Following reverse transcription of retrovirus RNA in virus-infected cells, the cytoplasmic preintegration complex (PIC) containing linear viral DNA and integrase (IN) is transported into the nucleus. Studies have shown that IN is necessary for the concerted integration of the viral DNA termini into the host chromosome. We have reconstituted an efficient, high-fidelity concerted (full-site) integration reaction with purified avian myeloblastosis virus (AMN) IN and model linear retrovirus like substrates that contain the long terminal repeat (LTR) at their ends. We will use the reconstitution assay to determine the roles that IN plays in the assembly, structural integrity and integration of potential of the PIC-like complexes. We will determine which LTR terminal inverted (IR) sequences are necessary for the full-site integration reaction. LTR IR mutations have allowed us to identify several major loss- and gain-of-function LTR mutants for full-site integration. Efficiencies for full-site catalysis between several IR mutants is approximately 20 fold. We will investigate the intermolecular communication mechanisms between wild type (wt) LTR termini complexed to IN and between major loss- and gain-of-function LTR mutants complex to IN. We will probe the interactions that occur between IN with wt and mutant LTR termini to determine their nucleoprotein organization in the absence and presence of target DNA. We will determine if IN is able to hold together two LTR substrates at the IR termini, the internal LTR IR and other IN """"""""active-site"""""""" consensus sequences located throughout the full-length LTR. Linear donor substrates (4.5 kbp) containing the natural 330 bp full-length LTR termini are efficiently utilized for full- site catalysis by IN. We have purified a bacterial recombinant Rous sarcoma virus PrA IN that has the same specific activity as AMV IN for full-site catalysis. We will try to crystallize the highly soluble PrA IN without or with wt, gain- or loss-of-function LTR IR oligonucleotides. The crystal structure of full-length IN is unknown. Selected amino acid substitutions in IN will permit us to investigate what residues are necessary for the full-site integration reaction both in vitro and in vivo. Understanding retrovirus integration is important for preventing retrovirus infection of humans and for human gene therapy using retroviruses.
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