Isolation and Functional Mapping of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein DNA-Damage Complexes

<p>Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral <em>trans</em>-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events.</p> <p>We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian cells using affinity purification followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, in order to identify both Tax-interacting proteins as well as post-translational modifications of the Tax protein itself. We identified the DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) as a novel Tax-interacting protein. Using bioinformatics analysis, we created a database of Tax-interacting proteins and, using this tool, identified DNA-PKcs as a predicted member of the Tax complex.</p> <p>Physical mapping of purified Tax protein revealed novel phosphorylation sites at T48, T184, T215 and S336. Mutational analysis demonstrated phosphorylation at T215 is associated with loss of Tax <em>trans</em>-activation of CREB and NF-κB-responsive promoters, while T48 preferentially affects NF-κB-responsive promoters, and T184 and 5336 have no effect on these Tax functions.</p> <p>We confirm the presence of DNA-PKcs and the regulatory protein Ku70 in the Tax complex by co-immunoprecipitation. Tax increases phosphorylation of DNA-PKcs and co-localizes with phosphor-DNA-PKcs within nuclear Tax Speckled Structures (TSS). Cytoplasmically-localized Tax deletion mutants cause a redistribution of phosphor-DNA-PK to the cytoplasm. Tax-expressing cells harbor significantly increased DNA-PK kinase activity, as measured in an <em>in vitro</em> kinase assay. Inhibition of DNA-PK activity dramatically reduces Tax-induced autophosphorylation of Chk2, a known DNA-PK substrate.</p> <p>Suppression of DNA-PKcs expression by siRNA has no significant effect on Tax-induced G2/M arrest. Tax shows no significant effect on cellular end-joining repair as measured by a plasmid-based <em>in vivo</em> end-joining assay. Tax-expressing cells show delayed dissolution of damage-induced nuclear speckles containing DNA-PK, <em>γ-</em>H2AX and Tax, suggesting a mechanism for impaired repair response.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Year dc:date.available

2006

Author dc:creator

• Durkin, Sarah Saionz

Contributors dc:contributor

• O. John Semmes
• Ann Campbell
• Richard Drake
• Julie Kerry

• DNA damage
• HTLV-1
• Oncoprotein
• Cell Biology
• Molecular Biology
• Virology

Identifier

9781109835014

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biomedicalsciences_etds-1028