HIV 1 transcription: Tat/novel cofactors/Mechanisms

描述（由申请人提供）：hiv - 1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理解Tat和细胞因子对HIV-1基因表达的控制。本研究将重点关注一种新的RNA Pol II复合物（Tat- SF），该复合物介导Tat增强转录（Tat功能）。Tat-SF含有几个因子（例如，Spt5/Spt4, P-TEFb）和新的多肽，但没有SRB/MED蛋白。这些Tat- sf辅因子是一般转录和Tat功能共同需要的。这项建议有四个具体目标。首先是确定Tat-SF的新成分并检查其作用机制。Tat-SF辅因子的多肽组成将通过色谱方法和生化互补试验进行表征。在新的Tat- sf辅因子通过质谱鉴定和克隆后，将研究它们与其他Tat- sf辅因子对Tat活性的相互作用。第二项研究将采用诱变技术来确定新型Tat- sf辅因子之间以及与Tat功能所需的其他转录因子之间的结构-功能关系。第三部分将讨论Tat- SF辅因子和SRB/MED复合物对Tat活性的相互作用。这些实验将包括详细表征在HIV-1启动子上形成的含有SRB/MED复合物的潜在支架，该支架可以被Tat-SF用于转录再起始。Tat和Tat- sf辅助因子对Tat- sf和RNA Pol II全酶介导的再起始的影响将被研究。最后，第四个目标将研究Tat在HIV-1染色质转录中的作用。在裸DNA上重组的Tat依赖转录系统将优化Tat在染色质上的功能。转录所需的已知因子对染色质的影响将首先进行检查，然后进一步筛选可能补充系统Tat功能的潜在活性。其他研究将确定Tat、Tat- sf辅助因子和SRB/MED复合物对染色质上转录再起始的影响。这些研究将为未来阐明HIV-1转录延伸和mRNA加工之间的联系建立一个独特的系统。总之，在分子水平上理解tat增强的HIV-1转录将有助于未来阻断病毒复制的治疗性抗病毒策略。

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.