We have established conditions for in vitro assembly of stable synaptic complexes of a pair of viral DNA ends with HIV-1 integrase. These nucleoprotein complexes are intermediates in the integration of HIV DNA into a target DNA. Furthermore, the association of integrase with viral DNA in these complexes mimics all the properties of the association of integrase with viral DNA in preintegration complexes (PICs) isolated from virus infected cells. The synaptic complexes contain a tetramer of integrase tightly bounds to a pair of viral DNA ends. Footprinting of the viral DNA ends within the complex reveals that less than 20 base pairs of terminal viral DNA sequence are protected by integrase. within the SSC, a conclusion that is also in agreement with atomic force microscope images of the stable nucleoprotein complexes. 20bp of terminal viral DNA end sequence are efficient substrates for half-site integration in vitro, and are the only protected region observed in footprinting experiments. However, several hundred base pairs of non-specific flanking DNA sequence are required for efficient SSC assembly, stability and concerted integration. We are probing the function of this non-specific DNA sequence in SSC assembly and stability and propose that non-specific interactions between IN and DNA (distinct from the stable association of a tetramer of IN with the viral DNA ends) are involved. Having established methodologies to assemble synaptic complexes in vitro with purified integrase and HIV-1 DNA substrate, we are attempting both low and high-resolution structural studies combined with biochemical approaches to understand the detailed mechanism of DNA integration. In collaboration with Emilios Dimidriadis and Svetlana Kotova we have confirmed by atomic force microscopy (AFM) that a tetramer of integrase bridges the pair of viral DNA ends in the SSC and obtained evidence that assembly of an integrase tetramer on one viral DNA end is an intermediate step in assembly of the SSC. AFM also reveals that the viral DNA ends are arranged in antiparallel orientation in the SSC. This arrangement of the viral DNA ends is also supported by fluorescence resonance energy transfer (FRET) studies. Cy3 and Cy5 fluorophores have been incorporated at various positions along the DNA substrate and the FRET signal measured upon assembly into the SSC. The results show a significant FRET signal in the SSC when fluorophores are positioned close to the ends of the viral DNA. FRET efficiency was reduced when the fluorophores were located away from the end. The potential role of cellular proteins in SSC assembly is under investigation. One cellular protein that has been implicated in playing an important role in HIV-1 DNA integration is Lens Epithelial Derived Growth Factor (LEDGF). We find that LEDGF does not stimulate assembly of the SSC and in fact inhibits complex assembly. LEDGF must therefore be acquired by the preintegration complex after the two viral DNA ends are engaged by integrase to form the SSC. We are preparing the groundwork higher resolution structural studies of integrase in complex with viral DNA substrates. At this stage a number of major obstacles must be overcome before such studies become feasible. The first obstacle is that SSCs aggregate in solution. Atomic force microscopy shows that aggregation occurs through protein-protein interactions between SSCs. This suggests that IN undergoes conformational changes upon SSC assembly. Extensive studies directed at finding solution conditions that do not permit aggregation have not proved fruitful. We are therefore pursuing an alternative strategy of screening large numbers of IN mutants with single amino acid changes to identify the protein surface responsible for this aggregation. A second strategy is the parallel study of closely related integrases. Human foamy virus integrase exhibits much better behavior in solution compared to its HIV-1 counterpart and preliminary studies suggest it is a promising candidate for structural studies of integrase in complex with DNA.
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