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Gene Therapy Aug 2013Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes...
Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes the retroviral and lentiviral vectors commonly used for deriving induced pluripotent stem cells (iPSCs). We previously used integrating foamy virus (FV) vectors expressing OCT4, SOX2, MYC and KLF4 to reprogram osteogenesis imperfecta mesenchymal stem cells (MSCs). Here, we have studied the effects of 10 FV vector proviruses on neighboring gene expression in four iPSC lines and their corresponding iPSC-derived MSC (iMSCs). Gene expression profiles in these iPSC lines showed that none of the 38 genes within 300 kb up- or downstream of integrated proviruses had a significant difference in mRNA levels, including five genes with proviruses in their transcription units. In the iMSCs derived from these iPSCs, the same type of analysis showed a single dysregulated transcript out of 46 genes found near proviruses. This frequency of dysregulation was similar to that of genes lacking nearby proviruses, so it may have been due to interclonal variation and/or measurement inaccuracies. While the number of integration sites examined in this paper is limited, our results suggest that integrated FV proviruses do not impact the expression of chromosomal genes in pluripotent human stem cells or their differentiated derivatives. This interpretation is consistent with previous reports that FV vectors have minimal genotoxicity, even when integrating near or within genes.
Topics: Cell Differentiation; Cell Line; Gene Expression Regulation, Developmental; Genetic Vectors; Humans; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Spumavirus; Virus Integration
PubMed: 23388702
DOI: 10.1038/gt.2013.6 -
Journal of Virology Jul 1998Five site-specific adeno-associated virus integrants generated in a model system with an Epstein-Barr virus- based shuttle vector have been characterized. The results...
Five site-specific adeno-associated virus integrants generated in a model system with an Epstein-Barr virus- based shuttle vector have been characterized. The results suggest a deletion-substitution mechanism of recombination.
Topics: Dependovirus; Plasmids; Recombination, Genetic; Virus Integration
PubMed: 9621089
DOI: 10.1128/JVI.72.7.6195-6198.1998 -
The Journal of General Virology Jun 1999The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA...
The brown alga Ectocarpus siliculosus frequently carries an endogenous virus, E. siliculosus virus (EsV-1), the genome of which is a circular, double-stranded DNA molecule of about 320 kbp. After infection, which occurs in the unicellular spores or gametes, the virus is present latently in all somatic cells of the host. Virus multiplication is restricted to cells of the reproductive organs. It has been an open question whether the latent viral DNA occurs as a free episome or becomes integrated into the host genome. PCR studies showed that viral DNA co-migrates with high molecular mass DNA in pulsed-field gel electrophoresis, which confirms that latent viral DNA is integrated into the host genome.
Topics: DNA, Plant; DNA, Viral; Electrophoresis, Gel, Pulsed-Field; Phaeophyceae; Phycodnaviridae; Polymerase Chain Reaction; Virus Integration
PubMed: 10374952
DOI: 10.1099/0022-1317-80-6-1367 -
Trends in Cell Biology May 2004Barrier-to-autointegration factor (BAF) is an essential protein that is highly conserved in metazoan evolution. BAF binds directly to double-stranded DNA, nuclear... (Review)
Review
Barrier-to-autointegration factor (BAF) is an essential protein that is highly conserved in metazoan evolution. BAF binds directly to double-stranded DNA, nuclear LEM-domain proteins, lamin A and transcription activators. BAF is also a host cell component of retroviral pre-integration complexes. BAF binds matrix, a retroviral protein, and facilitates efficient retroviral DNA integration in vitro through unknown mechanisms. New findings suggest that BAF has structural roles in nuclear assembly and chromatin organization, represses gene expression and might interlink chromatin structure, nuclear architecture and gene regulation in metazoans.
Topics: Amino Acid Sequence; Animals; Cell Nucleus; Chromatin; DNA-Binding Proteins; Humans; Molecular Sequence Data; Nuclear Proteins; Repressor Proteins; Retroviridae; Virus Integration
PubMed: 15130582
DOI: 10.1016/j.tcb.2004.03.004 -
Revue Scientifique Et Technique... Apr 2016Viruses and their hosts can co-evolve to reach a fragile equilibrium that allows the survival of both. An excess of pathogenicity in the absence of a reservoir would be... (Review)
Review
Viruses and their hosts can co-evolve to reach a fragile equilibrium that allows the survival of both. An excess of pathogenicity in the absence of a reservoir would be detrimental to virus survival. A significant proportion of all animal genomes has been shaped by the insertion of viruses that subsequently became 'fossilised'. Most endogenous viruses have lost the capacity to replicate via an infectious cycle and now replicate passively. The insertion of endogenous viruses has contributed to the evolution of animal genomes, for example in the reproductive biology of mammals. However, spontaneous viral integration still occasionally occurs in a number of virus-host systems. This constitutes a potential risk to host survival but also provides an opportunity for diversification and evolution.
Topics: Animals; Genome; Retroviridae; Virus Diseases; Virus Integration; Virus Replication
PubMed: 27217174
DOI: 10.20506/rst.35.1.2423 -
Journal of Virology Oct 2006When 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...
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.
Topics: Avian Sarcoma Viruses; Genetic Vectors; Integrases; Recombination, Genetic; Terminal Repeat Sequences; Virus Integration
PubMed: 17005708
DOI: 10.1128/JVI.00361-06 -
MicrobiologyOpen Feb 2017The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic...
The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis-lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, these two distinct pathways can be visualized by following the DNA movements in vivo. Surprisingly, we frequently observed an interesting "lyso-lysis" phenomenon in lytic cells, where phage integrates its DNA into the host, a characteristic event of the lysogenic pathway, followed by cell lysis. Furthermore, the frequency of lyso-lysis increases with the number of infecting phages, and specifically, with CII activity. Moreover, in lytic cells, the integration site attB on the E. coli genome migrates toward the polar region over time, leading to more spatial overlap with the phage DNA and frequent colocalization/collision of attB and phage DNA, possibly contributing to a higher chance for DNA integration.
Topics: Attachment Sites, Microbiological; Bacteriophage lambda; DNA, Viral; Escherichia coli; Lysogeny; Virus Integration
PubMed: 27530202
DOI: 10.1002/mbo3.395 -
Virus Research Dec 2013Human Immunodeficiency virus type 1 (HIV-1), as well as many other viruses that depend on nuclear entry for replication, has developed an evolutionary strategy to dock... (Review)
Review
Human Immunodeficiency virus type 1 (HIV-1), as well as many other viruses that depend on nuclear entry for replication, has developed an evolutionary strategy to dock and translocate through the nuclear pore complex (NPC). In particular, the nuclear pore is not a static window but it is a dynamic structure involved in many vital cellular functions, as nuclear import/export, gene regulation, chromatin organization and genome stability. This review aims to shed light on viral mechanisms developed by HIV-1 to usurp cellular machinery to favor viral gene expression and their replication. In particular, it will be reviewed both what is known and what is speculated about the link between HIV translocation through the nuclear pore and the proviral integration in the host chromatin.
Topics: HIV-1; Humans; Nuclear Pore Complex Proteins; Virus Integration
PubMed: 24051001
DOI: 10.1016/j.virusres.2013.09.003 -
Journal of Virology Nov 2002Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent...
Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.
Topics: CD4-Positive T-Lymphocytes; Cell Line; DNA, Viral; Gene Products, gag; HIV-1; Humans; Linear Models; Proviruses; Sensitivity and Specificity; Virus Integration
PubMed: 12368337
DOI: 10.1128/jvi.76.21.10942-10950.2002 -
Journal of Virological Methods May 2019Paired-end deep sequencing is a powerful tool to investigate integration sites of the HIV-1 genome in infected cells. Integration sites of HIV-1 proviral DNA carrying...
Paired-end deep sequencing is a powerful tool to investigate integration sites of the HIV-1 genome in infected cells. Integration sites of HIV-1 proviral DNA carrying intact LTR ends have been well documented. In contrast, integration sites of proviral DNA with aberrant ends, which emerge infrequently but can also induce replication-competent viruses, have not been extensively examined, in part, because of the lack of a suitable bioinformatics method for deep sequencing. Here, we report a novel bioinformatics protocol, named the VINSSRM, to search for integration sites of proviral DNA carrying intact and aberrant LTR ends using paired-end deep sequencing data. The protocol incorporates split-read mapping to assign viral and human genome parts within read sequences and overlapping paired-end read merging to construct long error-corrected sequences. The VINSSRM not only consistently detects integration sites similar to the conventional method but also provides information on additional integration sites, including those of proviral DNA with aberrant ends, which were mainly found in non-exonic regions of the human genome. Therefore, the VINSSRM may help us to understand HIV-1 integration, persistence of infected cells, and viral latency.
Topics: Computational Biology; DNA, Viral; Genome, Viral; HIV Infections; HIV-1; High-Throughput Nucleotide Sequencing; Humans; Proviruses; Sensitivity and Specificity; Virus Integration
PubMed: 30857886
DOI: 10.1016/j.jviromet.2019.03.004