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 Kiyoshi Mizuuchi, we have studied the condensation of DNA by BAF using total internal reflection fluorescence microscopy (TIRFM). We first constructed GFP-BAF fusions for this purpose, but these proteins exhibited aberrant DNA binding properties compared to the wild-type BAF (data not shown). We therefore instead labeled BAF with Alexa488 maleimide for the TIRFM experiments. Alexa488- BAF exhibits DNA binding and condensation properties indistinguishable from those of wild- type BAF. Together with biochemical studies, the TIRFM experiments demonstrate that BAF condenses DNA by a looping mechanism that is different from the compaction of DNA by wrapping proteins such as histones or charge-neutralizing proteins such as protamines. BAF loops DNA by bridging two DNA segments, one binding to each monomer within the BAF dimer. The slow timescale of dissociation of BAF and DNA extension observed in the TIRFM experiments suggests that the dissociation behavior of BAF from DNA may be sufficient to explain the dissociation of BAF from retroviral DNA within preintegration complexes as inferred from functional assays. This implies that the salt-stripped PIC likely still contains a limited amount of BAF that could partially condense viral DNA. Blocking of autointegration likely depends on the tight compaction of viral DNA by a relatively high stoichiometry of stably bound BAF/DNA in the complex. Different preparations of PICs from infected cells display different degrees of protection from autointegration, suggesting partial loss of BAF during preparation, but full protection can be recovered by addition of BAF. These observations can easily be explained on the basis of the DNA binding properties of BAF reported here. 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.
| Zhuang, Xiaolei; Semenova, Elena; Maric, Dragan et al. (2014) Dephosphorylation of barrier-to-autointegration factor by protein phosphatase 4 and its role in cell mitosis. J Biol Chem 289:1119-27 |
| Huang, Ying; Cai, Mengli; Clore, G Marius et al. (2011) No interaction of barrier-to-autointegration factor (BAF) with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C in absence of DNA. PLoS One 6:e25123 |
| Cai, Mengli; Huang, Ying; Craigie, Robert et al. (2010) Structural basis of the association of HIV-1 matrix protein with DNA. PLoS One 5:e15675 |
| Li, Min; Craigie, Robert (2009) Nucleoprotein complex intermediates in HIV-1 integration. Methods 47:237-42 |
