Structural Biophysics of HIV Alternative Splicing

描述（由申请人提供）：本提案的长期目标是开发一个创新的结构，生物物理和功能生化框架，以促进我们对HIV剪接机制的理解。HIV基因组合成为单个多顺反子RNA转录物，其经历调节剪接以确保mRNA稳态。病毒剪接机制的异常会损害HIV复制的保真度，在某些情况下会降低病毒的感染性。因此，HIV剪接途径代表了治疗干预的新靶点。剪接决定是由顺式病毒RNA控制元件的反式宿主因子的动态组装/拆卸决定的;然而，决定剪接位点是否被激活或抑制的潜在分子事件仍然难以捉摸。一个高度保守的剪接位点如何被两个跨宿主因子差异调节的物理和功能决定因素的知识提供了定义机制的机会。因此，该建议的直接目标是确定HIV用于增选相互拮抗因子hnRNP A1（A1）和ASF的分子原理，以调节其3'剪接位点A7（ssA 7）。此外，A1和ASF调节其他保守的HIV剪接位点，通过不良的特点机制，因此，这里提出的工作很可能有广泛的影响，我们的理解HIV剪接一般，并可能导致新的方法来阻止HIV复制。该项目的具体目标是：（1）确定结构对A1和ASF靶位点选择的贡献，（2）阐明A1和ASF之间相互拮抗的生物热力学基础，（3）研究ssA 7调节中协同性的分子起源。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to develop an innovative structural, biophysical, and functional biochemical framework to advance our understanding of HIV splicing mechanisms. The HIV genome is synthesized as a single polycistronic RNA transcript that undergoes regulated splicing to ensure mRNA homeostasis. Aberrations in the viral splicing machinery impair HIV replication fidelity and in some cases render the virus less infectious. Thus, HIV splicing pathways represent novel targets for therapeutic intervention. Splicing decisions are determined by the dynamic assembly/disassembly of trans host factors with cis viral RNA control elements; however, the underlying molecular events that determine whether a splice site will be activated or repressed remain elusive. Knowledge of the physical and functional determinants of how one highly conserved splice site gets differentially regulated by two trans host factors offers the opportunit to define a mechanism. Hence, the immediate goal of this proposal is to identify the molecular principles that HIV uses to co-opt the mutually antagonistic factors, hnRNP A1 (A1) and ASF, in order to regulate its 3' splice site A7 (ssA7). Furthermore, A1 and ASF regulate other conserved HIV splice sites via poorly characterized mechanisms; thus, the work proposed here is likely to have broad implications on our understanding of HIV splicing in general and may lead to new approaches to thwart HIV replication. The specific aims of this project are to: (1) Determine the contribution of structure to target site selection by A1 and ASF, (2) Elucidate the bio-thermodynamic underpinnings of mutual antagonism between A1 and ASF, and (3) Investigate the molecular origins of cooperativity in ssA7 regulation.