Han X M, Laras A, Rounseville M P, Kumar A, Shank P R
Department of Biochemistry and Molecular Biology, George Washington University, Washington, D.C. 20037.
J Virol. 1992 Jul;66(7):4065-72. doi: 10.1128/JVI.66.7.4065-4072.1992.
Protein kinase C (PKC) is involved in the mitogenic stimulation of cell proliferation and has recently been reported to be essential for Tat-mediated trans activation. We have determined that RNA binding of a cellular factor which specifically interacts with the trans-activation response region (TAR) is blocked in cells depleted of PKC activity by chronic phorbol myristate acetate stimulation. We also show that nuclear extracts can be depleted of the cellular TAR-binding factor by in vitro treatment with purified protein phosphatase 2A. Furthermore, TAR RNA-binding activity can be partially restored to depleted nuclear extracts in vitro by addition of PKC. Chimeric constructs in which the Tat protein is artificially tethered to viral RNA show PKC independence for Tat-mediated trans activation. Specific mutations in the TAR RNA stem region which cause reduced binding of host cell factor in vitro also cause reduced Tat-mediated trans activation in vivo. Together, these results suggest that phosphorylation-dependent binding of a cellular cofactor to TAR RNA is an essential step in Tat-mediated trans activation. Deciphering the regulation of Tat-mediated trans activation by phosphorylation will be critical in fully understanding the regulation of human immunodeficiency virus type 1 activation.
蛋白激酶C（PKC）参与细胞增殖的促有丝分裂刺激，最近有报道称其对Tat介导的反式激活至关重要。我们已经确定，通过慢性佛波酯肉豆蔻酸酯刺激使PKC活性耗竭的细胞中，一种与反式激活应答区域（TAR）特异性相互作用的细胞因子的RNA结合被阻断。我们还表明，通过用纯化的蛋白磷酸酶2A进行体外处理，核提取物中的细胞TAR结合因子可以被耗尽。此外，通过添加PKC，TAR RNA结合活性可以在体外部分恢复到耗尽的核提取物中。将Tat蛋白人工连接到病毒RNA的嵌合构建体显示Tat介导的反式激活不依赖PKC。TAR RNA茎区的特定突变在体外导致宿主细胞因子结合减少，在体内也导致Tat介导的反式激活减少。总之，这些结果表明细胞辅因子与TAR RNA的磷酸化依赖性结合是Tat介导的反式激活中的关键步骤。解读磷酸化对Tat介导的反式激活的调节对于全面理解1型人类免疫缺陷病毒激活的调节至关重要。
