Biochemical and biophysical analyses of concerted (U5/U3) integration |
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Authors: | Duane P. Grandgenett Sibes Bera Krishan K. Pandey Ajaykumar C. Vora Jacob Zahm Sapna Sinha |
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Affiliation: | aSaint Louis University Health Sciences Center, Institute for Molecular Virology, Doisy Research Center, Rm 621, 1100 South Grand, St. Louis, MO 63104-1009, USA |
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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; (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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Keywords: | Retrovirus concerted integration Integrase Preintegration complex Reconstituted system Synaptic complex |
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