Molle Dorothée, Maiuri Paolo, Boireau Stéphanie, Bertrand Edouard, Knezevich Anna, Marcello Alessandro, Basyuk Eugenia
IGMM-CNRS UMR 5535, Montpellier, France.
Retrovirology. 2007 May 30;4:36. doi: 10.1186/1742-4690-4-36.
HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.
HIV-1转录受到严格调控：在长期潜伏状态下处于沉默，而在急性感染细胞中高度活跃。转录由病毒蛋白Tat激活，Tat通过结合新生HIV-1 RNA中存在的TAR序列来招募延伸因子P-TEFb。在本研究中，我们通过在HIV-1转录位点进行荧光漂白恢复（FRAP）实验，分析了活细胞中TAR:Tat:P-TEFb复合物的动态变化。我们的结果表明，存在于这些位点的大部分Tat是由细胞周期蛋白T1招募的。我们发现，在有Tat存在的情况下，Cdk9与新生HIV-1 RNA结合71秒。相比之下，当在没有Tat的情况下由佛波酯（PMA）/离子霉素激活转录时，Cdk9快速周转，仅在HIV-1启动子上停留11秒。因此，反式激活机制决定了P-TEFb在HIV-1基因处的停留时间，这可能解释了为什么Tat是如此强大的转录激活剂。此外，我们观察到Tat在HIV-1转录位点占据55秒，这表明TAR:Tat:P-TEFb复合物在转录起始后从聚合酶上解离，并经历随后的结合/解离循环。
