HIV 1 transcription: Tat/novel cofactors/Mechanisms

描述(申请人提供)：艾滋病毒-L编码的TAT蛋白是一种有效的转录激活剂，对病毒复制至关重要。这项建议的长期目标是在分子水平上更好地了解TAT和细胞因子对HIV-1基因表达的控制。这项研究将集中在一个新的RNA POLII复合体(TAT-SF)，它介导TAT增强转录(TAT功能)。TAT-SF含有多种因子(如Spt5/Spt4、P-TEFb)和新的多肽，但不包括SRB/MED蛋白。这些TAT-SF辅助因子是一般转录和TAT功能共同需要的。这项提议有四个具体目标。第一个是确定TAT-SF的新成分并研究它们的作用机制。TAT-SF辅因子的多肽组成将通过层析和生化互补分析进行表征。在新的TAT-SF辅因子被质谱学鉴定和克隆后，将研究它们与其他TAT-SF辅因子对TAT活性的相互作用。第二个将利用突变来确定新的TAT-SF辅助因子之间的结构-功能关系，以及与TAT功能所必需的其他转录因子的结构-功能关系。第三部分将讨论TAT-SF辅助因子和SRB/MED复合体对TAT活动的相互作用。这些实验将包括对在HIV-1启动子上形成的潜在的含有SRB/MED复合体的支架的详细表征，TAT-SF可以利用该支架进行转录重新启动。我们将研究TAT和TAT-SF辅助因子对TAT-SFs和RNAPOLII全酶介导的再启动的影响。最后，第四个目标将研究TAT在HIV-1染色质转录中的作用。重新构建的裸DNA上TAT依赖转录系统将优化TAT在染色质上的功能。在进一步筛选可能补充TAT功能的潜在活性之前，将首先检查转录所需的已知因子对染色质的影响。其他研究将确定TAT、TAT-SF辅助因子和SRB/MED复合体对染色质转录重新启动的影响。这些研究将为未来阐述HIV-1转录延长和mRNA加工之间的联系建立一个独特的系统。总之，在分子水平上了解TAT增强的HIV-1转录将有助于未来阻止病毒复制的治疗抗病毒策略。

DESCRIPTION (provided by applicant): The HIV-l-encoded Tat protein is a potent transcriptional activator that is essential for viral replication. The long-term objective of this proposal is to gain a better understanding of the control of HIV-1 gene expression by Tat and cellular factors at the molecular level. This study will focus on a novel RNA Pol II complex (Tat- SF) that mediates Tat-enhanced transcription (Tat function). Tat-SF contains several factors (e.g., Spt5/Spt4, P-TEFb) and novel polypeptides, but none of the SRB/MED proteins. These Tat-SF cofactors are jointly required for general transcription and Tat function. This proposal has four specific aims. The first is to identify novel components of Tat-SF and examine their mechanisms of action. The polypeptide composition of the Tat-SF cofactors will be characterized by chromatographic methods and biochemical complementation assays. After the novel Tat-SF cofactors are identified by mass spectrometry and cloned, their interplay with other Tat-SF cofactors for Tat activity will be studied. The second will employ mutagenesis to determine the structure-function relationships of novel Tat-SF cofactors among themselves and with other transcription factors necessary for Tat function. The third will address the interplay of the Tat- SF cofactors and the SRB/MED complex for Tat activity. These experiments will include the detailed characterization of a potential SRB/MED complex-containing scaffold formed on the HIV-1 promoter that could be utilized by Tat-SF for transcription reinitiation. The effects of Tat and Tat-SF cofactors on Tat-SFand RNA Pol II holoenzyme-mediated reinitiation will be studied. Finally, the fourth aim will study the role of Tat in HIV-1 transcription on chromatin. A reconstituted system for Tat-dependent transcription on naked DNA will be optimized for Tat function on chromatin. The effects on chromatin of known factors necessary for transcription will be examined first, before further screening for potential activities that could complement the system for Tat function. Other studies will determine the effects of Tat, Tat-SF cofactors, and SRB/MED complex on transcription reinitiation on chromatin. These studies will establish a unique system for future elaboration of the link between HIV-1 transcription elongation and mRNA processing. Together, understanding Tat-enhanced HIV-1 transcription at the molecular level will be instrumental for future therapeutic antiviral strategies to block viral replication.