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

Mary K Lewinski,Heather Marshall,Angela Ciuffi,Masahiro Yamashita,Sridhar Hannenhalli,Charles C Berry,Jeremy Leipzig,Michael Emerman,Frederic D Bushman,Francis Collins,Paul Shinn,Joseph R Ecker,Gregory Crawford

doi:10.1371/journal.ppat.0020060

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.

Highlights

The selection of target sites for integration of retroviral DNA is central to the biology of retroviruses and the application of retroviral vectors to gene therapy
Studies of murine leukemia virus (MLV) integration targeting led to the suggestion that integration may be favored in open chromatin [8], since a positive correlation was detected between integration frequency and DNase I–hypersensitive sites [9,10]
(6) The provirus is flanked by repeated segments of the target DNA. (B) Chimeric Human immunodeficiency virus (HIV) derivatives containing segments of MLV

Summary

Introduction

The selection of target sites for integration of retroviral DNA is central to the biology of retroviruses and the application of retroviral vectors to gene therapy. The recent setbacks in human gene-therapy trials, in which a therapeutic retroviral vector integrated near the LMO-2 proto-oncogene and caused leukemia-like illness in three patients [1,2,3], have focused particular attention on the mechanisms responsible for integration targeting. Human immunodeficiency virus (HIV) integration sites are found predominantly in active transcription units [11,13]. Avian sarcoma-leukosis virus (ASLV) shows the most random pattern of integration-site selection—ASLV favors transcription units only weakly and does

Methods

Results

Conclusion