Moreover, we developed tools to visualize single integration events, VIS clusters, DNA elements proximal to VISs and virus–host interactions involved in ncRNA. The current version of VISDB contains a total of 77 632 integration sites of five DNA viruses and four RNA retroviruses. VISDB is currently the only active comprehensive VIS database, which provides broad usability for the study of disease, virus related pathophysiology, virus biology, host–pathogen interactions, sequence motif discovery and pattern recognition, molecular evolution and adaption, among others.

Retroviral Vector Integration Site Analysis in Rhesus Macaques Transplanted with Hematopoietic Stem Cells Transduced with Simian Immunodeficiency Virus Vectors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3255-3255
Author(s):  
Yan Shou ◽  
John Gray ◽  
Brian A. Agricola ◽  
Zhijun Ma ◽  
Derek A. Persons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Integration Site ◽  
Rhesus Macaques ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Integration Pattern ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Selection Of

Abstract Lentiviral vectors derived from the Simian Immunodeficiency Virus (SIV) mediate relatively efficient transduction of hematopoietic stem cells (HSCs) from rhesus macaques. While integration sites associated with onco-retroviral vectors have been extensively studied in primate transplantation experiments, much less in known about lentiviral vector integration site patterns. The existing literature is limited to one report showing that SIV vectors have a distinctive genomic integration pattern compared with onco-retroviral vectors (Hematti et al 2004). Here we report our results mapping 263 integration sites for SIV vectors in an autologous rhesus macaque transplantation model. Two SIV vectors were used that expressed either MGMT-P140K alone or MGMT-P140K together with HOXB4 from an internal MSCV promoter. Two rhesus macaques were transplanted with autologous CD34+ cells, half of which were transduced with the MGMT vector and half were transduced with MGMT-HOXB4 vector. The first animal was treated with 7 courses of temozolomide and 6-BG which has resulted in selection of transduced cells in vivo, both at the level of myeloid progenitors, and to a lesser degree, in HSCs. A total of 152 integration sites were identified from this animal based on LAM-PCR. Sequence analysis showed a favored preference for integration into transcription units, which comprised 70% of all integrations, with 64% integrations occurring within introns and 6% within exons. The highest density of SIV integration sites per Mbp were on chromosomes 17 and 19 (0.17 and 0.2 respectively). At different time points during drug treatment, multiple clones contributed to hematopoiesis and 24 clones were identified repetitively. The second animal was treated with two courses of TMZ/BG and two courses of BCNU/BG resulting in selection of transduced cells in all lineages. So far, a total of 111 integration sites have been identified in this animal and a similar general integration pattern was observed as seen in the first animal. Integration into transcription units was favored (71%) with 65% occurring within introns and 6% within exons. The three most gene-dense chromosomes 17, 19 and 22 had the highest density of SIV integration sites (0.11, 0.16 and 0.18 respectively). In this animal, 10 out 111 integration sites were identified repetitively during the drug treatments. Vector integrations near previously described oncogenes were identified in both animals (19 out 152 and 11 out of 111 integration sites for each animal respectively). However, no common integration sites (CIS) into a single oncogene were observed and no abnormal hematopoietic proliferation developed in either animal. Moreover, there were no integrations seen within the MDS/Evi locus that has been previously shown to be a CIS for onco-retroviral vectors. Our study shows that the SIV integration pattern is distinctly different from that obtained with murine oncoretroviral vectors and is consistent with the previous study. The lack of integrations within the MDS1/Evi locus represents a potential safety advantage, however further study will be necessary to determine whether the overall propensity for insertional mutagenesis and transformation is decreased. We also show that multiple clones contributed to hematopoiesis before and after MGMT-mediated selection suggesting that this approach is not necessarily associated with restrictions in clonal numbers contributing to hematopoiesis.

scholarly journals Porcine Endogenous Retrovirus Integration Sites in the Human Genome: Features in Common with Those of Murine Leukemia Virus

2006 ◽  
Vol 80 (22) ◽  
pp. 10980-10988 ◽  
Author(s):  
Yann Moalic ◽  
Yannick Blanchard ◽  
Hélène Félix ◽  
André Jestin
Keyword(s):  
Human Genome ◽  
Site Selection ◽  
Murine Leukemia Virus ◽  
Integration Site ◽  
Leukemia Virus ◽  
Porcine Endogenous Retrovirus ◽  
Zoonotic Risk ◽  
Integration Sites ◽  
Murine Leukemia

ABSTRACT Porcine endogenous retroviruses (PERV) are a major concern when porcine tissues and organs are used for xenotransplantation. PERV has been shown to infect human cells in vitro, highlighting a potential zoonotic risk. No pathology is associated with PERV in its natural host, but the pathogenic potential might differ in the case of cross-species transmission and can only be inferred from knowledge of related gammaretroviruses. We therefore investigated the integration features of the PERV DNA in the human genome in vitro in order to further characterize the risk associated with PERV transmission. In this study, we characterized 189 PERV integration site sequences from human HEK-293 cells. Data showed that PERV integration was strongly enhanced at transcriptional start sites and CpG islands and that the frequencies of integration events increased with the expression levels of the genes, except for the genes with the highest levels of expression, which were disfavored for integration. Finally, we extracted genomic sequences directly flanking the integration sites and found an original 8-base statistical palindromic consensus sequence [TG(int)GTACCAGC]. All these results show similarities between PERV and murine leukemia virus integration site selection, suggesting that gammaretroviruses have a common pattern of integration and that the mechanisms of target site selection within a retrovirus genus might be similar.

scholarly journals DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

2007 ◽  
Vol 81 (20) ◽  
pp. 11290-11303 ◽  
Author(s):  
Katsuya Inagaki ◽  
Susanna M. Lewis ◽  
Xiaolin Wu ◽  
Congrong Ma ◽  
David J. Munroe ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Integration Site ◽  
Marker Gene ◽  
Cpg Islands ◽  
Previous Method ◽  
Adeno Associated Virus ◽  
Transcription Start Sites ◽  
Vector Integration ◽  
Integration Sites

ABSTRACT Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

scholarly journals Integration Site Preference of Xenotropic Murine Leukemia Virus-Related Virus, a New Human Retrovirus Associated with Prostate Cancer

2008 ◽  
Vol 82 (20) ◽  
pp. 9964-9977 ◽  
Author(s):  
Sanggu Kim ◽  
Namshin Kim ◽  
Beihua Dong ◽  
David Boren ◽  
Serena A. Lee ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Integration Site ◽  
Leukemia Virus ◽  
Integration Sites ◽  
Cancer Tissues ◽  
Xmrv Infection ◽  
Murine Leukemia

ABSTRACT Xenotropic murine leukemia virus-related virus (XMRV) is a new human gammaretrovirus identified in prostate cancer tissue from patients homozygous for a reduced-activity variant of the antiviral enzyme RNase L. Neither a casual relationship between XMRV infection and prostate cancer nor a mechanism of tumorigenesis has been established. To determine the integration site preferences of XMRV and the potential risk of proviral insertional mutagenesis, we carried out a genome-wide analysis of viral integration sites in the prostate cell line DU145 after an acute XMRV infection and compared the integration site pattern of XMRV with those found for murine leukemia virus and two human retroviruses, human immunodeficiency virus type 1 and human T-cell leukemia virus type 1. Among all retroviruses analyzed, XMRV has the strongest preference for transcription start sites, CpG islands, DNase-hypersensitive sites, and gene-dense regions; all are features frequently associated with structurally open transcription regulatory regions of a chromosome. Analyses of XMRV integration sites in tissues from prostate cancer patients found a similar preference for the aforementioned chromosomal features. Additionally, XMRV integration sites in cancer tissues were associated with cancer breakpoints, common fragile sites, microRNA, and cancer-related genes, suggesting a selection process that favors certain chromosomal integration sites. In both acutely infected cells and cancer tissues, no common integration site was detected within or near proto-oncogenes or tumor suppressor genes. These results are consistent with a model in which XMRV may contribute to tumorigenicity via a paracrine mechanism.

scholarly journals Mouse Mammary Tumor Virus Integration Site Selection in Human and Mouse Genomes

2007 ◽  
Vol 82 (3) ◽  
pp. 1360-1367 ◽  
Author(s):  
Alexander Faschinger ◽  
Francoise Rouault ◽  
Johannes Sollner ◽  
Arno Lukas ◽  
Brian Salmons ◽  
...  
Keyword(s):  
Integration Site ◽  
Leukemia Virus ◽  
Transcription Start Sites ◽  
Mouse Mammary Tumor ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Tumor Virus ◽  
Mouse Cells ◽  
Human And Mouse ◽  
Integration Site Selection

ABSTRACT Based on integration site preferences, retroviruses can be placed into three groups. Viruses that comprise the first group, murine leukemia virus and foamy virus, integrate preferentially near transcription start sites. The second group, notably human immunodeficiency virus and simian immunodeficiency virus, preferentially targets transcription units. Avian sarcoma-leukosis virus (ASLV) and human T-cell leukemia virus (HTLV), forming the third group, show little preference for any genomic feature. We have previously shown that some human cells sustain mouse mammary tumor virus (MMTV) infection; therefore, we infected a susceptible human breast cell line, Hs578T, and, without introducing a species-specific bias, compared the MMTV integration profile to those of other retroviruses. Additionally, we infected a mouse cell line, NMuMG, and thus we could compare MMTV integration site selection in human and mouse cells. In total, we examined 468 unique MMTV integration sites. Irrespective of whether human or mouse cells were infected, no integration bias favoring transcription start sites was detected, a profile that is reminiscent of that of ASLV and HTLV. However, in contrast to ASLV and HTLV, not even a modest tendency in favor of integration within genes was observed. Similarly, repetitive sequences and genes that are frequently tagged by MMTV in mammary tumors were not preferentially targeted in cell culture either in mouse or in human cells; hence, we conclude that MMTV displays the most random dispersion of integration sites among retroviruses determined so far.

High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors

Blood ◽  
2010 ◽  
Vol 116 (25) ◽  
pp. 5507-5517 ◽  
Author(s):  
Claudia Cattoglio ◽  
Danilo Pellin ◽  
Ermanno Rizzi ◽  
Giulietta Maruggi ◽  
Giorgio Corti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Leukemia Virus ◽  
Regulatory Elements ◽  
Retroviral Vectors ◽  
Alternative Promoters ◽  
High Definition ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Integration Sites

Abstract Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)– and HIV-based retroviral vectors in the genome of human CD34+ multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.

scholarly journals Viral Determinants of Integration Site Preferences of Simian Immunodeficiency Virus-Based Vectors

2006 ◽  
Vol 80 (16) ◽  
pp. 8145-8150 ◽  
Author(s):  
Hella Monse ◽  
Stephanie Laufs ◽  
Seraphin Kuate ◽  
W. Jens Zeller ◽  
Stefan Fruehauf ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Integration Site ◽  
Active Regions ◽  
Retroviral Vectors ◽  
Site Preferences ◽  
Accessory Genes ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Viral Determinants ◽  
Viral Components

ABSTRACT Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes.

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