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 the absence of a suitable fluorescent fusion protein, we have studied BAF localization by immunofluorescence with BAF specific antibodies. To accopmplish 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 distribution of non-phosporylated BAF can be qualitively determined by the difference in staining pattern with these two antibodies. Indirect confocal immunofluorescence miscroscopy with antibody to total BAF shows that it is located in both the cytoplasm and nucleus during interphase, with a higher concentration at the nuclear periphery. This type of distribution is similar to the previously reported distribution of endogenous BAF in C. elegans. On the other hand, phosphorylated BAF is found mostly in the nucleus. Thus both forms of BAF are associated with the nucleus but the non-phosphorylated form also localizes in cytoplasm near nucleus during interphase. 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. The enzyme responsible for dephosphorylation of BAF is not known. We have established an assay for dephosphorylation of BAF and are using this to purify the factors responsible for dephosphorylation. HIV-1 matrix (MA) is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC), we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC. The basic N-terminal region of MA modulates multiple steps in HIV-1 replication and is required for maximal infectivity. It is not surprising that this positively charged surface can play roles in binding to RNA, DNA, and membranes, making the phenotypes of mutations in this region difficult to interpret. The interaction of MA with DNA is stable at least up to 200 mM NaCl and is therefore expected to occur under physiological conditions;indeed we found it necessary to include 500 mM NaCl in buffers during the early stages of purification to prevent co-purification with DNA. Although a function for MA as a predominant determinant of nuclear import is not supported by the preponderance of evidence, MA is a component of the HIV-1 PIC and can influence the fate of the viral DNA. The interaction of MA reported here is likely sufficient to account for the retention of MA in the PIC even in the absence of an interaction with other protein components.
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