Abstract
Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs) flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)- and human immunodeficiency virus (HIV)-derived vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications.
Highlights
Integration of viral cDNA into the host cell genome is an essential step in the retroviral life cycle
Human cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HSCs) were transduced under cytokine stimulation with Moloney leukemia virus (MLV)-derived RV vectors carrying a wild-type long terminal repeats (LTRs), a DU3 LTR, or an LTR from the spleen focus-forming RV (SFFV), and human immunodeficiency virus (HIV)-derived LV vectors carrying a wild-type LTR, a DU3 LTR or an LTR containing the MLV U3 enhancer (Figure 1)
A collection of 795 sequences randomly cloned by linker-mediated PCR (LM-PCR) and 100,000 computergenerated random insertion sites were used as control groups
Summary
Integration of viral cDNA into the host cell genome is an essential step in the retroviral life cycle. The RNA genome is reverse transcribed into double-stranded DNA, and assembled in pre-integration complexes (PICs) containing viral as well as cellular proteins. Retroviral PICs may actively enter the nucleus of non-dividing cells, as in the case of lentiviruses (LV), or gain access to chromosomal DNA during mitosis, as in gammaretroviruses (RV). PICs associate with the host cell chromatin, where the virally encoded integrase mediates proviral insertion into the genomic DNA [1]. Different retroviruses show significantly different integration preferences [2,3,4], implying that PICs recognize components or features of the host cell chromatin in a specific fashion [5,6,7]. Much less is known about the RV integrase, and the genetic and/or epigenetic determinants of RV target site selection remain poorly understood
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