Retroviruses integrate a DNA copy of their genome into host DNA as an obligatory step in their replication cycle. Our work focusses on the molecular mechanism of integration. The solution structure of the zinc-binding domain of HIV-1 integrase, the key enzyme involved in integration, has been solved in collaboration with the Gronenborn and Clore laboratories in LCP/NIDDK. We have also used photo-crosslinking methods to probe the interactions between HIV-1 integrase and DNA substrate. We have probed the nucleoprotein organization of Moloney murine leukemia virus (MLV) preintegration complexes using a novel footprinting technique. We find that the ends of the viral DNA are organized in a higher order nucleoprotein complex involving several hundred base pairs at each end of the viral DNA. This complex is not formed when preintegration complexes are made by infection with integrase-minus virus, demonstrating the involvement of integrase in the complex. Treatment with high salt disrupts the complex in parallel with loss of intermolecular integration activity; reconstitution of the functional complex requires a cellular factor. Finally, functional interference experiments demonstrate that the integrity of the complex is required for normal intermolecular integration into a target DNA. We have previously shown that Moloney murine leukemia virus preintegration complexes exhibit a barrier to self-destructive autointegration. We also demonstrated that the autointegration barrier could be destroyed by stripping factors from these complexes and subsequently restored by incubation with a host cell extract. We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH3T3 cells. Partial amino acid sequencing of the polypeptide led to cloning of the cDNA which contains an open reading frame for 89 amino acids that does not match any previously identified protein. The identity of the protein was confirmed by expressing it in Escherichia coli and demonstrating functional activity.
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