Structural Basis of -1 Programmed Ribosomal Frameshifting by the Human T-cell Lymphotropic Virus Type I RNA

程序性核糖体移码（Programmed ribosomal frameshifting，PRF）是一种常见的病毒机制，用于调节相对水平， 各种基因产物。RNA结构如何诱导PRF是一个与人类相关的基本问题 由于其在感染和引起人类疾病的逆转录病毒中的流行，因此对健康具有重要意义。人嗜t淋巴细胞 I型病毒（HTLV-I）的复制依赖于两个PRF事件，这两个事件发生在gag-pro和pro-pol打开时 阅读帧连接。在这些基因组位置的顺式作用RNA元件如何起作用以诱导 移帧是未知的。这项研究的长期目标是提高对病毒RNA是如何 结构操纵宿主翻译机器以确保成功的病毒复制。总体目标 本申请的目的是确定HTLV-1逆转录病毒中-1PRF的结构基础。我们的核心假设 每个移码位点结构中的热力学稳定性的特定区域对于 移码刺激所提出的研究的基本原理是，一旦-1的结构基础 如果理解了PRF，那么关于HTLV-I移码机制的知识库中的一个显著空白将是 充满了。我们提出了两个具体的目标：1）确定HTLV-I移码位点RNA结构，2）研究 每个结构的局部热力学稳定性和PRF效率之间的关系。在这一提议中， 局部热力学稳定性由直接位于mRNA外部的碱基对的稳定性定义 在移帧的时候进入频道。为了实现这些目标，RNA化学探测实验将 结合计算方法来确定每个移码位点的RNA二级结构。 将使用诱变和体外移码试验评价每个结构对-1PRF的重要性 并研究了局域热力学稳定性与移码效率的关系。结果 这项研究的意义重大，因为它们将大大增加对HTLV-I的了解。 结构刺激的程序性核糖体移码这些研究有望开启新的研究 视野，特别是在靶向HTLV-I移码位点作为破坏HTLV-I复制的手段。

Programmed ribosomal frameshifting (PRF) is a common viral mechanism used to regulate the relative levels of various gene products. How RNA structures induce PRF is a fundamental question of relevance to human health, due to its prevalence in retroviruses that infect and cause human diseases. Human T-cell lymphotropic virus type I (HTLV-I) replication depends on two -1 PRF events, which occur at the gag-pro and pro-pol open reading frame junctions. How the cis-acting RNA elements at these genomic locations function to induce frameshifting is unknown. The long-term goal of this research is to improve understanding of how viral RNA structures manipulate host-translational machinery to ensure successful viral replication. The overall objective of this application is to determine the structural basis of -1 PRF in the HTLV-I retrovirus. Our central hypothesis is that specific regions of thermodynamic stability within each frameshift site structure are fundamental to frameshift stimulation. The rationale that underlies the proposed research is that once the structural basis of -1 PRF is understood, a significant gap in the knowledge base about the HTLV-I frameshift mechanisms would be filled. We propose two specific aims: 1) Define the HTLV-I frameshift site RNA structures, and 2) Investigate the relationship between each structure's local thermodynamic stability and -1 PRF efficiency. In this proposal, local thermodynamic stability is defined by the stability of base-pairs positioned directly outside of the mRNA entry channel at the time of frameshifting. To accomplish these aims, RNA chemical probing experiments will be combined with computational methods to define the RNA secondary structures at each frameshift site. Mutagenesis and in vitro frameshift assays will be used to evaluate the importance of each structure to -1 PRF and to investigate the relationship between local thermodynamic stability and frameshift efficiency. The results of the proposed research are significant because they will substantially increase what is known about HTLV-I structure-stimulated programmed ribosomal frameshifting. These studies promise to open new research horizons, particularly in targeting HTLV-I frameshift sites as a means of disrupting HTLV-I replication.