Simian virus 40 minichromosomes as targets for retroviral integration in vivo.

Abstract

We present a method for studying multiple retroviral integration events into a small DNA target in vivo. Episomal simian virus 40 (SV40) genomes established by infection of CV-1 cells served as integration targets during subsequent infection with murine leukemia virus (MLV). Using a PCR-based assay for the abundance and distribution of integration events, nonrandom integration of MLV DNA into SV40 DNA is detectable as early as 4 hr and reaches a maximum level by 8 hr after MLV infection. The level of integration but not the distribution of integration sites is sensitive to the stage in the SV40 life cycle at which MLV infection is performed. Using a temperature-sensitive tumor (T) antigen mutant SV40 strain, we observed that active replication of the target DNA is not required for efficient integration in vivo. The distribution of integration sites in vivo is closely approximated by in vitro reactions with isolated SV40 minichromosomes as integration targets. However, the degree of bias between the most and least favored sites is greater in vivo than in vitro.To replicate, retroviruses must insert a DNA copy of their genome into the DNA of the host cell (for reviews, see refs. 1 and 2). Integration is site-specific with regard to the retroviral DNA-invariably occurring near the termini-but is relatively nonspecific with regard to the target DNA (for reviews, see refs. 1 and 3): integration occurs at many sites and in varied target sequences both in vivo and in vitro (4-8). Nevertheless, integration sites are not chosen randomly; the distribution of sites is nonuniform, even in naked DNA in vitro (8-10), and can be influenced further by more complex targets, such as DNA assembled into nucleosomes (7,8). In addition, integration in vivo shows a nonrandom tendency to occur near DNase I-hypersensitive sites and in transcriptionally active regions (11)(12)(13)(14) (Fig. 1A). We find that integration of MLV DNA into SV40 minichromosomes occurs frequently in vivo, that target site preference is similar to that seen when using isolated Time course of integration. Duplicate plates of cells were infected with MLV 20 hr after infection with SV40, and extrachromosomal DNA was prepared at the indicated times (2 to 16 hr) after MLV infection. MLV-SV40 recombinants were amplified by polymerase chain reaction (PCR) between an end-labeled MLV long-terminalrepeat primer (MoU5L26) and an unlabeled SV40 primer (SV273+). Extrachromosomal DNAs were also harvested at the last time point from plates infected with one of the two viruses and mock-infected with the other, either MLV (-M) or SV40 (-S), and were mixed before the PCR to demonstrate that PCR products are not generated unless the cells were coinfected (lane 14 9237The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Integration by design

“…A generalization that has emerged from studies conducted in several laboratories is that bending of DNA favors integration (18), as do hairpin structures (19). The former occurs in nucleosomes, which, contrary to expectations, were found to act as preferred targets over nonnucleosomal DNA, both in vitro and in vivo (20)(21)(22).…”

An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA

“…1A and B) (8,17,20). Although any accessible site in nonviral DNA can be used as the target for viral DNA insertion, preferences are noted in vitro and in vivo (7,19,32,33,39,43). Characteristics of cellular DNA that affect the susceptibility of target sites were reviewed recently (6,16,19).…”

Role of the Nonspecific DNA-binding Region and α Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration

“…However, certain sites within a genome can be used at a frequency several hundred-fold greater than chance (11,14,15). This finding suggests that there are hot and cold spots for the insertion of retroviral DNA and that integration is not a random process (16).…”