Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus that gives rise to Adult T-cell Leukemia (ATL) and a variety of other subneoplastic conditions such as HTLV-Associated Myelopathy/Tropical Spastic Paraperesis (HAM/TSP). In ATL, the transformation and immortalization of T-lymphocytes has been attributed to the expression and activity of a single HTLV-1 viral protein, namely the trans-activating protein Tax. Although the exact mechanism of Tax-mediated transformation is uncertain, current studies support a model in which Tax induces genomic instability in the host cell through interference with DNA repair mechanisms, dysregulation of cell cycle progression, transcriptional activation of cellular genes, and protein-protein interactions with cellular partners leading to perturbation of their functions. Tax has both nuclear and cytoplasmic activities and shuttles between the two compartments via defined nuclear localization and nuclear export signals (NLS and NES, respectively), but the mechanisms regulating nucleocytoplasmic shuttling and targeting of Tax to distinct subcellular regions have yet to be determined. In this study we identified regions in Tax that regulate nucleocytoplasmic shuttling and dictate subnuclear targeting.

We identified the region in Tax containing the sequence that targets the protein into discrete nuclear foci named Tax Speckled Structures (TSS). These TSS are protein complexes that partially overlap with the cellular marker of splicing SC35 and contain other cellular proteins such as DNA-PKcs and Chia. Targeting Tax to TSS places Tax in a centralized location to affect transcription, DNA damage recognition and other processes, and targeting to these foci is therefore crucial to Tax-mediated transformation. We identified the Tax speckle targeting signal (TSTS) as the Tax region containing amino acids 50-75. This sequence lies downstream from the Tax NLS and is completely separable from the NLS. We demonstrated that a mutant missing the NLS and a mutant missing the TSTS can interact with each other and rescue proper localization through complementation of the deleted domains.

We also determined that dimerization of Tax is required for nuclear localization. The previously defined Tax dimerization domain spans 150 amino acids which represent nearly one-half of the protein. Within this larger domain are three subdomains that were identified as regions required for Tax dimerization. We created Tax mutants deleted in individual dimerization subdomains and assayed their ability to dimerize and their subsequent subcellular localization. Tax mutants deleted in one of the three dimerization subdomains were unable to efficiently homodimerize and were retained in the cytoplasm. They were able to weakly dimerize with wildtype Tax which resulted in partial rescue of nuclear localization. A mutant deleted in two dimerization subdomains was unable to dimerize with itself or with wildtype Tax and remained in the cytoplasm. A Tax mutant that was induced to become a dimer was subsequently able to translocate into the nucleus.

Our studies further identified that cellular proteins including the ubiquitin ligase RNF4 affect the subcellular localization of Tax. Previous studies suggested that ubiquitylation of Tax is associated with its cytoplasmic localization, but the specific ubiquitin ligase involved had not been identified. We demonstrated that RNF4 was able to ubiquitylate Tax in vitro. This study is the first to identify a substrate protein for the ubiquitylation activity of RNF4. Overexpression of RNF4 led to an egress of Tax from the TSS and the nucleus. We co-purified Tax and RNF4 from transfected cell lystates and demonstrated that they are both present in a protein complex. Increasing RNF4 expression increased the cytoplasmic activity of Tax and decreased the nuclear activity of Tax in a dose-dependent manner, suggesting that RNF4's ubiquitylation of Tax affects its subcellular localization and subsequently affects Tax function.

Overall, in this study we have identified novel domains and interactions that contribute to the regulation of the subcellular localization of Tax. The knowledge gained through this work will provide a better understanding of Tax function and its role in cellular transformation.