Optimized chimeras between kinase-inactive mutant Cdk9 and truncated cyclin T1 proteins efficiently inhibit Tat transactivation and human immunodeficiency virus gene expression
Optimized chimeras between kinase-inactive mutant Cdk9 and truncated cyclin T1 proteins efficiently inhibit Tat transactivation and human immunodeficiency virus gene expression
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DOI：
10.1128/jvi.76.21.10873-10881.2002
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发表时间：
2002-11-01
影响因子：
5.4
通讯作者：
Peterlin, BM
中科院分区：
文献类型：
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作者：
Fujinaga, K;Irwin, D;Peterlin, BM
来自正转录延伸因子b （P-TEFb）的人类细胞周期蛋白T1 （hCycT1）蛋白结合来自人类免疫缺陷病毒1型（HIV）的反激活子Tat和反激活反应（TAR） RNA干环。这个复合体激活病毒转录的延伸。为了创造有效的Tat抑制剂和HIV复制，我们构建了突变型hCycT1蛋白，该蛋白在结合其激酶伙伴Cdk9或TAR时存在缺陷。虽然这些突变的hCycT1蛋白在小鼠细胞中没有增加Tat的反活化，但它们在人类细胞中的主要负作用很小。当hCycT1与突变体Cdk9蛋白融合时，获得了更高的抑制效果。由于Cdk9的C端自磷酸化是形成P-TEFb、Tat和TAR之间稳定复合物所必需的，因此这些丝氨酸和苏氨酸在激酶失活的Cdk9蛋白中被转变为谷氨酸。这种嵌合体抑制了人类细胞中的Tat转录激活和HIV基因表达。因此，这个显性阴性激酶失活突变体Cdk9。hCycT1嵌合体可用于抗病毒基因治疗。
The human cyclin T1 (hCycT1) protein from the positive transcription elongation factor b (P-TEFb) binds the transactivator Tat and the transactivation response (TAR) RNA stem loop from human immunodeficiency virus type 1 (HIV). This complex activates the elongation of viral transcription. To create effective inhibitors of Tat and thus HIV replication, we constructed mutant hCycT1 proteins that are defective in binding its kinase partner, Cdk9, or TAR. Although these mutant hCycT1 proteins did,not increase Tat transactivation in murine cells, their dominant-negative effects were small in human cells. Higher inhibitory effects were obtained when hCycT1 was fused with the mutant Cdk9 protein. Since the autophosphorylation of the C terminus of Cdk9 is required for the formation of the stable complex between P-TEFb, Tat, and TAR, these serines and threonines were changed to glutamate in a kinase-inactive Cdk9 protein. This chimera inhibited Tat transactivation and HIV gene expression in human cells. Therefore, this dominant-negative kinase-inactive mutant Cdk9.hCycT1 chimera could be used for antiviral gene therapy.