HIV 1 transcription: Tat/novel cofactors/Mechanisms

HIV 1 转录：Tat/新型辅助因子/机制

• 批准号: 6892475
• 负责人: CAMILO A PARADA
• 金额: $ 21.77万
• 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/6892475
• 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(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了解TAT和细胞因子对HIV-1基因表达的控制。本研究将集中于一种新的RNA POLII复合体(TAT-SF)，它能有效地介导TAT增强的转录(TAT功能)。TAT-SF含有P-TEFb、Spt5/Spt4、TAT-SF1和新的多肽，但没有发现与RNA PolII全酶相关的酵母共激活因子SRB/MED样蛋白。值得注意的是，这些TAT-SF辅助因子可以共同补充转录起始活性RNA POL II全酶，从而有效地转录延伸、重新启动和TAT反式激活，表明TAT-SF含有关键的转录因子。TAT-SF和RNA POLII全酶共同介导TAT增强的HIV-1转录的机制尚不清楚。这项提议有三个具体目标。首先将测定和比较来源于HeLa细胞质、核提取液和核小球(染色质)的TAT-SF和TAT-SF辅助因子的多肽组成。第二个目标将研究TAT-SF辅助因子介导TAT增强的HIV-1转录的机制。这些研究将为深入了解TAT-SF辅助因子本身以及与其他转录因子和蛋白质修饰(如磷酸化、乙酰化、甲基化)之间的协同作用而有效地激活TAT。第三个目标将专门研究TAT-SF和SRB/MED复合体之间的相互作用，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高效转录。TAT对TAT-SF-和RNA POLII全酶介导的转录延长和重新启动的影响也将被检测。总的来说，这些研究将确定对HIV-1单轮和多轮转录至关重要的新因素和新机制。他们还将建立一个独特的、定义明确的体外系统，用于未来详细阐述HIV-1转录延伸和在裸露或染色质DNA模板上的mRNA处理之间的联系。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抗病毒策略。