Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Abstract

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virusbased retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.The RNA genome of retroviruses is reverse transcribed into a double-stranded DNA copy, which is then integrated into the host chromosome as a provirus. Viral elements, such as integrase (IN) and the terminal structures of viral DNA, that are required for retroviral integration have been extensively characterized. It has also been shown that selection of the proviral integration targets is nonrandom (15, 29-31, 37, 47) and that the central domain of IN plays a role in determining the target specificity (3, 36). The efficiency of chromosomal sites to become a preferred integration target appears to be affected by several factors, such as transcriptional activity (35, 46), DNase I hypersensitivity (9,32,33,45), methylation (15), GC content (5, 16, 34), nuclear scaffold attachment (20), nucleosome structure (27,28,30,31), and DNA structure of a higher order (14,22,24,27,28). However, these results were obtained by in vitro studies using artificial target DNA or analysis of a small number of in vivo integration sites, and it is still unclear why proviral integration takes place at certain target sites more often than others. To elucidate the mechanism of target selection for proviral integration, it may be useful to compile and analyze a large number of nucleotide sequences corresponding to proviral integration sites in the context of actual cellular chromosome. Traditionally, cellular flanking DNA of a provirus was cloned by the time-consuming method o...

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation

“…20 For example, in one successful example of inverse PCR, highly efficient retroviral integration was examined and cells containing integrations were selected using thymidine kinase/HAT selection. 21 Owing to the direct-PCR approach, lack of ligation steps and high sensitivity, repeat-anchored (eg, Alu) PCR is generally preferred over inverse PCR. 20 The RAIC method described in the present report simply augments the sensitivity of repeat-anchored PCR by also employing the advantages of biotin-capture PCR.…”

Analysis of Wild-Type and Mutant SL3-3 Murine Leukemia Virus Insertions in the c-mycPromoter during Lymphomagenesis Reveals Target Site Hot Spots, Virus-Dependent Patterns, and Frequent Error-Prone Gap Repair

“…Mo-MLV integration in vitro and in cell culture (39,57) and for human T-lymphotropic virus type 1 insertions isolated from patient samples (11,45). Based on these observations, both SL3-3 and HIV-1 INs target rotationally symmetric sequences, and both enzymes prefer a combination of T and A at the central positions of the targeted stretch of host DNA.…”

“…The repair of the two resulting gaps includes DNA synthesis, removal of the two viral 5Ј dinucleotide overhangs generated during 3Ј processing, and ligation of the resulting nicks (for a review, see reference 21). In general, a virus-specific stretch of 4 to 6 bp of the host DNA is duplicated at the integration site; however, atypical virus-host DNA junctions, including alterations in repeat length and point mutations, are occasionally generated (14,30,39,52,71,72).…”

“…The repair of the two resulting gaps includes DNA synthesis, removal of the two viral 5Ј dinucleotide overhangs generated during 3Ј processing, and ligation of the resulting nicks (for a review, see reference 21). In general, a virus-specific stretch of 4 to 6 bp of the host DNA is duplicated at the integration site; however, atypical virus-host DNA junctions, including alterations in repeat length and point mutations, are occasionally generated (14,30,39,52,71,72).EthelbergIn contrast to the retrovirus-like Ty retrotransposons in yeast, which are very selective in the choice of integration sites (as reviewed in reference 9), retroviruses integrate throughout the chromosomes (10,22,33,37,42,43,46,49,50,63,65,67,70,75,76). Although studied in vivo and by use of simplified in vitro models during the last decades (for a review, see references 8 and 36), integration site selection still remains poorly understood.…”

Detection of SRS19-6MuLV in mouse dendritic cell sarcoma and its tumorigenesis

“…Using PCR and inverse PCR (IPCR) (8)(9)(10), the existence of SRS19-6MuLV in different cells and tumors was explored and multiple integration sites were identified.…”