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基因组被合成为一个单一的多顺反子RNA转录物，经过调节剪接以确保mRNA的稳态。病毒剪接机制的畸变损害了HIV复制的保真度，在某些情况下使病毒的传染性降低。因此，HIV剪接途径代表了治疗干预的新靶点。剪接决策是由反宿主因子与顺式病毒RNA控制元件的动态组装/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。了解一个高度保守的剪接位点如何受到两个反宿主因子的差异调节的物理和功能决定因素，为定义机制提供了机会。因此，本提案的直接目标是确定HIV利用相互拮抗因子hnRNP A1 （A1）和ASF来调节其3'剪接位点A7 （ssA7）的分子原理。此外，A1和ASF通过未知的机制调节其他保守的HIV剪接位点；因此，这里提出的工作可能会对我们对HIV剪接的理解产生广泛的影响，并可能导致阻止HIV复制的新方法。本项目的具体目的是：(1)确定A1和ASF的结构对目标位点选择的贡献；(2)阐明A1和ASF相互拮抗的生物热力学基础；(3)研究ssA7调控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.