Integration of the retrovirus DNA genome is essential in the life cycle of the virus. Following reverse transcription of the RNA in virus-infected cells, the cytoplasmic preintegration complexes (PIC) containing the linear viral DNA genome and integrase (IN) are transported into the nucleus. We have reconstituted nucleoprotein complexes (intasomes) with recombinant Rous sarcoma virus (RSV) or avian myeloblastosis virus IN and 3.6 kbp linear viral DNA substrates. The reconstituted intasomes are equivalent to purified PIC for efficient concerted DNA integration in vitro, attachment (att) site requirements, high fidelity for host site duplications, stability, low IN concentration dependency (<10 nM), and DNasel protection by IN up to approximately 20 bp from the viral ends. We propose to use genetic, molecular, and biophysical approaches to further our understanding of the protein-protein and protein-DNA interactions occurring between avian retrovirus IN and the long terminal repeats (LTR) terminal att sites for concerted integration in vitro and in vivo. We will investigate the mechanisms involved in forming synaptic complexes of two individual IN-viral DNA complexes and determine the multimeric structure of IN required for concerted DNA integration in vitro. Mass spectrometry of chemically crosslinked peptides in intasomes will allow us to identify the intermolecular interactions occurring between the different subunits involved in the synaptic complexes. We will study the functional protein interfaces between synapsed IN subunits bound to viral att DNA by site-directed mutagenesis of recombinant RSV IN and by complementation assays of IN mutants using a viral att DNA complementation protocol. We will determine the in vivo effects of functional mutations in RSV IN on viral replication and integration using a classical genetic approach and characterization of isolated avian retrovirus PIC from virus-infected cells. Using biophysical approaches, we will determine the distance (in angstroms) between two LTR sites bound by IN necessary for concerted integration as well as the orientation of the att DNA in intasomes by luminescence resonance energy transfer (LRET). Our proposal will illuminate critical steps in retrovirus integration; the knowledge gained may help prevent replication of retroviruses in humans that cause diseases and provide insights into the possiblities of human gene therapy with retroviruses.
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