HIV 1 transcription: Tat/novel cofactors/Mechanisms

描述（由申请方提供）：HIV-1编码的达特蛋白是一种有效的转录激活因子，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达特和细胞因子对HIV-1基因表达的控制。本研究将集中在一种新的RNA Pol II复合物（达特- SF），有效地介导Tat增强转录（达特功能）。Tat-SF含有P-TEFb、Spt 5/Spt 4和Tat-SF 1以及新的多肽，但没有发现与RNA Pol II全酶相关的酵母共激活剂SRB/MED样蛋白。值得注意的是，这些Tat-SF辅因子可以共同补充转录起始能力RNA Pol II全酶有效的转录延伸，再起始，和达特反式激活，表明Tat-SF包含关键的转录因子。Tat-SF和RNA Pol II全酶共同介导有效的Tat增强的HIV-1转录的机制知之甚少。这项建议有三个具体目标。第一个将确定和比较Tat-SF和Tat-SF辅因子来源于HeLa细胞质，核提取物和核沉淀（染色质）的多肽组成。第二个目标是研究Tat-SF辅因子介导Tat增强HIV-1转录的机制。这些研究将提供对Tat-SF辅因子自身内以及与其他转录因子和蛋白质修饰（例如磷酸化、乙酰化、甲基化）的协同作用的深入了解，以有效激活达特。第三个目标将专门研究Tat-SF和SRB/MED复合物的相互作用，以实现有效的一般转录再起始和延伸。这些实验将包括一个潜在的SRB/MED复合物的支架上形成的HIV- 1启动子，可以利用Tat-SF有效转录的详细表征。还将检查达特对Tat-SF-和RNA Pol II全酶介导的转录延伸和再起始的影响。总的来说，这些研究将确定新的因素和机制，是重要的单轮和多轮的HIV-1转录。他们还将建立一个独特的定义明确的体外系统，用于未来在裸DNA模板或染色质DNA模板上阐述HIV-1转录延伸与mRNA加工之间的联系。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-1-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 efficiently mediates Tat-enhanced transcription (Tat function). Tat-SF contains P-TEFb, Spt5/Spt4, and Tat-SF1 and novel polypeptides, but none of the yeast coactivator SRB/MED-like proteins found associated with RNA Pol II holoenzyme. Notably, these Tat-SF cofactors can jointly complement the transcription initiation competent RNA Pol II holoenzyme for potent transcription elongation, reinitiation, and Tat transactivation, indicating that Tat-SF contains critical transcription factors. The mechanisms by which Tat-SF and RNA Pol II holoenzyme collectively mediate potent Tat-enhanced HIV-1 transcription are poorly understood. This proposal has three specific aims. The first will determine and compare the polypeptide composition of Tat-SF and Tat-SF cofactors derived from HeLa cytoplasm, nuclear extract, and nuclear pellet (chromatin). The second aim will examine the mechanisms by which Tat-SF cofactors mediate potent Tat-enhanced HIV-1 transcription. These studies will provide insights into synergisms of Tat-SF cofactors within themselves and with other transcription factors and protein modifications (e.g. phosphorylation, acetylation, methylation) for potent Tat activation. The third aim will specifically study the interplay of Tat-SF and the SRB/MED complex for efficient general transcription reinitiation and elongation. 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 efficient transcription. The effects of Tat on Tat-SF- and RNA Pol II holoenzyme-mediated transcription elongation and re-initiation will also be examined. Collectively, these studies will identify novel factors and mechanisms that are important for single and multiple round of HIV-1 transcription. They will also establish a unique well-defined in vitro system for future elaboration of the link between HIV-1 transcription elongation and mRNA processing either on naked or chromatin DNA templates. Together, understanding Tat-enhanced HIV-1 transcription at the molecular level will be instrumental for future therapeutic antiviral strategies to block viral replication.