Orsini M J, Debouck C M
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
J Virol. 1996 Nov;70(11):8055-63. doi: 10.1128/JVI.70.11.8055-8063.1996.
We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.
我们将各种突变引入人免疫缺陷病毒1型（HIV-1）Tat的激活域和RNA结合域，以开发一种新型强效的反式显性Tat蛋白，并阐明其作用机制。对不同的突变Tat蛋白进行了表征，以确定它们激活HIV长末端重复序列（LTR）以及反式抑制野生型Tat功能的能力。一种缺失碱性结构域（Tat（Δ）49-57）的Tat蛋白仅定位于转染人细胞的细胞质中，无功能，并以反式显性方式抑制HIV-1和HIV-2 Tat的功能。在富含半胱氨酸和核心结构域含有突变的Tat蛋白无功能，但不能反式抑制Tat功能。当Tat核定位信号或核仁定位信号与Tat（Δ）49-57的羧基末端融合时，嵌合蛋白分别定位于细胞核或核仁，并仍能以反式显性方式发挥作用。在各种反式显性Tat蛋白的富含半胱氨酸和核心结构域引入二级突变，完全消除了它们以反式显性方式发挥作用的能力。我们的数据最有力地支持了一种机制，即这些反式显性Tat蛋白抑制了一种或多种与Tat激活域相互作用且Tat发挥功能所必需的细胞因子。
