Studies On The Mechanism Of Retroviral Dna Integration

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications, trials & patents

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. Integrase is the viral enzyme that carries out the key DNA cutting and joining steps in the integration reaction. The structures of each of the individual domains of HIV-1 integrase have been determined, but their spatial arrangement in the active complex with DNA substrate is unknown. All structures of the catalytic domain of HIV-1 integrase determined to date are in the absence of DNA. However, the presence of DNA in the active site has been shown to strongly influence the binding of inhibitors. It is therefore likely that high-resolution structures of inhibitors bound to an active site engaged with DNA substrate will be required to understand their binding in detail and serve as the basis for the design of better derivatives. A major obstacle to directly determining the structure of HIV integrase in complex with DNA substrate is the non-specific nature of the binding of integrase to DNA. We are attempting to circumvent this problem by tethering DNA substrate to integrase by means of disulfide bonds. HIV integrase contains 6 cysteine residues. The two cysteines in the N-terminus co-ordinate zinc and are therefore relatively unreactive. By site directed mutagenesis we have removed the remaining four cysteines while retaining catalytic activity. We are now introducing cysteines at specific positions in HIV integrase based on the available structural data. DNA substrates have also been made with SH groups at specific positions. The aim is to find combinations of modified protein and DNA substrate that retain activity after disulfide bond formation. This will allow map the path of viral DNA on the surface of HIV integrase and provide complexes for crystallization trials. We have also initiated an NMR collaboration to probe the interaction of the catalytic domain with short DNAs and with inhibitors. Although integrase is the key enzyme in the integration process, integrase does not function in isolation in vivo. Rather, it forms part of a large nucleoprotein complex, the preintegration complex, derived from the core of the infecting virion. We are studying Moloney murine leukemia virus preintegration complexes to elucidate the role of other viral and cellular proteins in retroviral DNA integration. In order to successfully integrate into the host genome, retroviruses must avoid self-destructive integration into their own DNA (autointegration). We have identified a cellular protein (BAF) that blocks self-destructive autointegration of retroviral DNA. BAF is non-specific DNA binding protein that is highly conserved among multicellular eukaryotes. It has the unusual property of bridging together double strand DNA molecules; such DNA bridging results in intramolecular compaction at low DNA concentration and intermolecular aggregation at high DNA concentration. We have previously hypothesized that BAF blocks autointegration by compacting the viral DNA, thereby making it inaccessible as a target for integration. We have now directly demonstrated that BAF is indeed responsible for compacting the viral DNA within the preintegration complex. In addition to blocking autointegration, BAF may also enhance intermolecular integration by promoting target DNA capture.