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Updated: Aug 21, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
Identification of TAZ as a binding partner of the polyomavirus T antigens
1Department of Pathology, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02115, USA.
Abstract:
A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Delta2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Delta2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D-->NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Delta2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2alpha. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.
Insights
Polyomavirus T antigens bind the transcriptional coactivator TAZ, with a specific N-terminal deletion preventing this interaction. TAZ binding is crucial for viral DNA replication and T antigen function.
Area of Science:
- Virology
- Molecular Biology
- Oncology
Background:
- Polyomaviruses encode T antigens that regulate viral replication and oncogenesis.
- Cellular proteins interacting with T antigens are key to understanding viral mechanisms.
- The TAZ protein is a transcriptional coactivator involved in cellular processes.
Purpose of the Study:
- To identify cellular proteins that bind to wild-type polyomavirus T antigens but not to a specific mutant.
- To elucidate the role of TAZ in polyomavirus replication and T antigen function.
Main Methods:
- Yeast two-hybrid screening using wild-type small T antigen as bait.
- In vivo binding assays to assess T antigen-TAZ interactions.
- Analysis of viral DNA replication and transcriptional regulation.
Main Results:
- TAZ was identified as a binding partner for all three polyomavirus T antigens (large T, middle T, small T).
- A polyomavirus mutant with an N-terminal deletion (Delta2-4) failed to bind TAZ.
- TAZ binding, mediated by its WW domain, is essential for viral DNA replication.
- Polyomavirus T antigens inhibit TAZ transactivation activity.
Conclusions:
- TAZ is a novel cellular binding partner for polyomavirus T antigens.
- The interaction between TAZ and T antigens is critical for viral DNA replication.
- Polyomavirus T antigens modulate TAZ function to promote viral propagation and potentially oncogenesis.
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