Interaction of HIV-1 Tat with Puralpha in nuclei of human glial cells: characterization of RNA-mediated protein-protein binding.

A complex between the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), and the cellular protein, Puralpha, has been implicated in activation of the late promoter of JC virus (JCV) and in enhancement of JCV DNA replication. JCV is the causative agent of progressive multifocal leukoencephalopathy (PML), an acquired immunodeficiency syndrome (AIDS) opportunistic infection of the brain. Puralpha also binds the HIV-1 TAR RNA element and activates HIV-1 transcription, suggesting a role for RNA binding in the action of this protein. Using immunoelectron microscopy, we find that in human glial cells expressing both proteins, Tat and Puralpha are colocalized in extranucleolar chromatin structural elements. The colocalized Puralpha and Tat are nearly exclusively nuclear, although individual proteins can be seen in both nucleus and cytoplasm, suggesting a preferential tropism of the complex for the nucleus. Analysis of the interaction between purified proteins indicates that the Tat-Puralpha interaction is strongly enhanced by the presence of RNA. Tat amino acids from 37-48 are essential for Tat binding. Residues 49-72, including the TAR RNA-binding domain, are critical for binding to Puralpha, while Cys(22), in the Tat transactivation domain, is responsible for an important global effect. Puralpha repeat II domains are involved in the interaction, and a polypeptide based on one such sequence inhibits binding. After RNase treatment of Puralpha enhancement of Tat binding can be partially restored by addition of a single-stranded JCV DNA PUR element, to which Tat does not bind. The results indicate that the Tat-Puralpha interaction is direct, rather than through an RNA link, and that RNA binding configures Puralpha for optimal interaction with Tat.

Association of HIV-1 Tat with the cellular protein, Puralpha, is mediated by RNA.

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Puralpha and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Puralpha includes the region demonstrated to bind Tat's viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Puralpha derived from human U-87MG cells plays an important role in the Puralpha:Tat interaction as examined by an in vitro reconstitution assay. Furthermore, expression of the Puralpha-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Puralpha.

Alterations in Pur(alpha) levels and intracellular localization in the CV-1 cell cycle.

Levels of the single-stranded DNA-binding protein Pur(alpha), previously implicated in control of both DNA replication and gene transcription, are altered during the CV-1 cell cycle. Just prior to the onset of S phase, Pur(alpha) levels drop precipitously, after which they recover nearly 8-fold throughout S and G2 to peak just after mitosis. As observed previously, Pur(alpha) binds the hypophosphorylated form of the retinoblastoma protein, Rb. Coimmunoprecipitation of Pur(alpha) and Rb reveals that the complex declines as cells enter S phase and does not reform as Pur(alpha) levels recover in S and G2. As Pur(alpha) levels recover, the protein is localized to nuclear foci containing newly replicated DNA, as determined by immunoelectron microscopy using different sized gold beads and antibodies against Pur(alpha) and bromodeoxyuridine-labeled DNA. These foci also contain cyclin A, and Pur(alpha) coimmunoprecipitates with cyclin A from extracts of cells in S and G2 phases. Pur(alpha) remains with these foci throughout G2, after the bulk of DNA synthesis has ceased. Changing levels of Pur(alpha) may affect Pur(alpha) functions at the onset of S phase and during progression to mitosis.