Retroviral DNA Integration: Viral and Cellular Determinants of Target-Site Selection

Abstract

Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I–hypersensitive sites (i.e., +/− 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

Integrase, LEDGF/p75 and HIV replication

“…These integration pattern results have been confirmed and extended in two new studies that are described in more detail below [101,110]. Since IN is the viral determinant of retroviral targeting specificity [149], the data are consistent with the exclusive interaction of p75 with lentiviral IN proteins [75,87,100]. In vitro, chimeric lambda repressor-IBD proteins were also found to increase recombinant IN-mediated strand transfer near lambda repressor-binding sites in a DNA target [150].…”

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…SC-1 cells transduced with GV vectors had a B1.7-fold higher MFI for eGFP and mCherry than their LV counterparts. As GV tend to integrate in the vicinity of promoters, DNaseI hypersensitive sites as well as CpG islands and LV within active transcription units, 53 the different expression levels observed for both vector families could reflect a better accessibility of transcription factors to the GV promoter/enhancer complex. The integration of bidirectional vectors within actively transcribed genes might be problematic considering that the cellular RNA running over the viral LTR boundaries will ultimately result in the formation of dsRNA that can trigger nuclear and cytoplasmic dsRNA responses and subsequent A-I editing of the message, reduced cap-dependent translation or cell death.…”

Absence of retroviral vector-mediated transformation of gene-modified T cells after long-term engraftment in mice

“…Thus, although integration site analysis can provide interesting data when malignancy arises from genetic modification, 45 it was unlikely to provide any meaningful data from the polyclonal populations of T cells in this study, as there would likely be many sites involved consistent with the largely random integration of retroviral vectors. 18 To realize the promise of gene therapy for the treatment of many diseases and disorders, there is a need for vectors with a demonstrated safety record for each particular application. We have demonstrated that despite the potential of Moloney murine leukemia virusbased vectors to lead to transformation of stem cell progeny in mice and humans, these vectors can be used with relative safety to genetically modify mature T cells.…”

“…[8][9][10] Retroviral vectors have also been used successfully in human trials to correct gene deficiencies in diseases including ADA-SCID, [11][12][13][14] X-linked SCID 15,16 and chronic granulomatous disease. 17 Retroviruses insert their genes in a semirandom manner into host chromosomes, 18 and it is thought that retroviruses may integrate preferentially into transcriptionally active sites. 19 There is therefore potential to dysregulate expression of genes including proto-oncogenes, which may lead to malignant transformation of gene-modified cells.…”