Biochemical and biophysical analyses of concerted (U5/U3) integration

Abstract

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.

The HIV-1 Integrase Monomer Induces a Specific Interaction with LTR DNA for Concerted Integration

“…We have observed that HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO 4 in a HEPES buffer (pH 6.8)(11, 19) is a monomer in solution and is converted to a tetramer in the presence of Zn ++ (Figure 2). IN was concentrated to 15 μM (as monomers) by an ultrafiltration device and applied to a Superdex 200 column.…”

HIV-1 Integrase Strand Transfer Inhibitors Stabilize an Integrase–Single Blunt-Ended DNA Complex

“…Recombinant wt HIV IN (pNY strain) 9; 48 and IN possessing the single N155H drug-resistant mutation were used in this study. Proteins were expressed in Escherichia coli BL21(DE3) cells and purified to near-homogeneity 48.…”

“…Proteins were expressed in Escherichia coli BL21(DE3) cells and purified to near-homogeneity 48. Purified IN (pNY) was used unless indicated.…”

Functional analyses of mutants of the central core domain of an Avian Sarcoma/Leukemia Virus integrase

“…Hydrogen-bonds are shown by a dashed line. The in vitro retroviral concerted integration assay has been previously described for ASLV by others (Aiyar et al, 1996;Grandgenett, 2000, 2003;Grandgenett et al, 2009;Hindmarsh et al, 1999Hindmarsh et al, , 2001Vora et al, 2004;Vora and Grandgenett, 2001) and by us (Moreau et al, 2003(Moreau et al, , 2004(Moreau et al, , 2009 as well as for HIV-1, Simian Immunodeficiency Virus (SIV) and PFV. Briefly, the system is composed of a linear donor DNA (D), a plasmid acceptor DNA, and recombinant IN.…”