Laboratory of Experimental Virology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
J Virol. 2011 Sep;85(18):9506-16. doi: 10.1128/JVI.00650-11. Epub 2011 Jul 13.
It is generally acknowledged that the Tat protein has a pivotal role in HIV-1 replication because it stimulates transcription from the viral long terminal repeat (LTR) promoter by binding to the TAR hairpin in the nascent RNA transcript. However, a multitude of additional Tat functions have been suggested. The importance of these functions is difficult to assess in replication studies with Tat-mutated HIV-1 variants because of the dominant negative effect on viral gene expression. We therefore used an HIV-1 construct that does not depend on the Tat-TAR interaction for transcription to reevaluate whether or not Tat has a second essential function in HIV-1 replication. This HIV-rtTA variant uses the incorporated Tet-On gene expression system for activation of transcription and replicates efficiently upon complete TAR deletion. Here we demonstrated that Tat inactivation does nevertheless severely inhibit replication. Upon long-term culturing, the Tat-minus HIV-rtTA variant acquired mutations in the U3 region that improved promoter activity and reestablished replication. We showed that in the absence of a functional TAR, Tat remains important for viral transcription via Sp1 sequence elements in the U3 promoter region. Substitution of these U3 sequences with nonrelated promoter elements created a virus that replicates efficiently without Tat in SupT1 T cells. These results indicate that Tat has a versatile role in transcription via TAR and U3 elements. The results also imply that Tat has no other essential function in viral replication in cultured T cells.
人们普遍认为，Tat 蛋白在 HIV-1 复制中起着关键作用，因为它通过与新生 RNA 转录本中的 TAR 发夹结合，刺激来自病毒长末端重复（LTR）启动子的转录。然而，已经提出了许多其他 Tat 功能。在使用 Tat 突变的 HIV-1 变体进行复制研究时，评估这些功能的重要性是困难的，因为它们对病毒基因表达具有显性负效应。因此，我们使用了一种不依赖 Tat-TAR 相互作用进行转录的 HIV-1 构建体，重新评估 Tat 是否在 HIV-1 复制中具有第二个必需功能。这种 HIV-rtTA 变体使用整合的 Tet-On 基因表达系统激活转录，并在完全缺失 TAR 后有效地复制。在这里，我们证明 Tat 的失活确实严重抑制了复制。经过长期培养，Tat 缺失的 HIV-rtTA 变体在 U3 区域获得了突变，提高了启动子活性并重新建立了复制。我们表明，在没有功能性 TAR 的情况下，Tat 通过 U3 启动子区域中的 Sp1 序列元件仍然对病毒转录很重要。用非相关的启动子元件替代这些 U3 序列，创建了一种在 SupT1 T 细胞中无需 Tat 即可有效复制的病毒。这些结果表明 Tat 在通过 TAR 和 U3 元件进行转录中具有多功能作用。结果还表明，Tat 在培养的 T 细胞中的病毒复制中没有其他必需功能。
