Genome-wide analysis of retroviral DNA integration

Retroviral vectors are often used to introduce therapeutic sequences into patients' cells. In recent years, gene therapy with retroviral vectors has had impressive therapeutic successes, but has also resulted in three cases of leukaemia caused by insertional mutagenesis, which has focused attention on the molecular determinants of retroviral-integration target-site selection. Here, we review retroviral DNA integration, with emphasis on recent genome-wide studies of targeting and on the status of efforts to modulate target-site selection.     

Moving DNA around: DNA transposition and retroviral integration

Mobile DNA elements are found in all kingdoms of life, and they employ numerous mechanisms to move within and between genomes. Here we review recent structural advances in understanding two very different families of DNA transposases and retroviral integrases: the DDE and Y1 groups. Even within the DDE family which shares a conserved catalytic domain, there is great diversity in the architecture of the synaptic complexes formed by the intact enzymes with their cognate element-end DNAs. However, recurring themes arise from comparing these complexes, such as stabilization by an intertwined network of protein-DNA and protein-protein contacts, and catalysis in trans, where each active subunit catalyzes the chemical steps on one DNA segment but also binds specific sequences on the other.     

Relationship between gene expression and observed intensities in DNA microarrays--a modeling study

A theoretical study of the physical properties which determine the variation in signal strength from probe to probe on a microarray is presented. A model which incorporates probe-target hybridization, as well as the subsequent dissociation which occurs during stringent washing of the microarray, is introduced and shown to reasonably describe publicly available spike-in experiments carried out at Affymetrix. In particular, this model suggests that probe-target dissociation during the stringent wash plays a critical role in determining the observed hybridization intensities. In addition, it is demonstrated that non-specific hybridization introduces uncertainties which significantly limit the ability of any model to accurately quantify absolute gene expression levels while, in contrast, target folding appears to have little effect on these results. Finally, for data from target spike-in experiments, our model is shown to compare favorably with an existing statistical model in determining target concentration levels.     

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.     

Approaches to site-directed DNA integration based on transposases and retroviral integrases

The ability of retroviruses and transposons to insert their genome into the host cell makes them attractive objects for constructing gene therapy vectors. However, enzymes that insert genetic material do not possess any selectivity relative to target nucleotide sequences, which results in almost random DNA insertion into the recipient cell genome. This leads to mutations that in turn may cause certain undesirable consequences and sometimes neoplastic cell transformation. For successful functioning, it is a primary necessity to modify a retrovirus and transposon based genetic therapy systems in such a way that the directed vector integration into a target sequence in the human genome can be achieved. In this review, the approaches to date that have been developed for highly specific modification of the genome using fusion protein construction based on retroviral integrases and transposases are discussed, as well as cellular factors interacting with these enzymes.     

Improved retroviral vectors for gene transfer and expression

We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.     

Relationship between gene expression and observed intensities in DNA microarrays—a modeling study

A theoretical study of the physical properties which determine the variation in signal strength from probe to probe on a microarray is presented. A model which incorporates probe-target hybridization, as well as the subsequent dissociation which occurs during stringent washing of the microarray, is introduced and shown to reasonably describe publicly available spike-in experiments carried out at Affymetrix. In particular, this model suggests that probe-target dissociation during the stringent wash plays a critical role in determining the observed hybridization intensities. In addition, it is demonstrated that non-specific hybridization introduces uncertainties which significantly limit the ability of any model to accurately quantify absolute gene expression levels while, in contrast, target folding appears to have little effect on these results. Finally, for data from target spike-in experiments, our model is shown to compare favorably with an existing statistical model in determining target concentration levels.     

In vitro murine leukemia retroviral integration and structure fluctuation of target DNA

Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.     

Approaches towards directed DNA integration by the use of retroviral integrases and transposases

The ability of retroviruses and transposases to insert own genome into a host-cell allow us to consider them as a preferable object for constructing gene therapy vectors. However, enzymes that perform the insertion of the genetic material do not display a selectivity towards target nucleotide sequences that results in an almost random DNA introduction into the recipient cell genome. Random insertion leads to mutations which might cause a number of undesirable consequences including neoplastic transformation in the cell. Thereby, in order to achieve a successful functioning of retroviral and trasposonal genetic therapy systems, it is essential to modify them in such a way that directed integration of the vector in a target sequence in the human genome could be achieved. In the review approaches that have been developed for a high specific modification of genome, including the construction of hybrid proteins on the basis of retroviral integrases, transposases, as well as cellular factors interacting with these enzymes, are presented.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

Sites of retroviral DNA integration: From basic research to clinical applications

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.     

Mobile gene cassettes and DNA integration elements

Integrons are unique natural systems for capturing and spreading the antibiotic resistance genes among Gram-negative bacteria. Gene transfer into small genomes and into plasmids is via site-specific recombination. Integrons act as receptors of antibiotic resistance cassettes. There are known more than 50 cassettes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptomycin, and other antibiotics. The structure of integrons and of gene cassettes are described and the mechanisms of capture, mobilization, and expression of cassettes considered.     

Relationship between plasmid loss and gene expression in Bacillus thuringiensis

The large genome of Bacillus thuringiensis was shortened by provoking plasmid loss, and the effect of such loss on the expression of both chromosomal and plasmid-harboured genes was studied. It was evidenced that the genomic material shortening of B. thuringiensis allows for the improvement of the expression of some of the constructive genes. Indeed, it was shown that curing was an efficient tool to obtain mutant strains with better expression of chromosomal genes. In fact, totally cured strains, HD1Cry-B and BNS3Cry, showed higher chitinolytic and/or proteolytic activities than the corresponding wild and/or partially cured ones. Although total curing is drastic for plasmid-harboured gene expression, the authors obtained partially cured strains, Cur255 and CurHD1, with a doubled level of delta-endotoxin production, and a 1.5-fold improvement of the bacteriocin-specific activity, respectively. In addition, the latter strains showed better relative proteolytic activities.