Human transcriptional coactivator p75/lens epithelium-derived growth factor (LEDGF) binds human immunodeficiency virus type 1 (HIV-1) integrase (IN). We studied the effects of LEDGF on the assembly and activity of HIV-1 synaptic complexes, which, upon association with a target, mediate concerted integration of viral DNA substrates in vitro. We found that while augmenting single-ended viral DNA integration into target DNA, the host factor was able to either stimulate or abrogate concerted integration in a concentrationdependent manner. LEDGF modestly stimulated (two-to threefold) concerted integration at low molar ratios to IN (

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Retrovirus preintegration complexes (PIC) in virus-Integration of the retrovirus linear DNA genome into host chromosomes is essential for replication of human immunodeficiency virus type 1 (HIV-1). Preintegration complexes (PIC) are formed in the cytoplasm after reverse transcription of the viral RNA. Integrase (IN) in the PIC catalyzes the excision of two nucleotides adjacent to the phylogenetically conserved CA dinucleotide from the 3Ј-OH blunt ends of the viral DNA (7). After nuclear transport of the PIC, the two viral DNA ends are inserted by IN into the host genome in a concerted fashion, here termed "full-site integration" (8,25,44). The unpaired dinucleotides at the 5Ј ends of the inserted viral DNA are removed, and the single-stranded gaps are repaired by a host cell DNA repair pathway (35). This process results in a short duplication of cell DNA ranging in size from 4 to 6 bp depending on the retrovirus species.Significant progress in our understanding of retrovirus fullsite integration was initiated by isolating PIC from virus-infected cells (8). Purified HIV-1 and murine leukemia virus (MLV) PIC are capable of incorporating ϳ15 to 50% of their viral DNA into an exogenously supplied DNA target as fullsite integration products after 45 to 90 min of incubation at 37°C (6,11,13,21,39,48). One report using HIV-1 PIC showed an ϳ95% incorporation of viral DNA into a target substrate where incorporation was essentially over after 45 min of incubation at 37°C (21). Cellular factors like barrier-toautointegration factor (BAF) appear to play a role in maintaining stable MLV and HIV-1 PIC structures, thus promoting full-site integration by preventing integration of the viral DNA into itself, termed "autointegration" (12,33,48,55).Simplified integration assays using model linear retrovirus DNA with terminal U5 and U3 long terminal repeat (LTR) sequences, purified recombinant IN, and a DNA target were also developed (9,16,32,45). These studies established that IN was the only viral protein necessary for both 3Ј-OH processing and DNA strand transfer activities.Further progress in reconstituting the HIV-1 full-site integration process was made using IN derived from nonionic detergent lysates of virus particles (10,22,23). The 3Ј-OH recessed ends containing attachment (att) site sequences from two different viral donors (480 bp) were integrated by IN in a concerted manner into a circular DNA target (bimolecular

Retrovirus integrase (IN) integrates the viral linear DNA genome (~10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable: 1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; 2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; 3) the determination of the multimeric state of IN within these complexes; 4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); 5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; and 6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.

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The genotoxic and cytotoxic effects of a widely used herbicide, fluchloralin, were assessed using cultured mammalian cells. Treatment of cells for 8–12 hr with fluchloralin resulted in a significant increase in the frequency of metaphase cells with chromosomal damage. At higher concentrations, the herbicide also induced an increase in the frequency of sister chromatid exchange. A 50% loss in viability was observed when cells were exposed to the herbicide for 72 hr. To understand the mechanism of cell death caused by fluchloralin, its effect on DNA synthesis and its ability to induce apoptosis were investigated. Even short (6 hr) treatment of cells with fluchloralin resulted in a 30–50% inhibition of DNA synthesis. Agarose gel electrophoresis of DNA from herbicide‐treated cells and cytochemical staining indicate the induction of apoptosis by fluchloralin. Environ. Mol. Mutagen. 31:257–262, 1998 © 1998 Wiley‐Liss, Inc.

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