Chen D, Fong Y, Zhou Q
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, USA.
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2728-33. doi: 10.1073/pnas.96.6.2728.
Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction.
Tat对HIV-1转录延伸的刺激具有物种特异性，据信这需要许多人类和灵长类细胞中存在的一种特定细胞辅因子，而在非允许性啮齿动物细胞中则不存在。由Cdk9和细胞周期蛋白T1组成的人类P-TEFb是一种一般转录延伸因子，可使RNA聚合酶II的C末端结构域磷酸化。先前的研究也表明P-TEFb是一种Tat特异性细胞辅因子，特别是人类细胞周期蛋白T1负责物种特异性的Tat激活。为了获得支持这些假设的功能证据，我们生成并检测了人-啮齿动物“杂交”P-TEFb复合物的活性。我们发现，含有与人类或啮齿动物Cdk9复合的人类细胞周期蛋白T1的P-TEFb复合物支持Tat反式激活，并与Tat激活结构域和HIV-1 TAR RNA元件相互作用，形成依赖TAR环的核糖核蛋白复合物。虽然含有啮齿动物细胞周期蛋白T1和人类Cdk9的稳定复合物能够磷酸化CTD并介导基础HIV-1延伸，但它未能与Tat相互作用并介导Tat反式激活，这表明P-TEFb支持基础延伸和Tat激活的能力可以分开。总之，我们的数据表明，人类P-TEFb与Tat/TAR的特异性相互作用，主要通过细胞周期蛋白T1，对于P-TEFb介导HIV-1转录的Tat特异性和物种限制性激活至关重要。人类Cdk9特有的氨基酸残基也部分有助于P-TEFb-Tat-TAR复合物的形成。此外，细胞周期蛋白T1的细胞周期蛋白框及其紧邻的侧翼区域在很大程度上负责P-TEFb-Tat的特异性相互作用。
