Structural Biophysics of HIV Alternative Splicing

描述(申请人提供)：这项建议的长期目标是开发一种创新的结构、生物物理和功能生化框架，以促进我们对艾滋病毒剪接机制的理解。HIV基因组被合成为单个多顺反子RNA转录本，它经历了受调控的剪接，以确保mRNA的动态平衡。病毒剪接机制中的异常会损害艾滋病毒复制的保真度，在某些情况下会降低病毒的传染性。因此，HIV剪接通路是治疗干预的新靶点。剪接决定是由反式宿主因子与顺式病毒RNA控制元件的动态组装/拆解决定的；然而，决定剪接位点是被激活还是被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反式宿主因子差异调控的物理和功能决定因素的知识为确定一种机制提供了机会。因此，这一建议的直接目标是确定HIV用来共同选择相互拮抗的因子hnRNP A1(A1)和ASF的分子原理，以调节其3‘剪接位点A7(SsA7)。此外，A1和ASF通过特征不佳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对我们对HIV剪接的总体理解具有广泛的影响，并可能导致阻止HIV复制的新方法。本项目的具体目的是：(1)确定结构对A1和ASF靶点选择的贡献，(2)阐明A1和ASF相互拮抗的生物热力学基础，以及(3)探讨ssA7调控中协同作用的分子起源。

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.