Simulating Ion Modulated Stability of Retroviral Kissing-Loop Complexes

描述（由申请人提供）：本项目的目标是揭示逆转录病毒RNA吻环复合物离子介导相互作用的物理基础，并解释最大机械稳定性的不寻常序列要求;特别是HIV和莫洛尼白血病病毒（MMLV）的二聚化起始位点（DIS）。众所周知，这种高稳定性对于逆转录病毒基因组二聚化至关重要，因为DIS环的突变总是会导致体内病毒复制和感染率大大降低。因此，干扰Kissing-loop介导的基因组二聚化可能被证明是设计新的抗逆转录病毒疗法的成功途径。然而，目前靶向该界面的尝试实际上导致了增加的吻环稳定性，而没有可检测到的病毒复制抑制。因此，它将是有用的，以确定增强的接吻环稳定性的物理基础，以告知未来的尝试在设计有针对性的抑制剂。突变分析表明，环残基侧翼的几个碱基对于高复合物稳定性至关重要，但结构和化学作图实验都证实这些碱基不是碱基配对的，不参与分子内或分子间氢键，实际上似乎被翻转到溶液中。最后，在过渡态观察到的分离距离太大，不能解释部分碱基配对或间隙水分子的存在。我们推测侧翼残基通过调节局部离子环境来影响邻近碱基对的解离动力学。我们还预测，部分脱水离子在过渡态的释放构成了接吻环解离的限速步骤。使用显式离子，隐式溶剂蒙特卡罗模拟，接吻环解离的动力学途径将被确定。一个马尔可夫状态模型的主要解离途径将被创建，然后详细检查使用大量的短，全原子分子动力学模拟转换沿着解离中间体。这些模拟将利用施加的外力来增强解离，类似于单分子拉伸实验。模拟的准确性将通过比较预测的力-延伸曲线、过渡态分离、临界力和解离速率与实际实验测量值来确定。以这种方式，将确定未配对的侧翼残基通过离子介导的环-环界面处碱基配对动力学的调节间接有助于复合物的总体机械稳定性的程度。了解离子介导的复合物形成的驱动力应该允许更好地预测稳定和不稳定的突变，以及确定特定的离子介导的相互作用，这可能是利用小分子抑制剂的设计。 公共卫生相关性：这项研究的目的是提供一个未知的接吻环基序的强度，这是绝对需要复制的艾滋病毒逆转录病毒的物理见解。识别产生增强的吻环稳定性的特定相互作用应有助于设计新的抗逆转录病毒药物。

DESCRIPTION (provided by applicant): The goal of this project is to uncover the physical basis for the ion-mediated interactions of retroviral RNA kissing-loop complexes and to explain the unusual sequence requirements for maximum mechanical stability; specifically that of the Dimerization Initiation Site (DIS) of HIV and Moloney Leukemia Virus (MMLV). This high stability is known to be crucial for retroviral genome dimerization, as mutations to the DIS loop always result in greatly reduced virus replication and infectivity rates in vivo. Therefore, interfering with kissing-loop mediated genome dimerization may prove to be a successful route to designing new anti-retroviral therapeutics. However, current attempts to target this interface have actually resulted in increased kissing-loop stability with no detectible inhibition of viral replication. It would therefore be useful to determine the physical basis of the enhanced kissing loop stability in order to inform future attempts at designing targeted inhibitors. Mutational analysis has shown that several bases flanking the loop residues are crucial for high complex stability, but both structural and chemical mapping experiments confirm that these bases are not base paired, do not participate in intra or inter-molecular hydrogen bonds, and actually appear to be flipped out into solution. Lastly, the observed separation distances at the transition state are too large to be explained by partial base-pairing or the presence of interstitial water molecules. We hypothesize that the flanking residues effect neigboring base pair dissociation kinetics through modulation of the local ionic environment. We also predict that the release of partially dehydrated ions at the transition state constitutes the rate-limiting step for kissing-loop dissociation. Using explicit ion, implicit solvent Monte Carlo simulations, the kinetic pathways of kissing loops dissociation will be determined. A Markov state model of the dominant dissociation pathway will be created, and then examined in detail using large numbers of short, all-atom molecular dynamics simulations of transitions along dissociation intermediates. These simulations will utilize an applied external force to enhance dissociation, analagous to single-molecule pulling experiments. The accuracy of the simulations will be ascertained by comparison of the predicted force-extension curves, separation at the transition state, critical force, and dissociation rates with the actual experimental measurements. In this way, the extent to which the unpaired flanking residues indirectly contribute to the overall mechanical stability of the complex through ion-mediated modulation of base pairing kinetics at the loop-loop interface will be ascertained. Understanding ion-mediated driving forces for complex formation should allow better prediction of stabilizing and destabilizing mutations, as well as identify specific ion-mediated interactions which may be exploitable in the design of small molecule inhibitors. PUBLIC HEALTH RELEVANCE: This aims of this research is to provide physical insight into the unexplained strength of a kissing- loop motif that is absolutely required for replication of the HIV retrovirus. Identification of the specific interactions that give rise to enhanced kissing-loop stability should aid in the design of new anti-retroviral drugs.