HIV 1 transcription: Tat/novel cofactors/Mechanisms

HIV 1 转录：Tat/新型辅助因子/机制

• 批准号: 7008196
• 负责人: CAMILO A PARADA
• 金额: $ 26.29万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7008196
• 关键词: DNA directed RNA polymerase; affinity chromatography; chromatin; cofactor; enzyme complex; enzyme mechanism; enzyme structure; genetic manipulation; genetic transcription; human immunodeficiency virus 1; peptides; posttranslational modifications; protein purification; protein structure function; tissue /cell culture; transcription factor; transfection; virus genetics; virus protein; virus replication; western blottings

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.

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，对于病毒复制至关重要。该提案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。本研究将重点关注一种新型 RNA Pol II 复合物 (Tat-SF)，该复合物可有效介导 Tat 增强转录（Tat 功能）。 Tat-SF 含有 P-TEFb、Spt5/Spt4 和 Tat-SF1 以及新的多肽，但没有发现与 RNA Pol II 全酶相关的酵母辅激活剂 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活性 RNA Pol II 全酶，以实现有效的转录延伸、重新起始和 Tat 反式激活，表明 Tat-SF 含有关键的转录因子。 Tat-SF 和 RNA Pol II 全酶共同介导有效的 Tat 增强的 HIV-1 转录的机制尚不清楚。该提案具有三个具体目标。第一个将确定并比较来自 HeLa 细胞质、核提取物和核沉淀（染色质）的 Tat-SF 和 Tat-SF 辅因子的多肽组成。第二个目标是研究 Tat-SF 辅助因子介导有效 Tat 增强的 HIV-1 转录的机制。这些研究将深入了解 Tat-SF 辅因子本身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，以有效激活 Tat。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重新启动和延伸。这些实验将包括在 HIV-1 启动子上形成的潜在 SRB/MED 复合物支架的详细表征，Tat-SF 可利用该支架进行有效转录。还将检查 Tat 对 Tat-SF- 和 RNA Pol II 全酶介导的转录延伸和重新启动的影响。总的来说，这些研究将确定对单轮和多轮 HIV-1 转录很重要的新因素和机制。他们还将建立一个独特的、明确的体外系统，以便将来在裸DNA或染色质DNA模板上详细阐述HIV-1转录延伸和mRNA加工之间的联系。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性抗病毒策略。