Tian Yu, Li Dawei, Dahl Jean, You John, Benjamin Thomas
Department of Pathology, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02115, USA.
J Virol. 2004 Nov;78(22):12657-64. doi: 10.1128/JVI.78.22.12657-12664.2004.
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.
通过肿瘤宿主范围选择程序分离出的一种多瘤病毒突变体（19），其T抗原的共同N末端有一个三氨基酸缺失（Δ2 - 4）。为了寻找与野生型T抗原结合但不与突变型T抗原结合的细胞蛋白，以N末端与Gal4的DNA结合结构域融合的野生型小T抗原（sT）为诱饵，对小鼠胚胎cDNA文库进行了酵母双杂交筛选。TAZ是一种具有WW结构域和PDZ结合基序的转录共激活因子（17），被鉴定为结合伴侣。TAZ在体内与所有三种T抗原结合，不同的表观亲和力估计为1:7:100（大T抗原[lT]：中T抗原[mT]：sT）。Δ2 - 4突变型T抗原未显示出可检测到的结合。宿主范围转化缺陷型（hr - t）突变体NG59的sT和mT在共同的sT/mT区域发生改变（179 D→NI）且N末端正常，也未能结合TAZ，而未改变的lT能结合，但与野生型病毒感染相比亲和力降低。TAZ的WW结构域而非PDZ结合基序对T抗原结合至关重要。Δ2 - 4突变体在病毒DNA复制方面存在缺陷。强制过表达TAZ以一种依赖于多瘤病毒增强子结合蛋白2α结合位点的方式阻断野生型DNA复制。野生型多瘤病毒T抗原有效阻断TAZ的反式激活。讨论了TAZ与多瘤病毒T抗原相互作用的功能意义。
