The key DNA cutting and joining steps responsible for integration of HIV-1 DNA into cellular DNA are carried out by the viral integrase protein. However, cellular proteins play important accessory roles in the integration process. A focus of our research on cellular factors has been the mechanism that prevents integrase using the viral DNA as a target for integration. Such autointegration would result in destruction of the viral DNA. We previously identified a cellular protein, which we called barrier-to-autointegration factor (BAF) that prevents integration of the viral DNA into itself. BAF is a DNA bridging protein that bridges together segments of double stranded DNA. At high DNA concentration this would result in aggregation. However, at low DNA concentration, such as the few copies of viral DNA in the cytoplasm of an infected cell, the DNA bridging property of BAF results in intracellular compaction. Our model is that that compaction of the viral DNA by BAF makes it inaccessible as a target for integration. In collaboration with Fred Dyda, the structural basis of DNA bridging by BAF was determined. BAF is a dimer and each monomer within the dimer binds one DNA duplex. The binding surface is a helix-hairpin-helix, a motif that binds DNA without any specific contacts thus accounting for absence of DNA sequence specificity of DNA compaction by BAF.? BAF interacts with a family of nuclear proteins termed LEM domain proteins. One LEM domain protein, LAP2alpha was found to be associated with the Moloney murine leukemia virus preintegration complex. NMR studies in collaboration with Marius Clore and Mengli Cai established the interaction surfaces between BAF and the LEM domain.? We have put a considerable effort into fluorescently labeling BAF for both in vitro and in vivo studies. Conventional fusion to green fluorescent protein (GFP) resulted in protein folding problems. Tethering BAF to GFP via a long linker proved more promising but the DNA binding properties of BAF were compromised by this fusion, Finally, we resorted to coupling a small fluorophore to BAF by a maleimide linkage. To accomplish this, two partially reactive cysteines were mutated to alanine and a reactive cysteine was introduced for coupling. The result is a well behaved fluorescently labeled protein that is not compromised in a biochemical or structural way. In collaboration with Kiyoshi Mizuuchi, this protein has been used to study the interaction of BAF with DNA at the single molecule level by total internal reflection fluorescence microscopy.? Lens epithelial derived growth factor (LADGF) is a cellular protein that has been implicated in targeting HIV-1 DNA integration to active regions of chromatin. Using an in vitro system we have developed for assembling synaptic complexes of HIV-1 integrase and viral DNA ends, we have investigated the effect of LEDGF on complex assembly. Surprisingly, we find that LEDGF potently inhibits assembly. We conclude that LEDGF must be recruited after synaptic complex assembly or is recruited by additional protomers of integrase that do not participate in catalysis.
| Li, Min; Craigie, Robert (2009) Nucleoprotein complex intermediates in HIV-1 integration. Methods 47:237-42 |
| Suzuki, Youichi; Craigie, Robert (2007) The road to chromatin - nuclear entry of retroviruses. Nat Rev Microbiol 5:187-96 |
| Bradley, Christina Marchetti; Jones, Sarah; Huang, Ying et al. (2007) Structural basis for dimerization of LAP2alpha, a component of the nuclear lamina. Structure 15:643-53 |
| Cai, Mengli; Huang, Ying; Suh, Jeong-Yong et al. (2007) Solution NMR structure of the barrier-to-autointegration factor-Emerin complex. J Biol Chem 282:14525-35 |
| Li, Min; Craigie, Robert (2006) Virology: HIV goes nuclear. Nature 441:581-2 |
