Studies On The Mechanism Of Retroviral DNA Integration

Diabetes, Digestive, Kidney Diseases

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Abstract

Retroviruses integrate a DNA copy of their genome into host DNA as an obligatory step in their replication cycle. Our work focuses on the molecular mechanism of integration, and in particular on the structure and function of HIV integrase and other proteins involved in retroviral DNA integration. Although integrase carries out the key DNA cutting and joining steps of integration in vitro, integration in the cell is carried out by a large nucleoprotein complex, the preintegration complex (PIC), that is derived from the core of the infecting virion. The PIC contains the viral DNA, integrase and a number of other viral and cellular proteins. The major impediment to further structural, biophysical and further biochemical studies of retroviral DNA integration is that it has not yet been possible to reconstitute a nucleoprotein complex from purified integrase and DNA substrate that exhibits all the properties of PICs isolated from infected cells. Under most in reaction conditions HIV-1 integrase integrates only one viral DNA end into one strand of target DNA in vitro (half-site reaction), a reaction that would not lead to integration in vivo. This contrasts with the concerted integration of pairs of DNA ends (full-site reaction) that occurs in the cell and with PICs in vitro. We have put much effort into reconstituting an in vitro reaction system for concerted DNA integration. Currently we are able to achieve up to approximately 5% conversion of DNA substrate into concerted integration product. We are currently investigating the factors that limit the reaction efficiency. The reaction requires the presence of PEG and it is likely that aggregation of reaction components results in the trapping of DNA substrate in a non-productive complex. Surprisingly concerted DNA integration requires a viral DNA substrate several hundred base pairs in length, whereas 20 bp oligonucleotides are sufficient for the half-site reaction. The reason for this difference is currently unknown. We are screening known protein components of the PIC and fractionated cellular extracts for factors that may enhance the efficiency of concerted integration. Preliminary experiments have identified one cellular protein and the mechanism of stimulation is under investigation. Integrase is tightly associated with viral DNA in the PIC and remains associated and active even after treatment with greater than 0.5M NaCl. In contrast, in simple DNA binding assays and conditions that have typically used for in vitro assays, integrase is dissociated from viral DNA by 200 mM NaCl. We have found that under reaction conditions that promote concerted DNA integration a highly stable complex is formed in which a tetramer of integrase is bound to a pair of viral DNA ends. Once this complex is formed concerted integration is highly efficient and essentially 100% of this stable synaptic complex (SSC) is converted to product. Although the initial binding of integrase to viral DNA is quite weak and non-specific, under appropriate conditions it is able to ?lock-in? to a tight complex that mirrors the association of integrase with viral DNA in PICs isolated from virus infected cells. We are attempting to improve this reaction system with the aim of obtaining material that is suitable for structural and biophysical studies.[unreadable] We have examined the viral DNA terminal sequence requirements for SSC assembly. The C at position 2 on the non-transferred strand is essential whereas the terminal A on this strand is not required. The first three nucleotides on the transferred stand are not required for SSC assembly, including the A that is essential for the DNA strand transfer reaction. This information provides a tool to block the integration reaction at the SSC step and accumulate this intermediate. As we have previously shown, a preprocessed substrate with terminating with di-deoxy A on the transferred stand is also an effective tool for trapping the SSC. A major problem with high throughput screens for integrase inhibitors is the large number false positives. In contrast, in vitro screening with PICs isolated from infected cells is far more selective. However, assaying HIV-1 DNA integration using PICs is not practical beyond the small laboratory scale because of the difficulty of obtaining sufficient material. Our finding that stable complexes of HIV-1 integrase with viral DNA substrate can be assembled in vitro, and that the properties of these complexes mirrors those of the association of integrase with PICs isolated from cells, potentially offers a simpler selective screen. As a preliminary test we have assayed a set of compounds with known differential activities in conventional in vitro assays with oligonucleotides and PIC assays. When added after assembly of the SSC, the patern of inhibition in most cases exhibits that seen with PICs. We are developing this assay as a simpler alternative to using PICs for secondary screening. We have previously identified a cellular protein (BAF) that blocks self-destructive autointegration of retroviral DNA. We have focused on the mechanism by which BAF blocks autointegration and obtained evidence that supports our hypothesis that BAF blocks autointegration by compacting the viral DNA within the preintegration complex. By gel filtration and equilibrium ultracentrifugation analysis, we have demonstrated that a 7 bp DNA is sufficient to make a complex with BAF. The structure of the complex, solved in collaboration with Dr. Dyda?s group, revealed that it consists of a dimer of BAF with DNA bound to each side of the dimer. Thus the bridging of DNA by BAF can be explained by a dimer with one DNA binding site on each monomer. The structure also explains why the interaction of BAF with DNA is sequence non-specific. However, the association of BAF with DNA is less stable than the association of BAF with the PIC. We find that an interacting partner of BAF, lamina-associated-protein-2-alpha (LAP2alpha) is also present in the PIC and that these two proteins collaborate to make a stable complex with DNA. The details of the interactions between BAF, LAP2alpha and DNA are under investigation.