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 continued to study the mechanism by which BAF compacts DNA. Previous structural studies in collaboration with the Dyda laboratory established that the structural basis for DNA condensation by BAF is binding of one DNA segment to each of the DNA binding sites on opposite faces of the BAF dimer. In order to study the DNA binding mode in more detail we engineered fluorescently labeled BAF by coupling Alex maleimide to a newly introduced reactive cysteine. We confirmed that the coupled protein retains all the biochemical properties of the unmodified protein. In collaboration with Kiyoshi Mizuuchi the binding of this fluorescently labeled BAF to DNA was studied in single molecule experiments employing total internal reflection fluorescent microscopy. The results reveal that BAF binds DNA in two modes that differ greatly in their dissociation rates. We interpret the more slowly dissociating form to be stabilized by looping between the two bound DNA segments. BAF can condense DNA into tight spheres as visualized by microscopy and these spheres likely reflect the association of BAF with the preintegration complex.? BAF is distributed throughout the cytoplasm during interphase as revealed by fluorescence microscopy. In order to further study the role of BAF in the retroviral replication cycle we are in the process of constructing a conditional knockout cell line and cell lines in which the phosphorylation of BAF can be manipulated. Phosphorylation of BAF by vaccinia related kinase and its homologues abrogates DNA binding of BAF. We are investigating the effect of down-regulation and up-regulation of vaccinia related kinase on viral replication.
| 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 |
