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. Together with biochemical studies, the total internal internal fluorescence microscopy (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. We have identified the PP4CR2 complex as a major phosphatase responsible for dephosphorylation of BAF. Knockdown of PP4C or R2 resulted in a major increase in the population of phosphorylated BAF. It has been reported that PP2A can also dephosphorylate BAF and influence the recruitment of BAF to chromatin. However, our results do not support the conclusion that knockdown of PP2A enhances the phosphorylation of BAF at Ser-4. BAF is distributed throughout the cytoplasm during interphase as revealed by fluorescence microscopy. In the absence of a suitable fluorescent fusion protein, we have studied BAF localization by immunofluorescence with BAF specific antibodies. To accomplish this, we have established protocols that that can distinguish the phosphorylated and unphosphorylated forms of BAF. A commercial monoclonal antibody was found to detect both unphosphorylated BAF and phosphorylated BAF with similar efficiency in Western blotting experiments. Antibody specific for phosphorylated BAF was generated by immunizing rabbits with a peptide comprising the first 20 aa of BAF with phosphorylation of serine 4, and affinity purification with phosphorylated BAF.The distinct localization of phosdphorylated BAF and unphosphorylated BAF was studied by direct immunostaining with antibodies and further confirmed by the change in the BAF distribution patterns in response to VRK1 or PP4C/R2 knockdown. The distribution of endogenous total BAF detected by direct immunostaining is largely similar to the previously reported pattern of exogenously expressed BAF fused with fluorescent protein. By comparing the immunostaining pattern obtained with antibodies specific for total BAF and phosphorylated BAF, the distribution of unphosphorylated BAF throughout the cell cycle was deduced. Unphosphorylated BAF was located throughout cells with no distinct pattern during early mitosis, but abundantly colocalized with newly synthesized nuclear envelope at late telophase. Phosphorylated BAF was preferentially associated with the centrosome during the early stages of mitosis and strictly colocalized with the centrosome marker γ-tubulin. Phosphorylated BAF accumulated at the core location of chromosome at early telophase and redistributed to the cytoplasm at late telophase. The binding of BAF to spindle microtubules may account for the localization of phosphorylated BAF at centrosomes, which is an organizing center for spindle microtubules. However, the possibility that BAF has other as yet unidentified partners still cannot be ruled out. Attempts to fish out interacting partners of BAF from cell extracts by biochemical means result in a very large number of candidates making it hard to discriminate partners that may be biologically relevant. This is not surprising because any DNA binding protein is likely to be fished out through indirect interactions through DNA. In fact, some of the previously reported interacting partners of BAF only interact in the presence of DNA. BAF has an important function in NE assembly. The core localization of the NE protein emerin largely depends on BAF phosphorylation. Modifying the BAF phosphorylation state by VRK1 siRNA or PP4C/R2 siRNA resulted in an abnormal NE phenotype in which there were extensive invaginations of the NE into the nucleus and mislocalization of NE proteins. This phenotype is strikingly similar to that observed in fibroblasts of individuals with an hereditary progeroid syndrome. The syndrome results from an Ala12Thr mutation in BAF, which likely destabilizes BAF and results in a reduced intracellular concentration.
|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|