Simulating Ion Modulated Stability of Retroviral Kissing-Loop Complexes

描述（由申请人提供）：该项目的目标是揭示逆转录病毒RNA亲吻环复合物离子介导的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Moloney白血病病毒（MMLV）二聚化起始位点（DIS）。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环的突变总是导致体内病毒复制和传染性大大降低。因此，干扰接吻环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，目前针对该界面的尝试实际上导致了接吻环稳定性的增加，但没有检测到病毒复制的抑制作用。因此，确定增强的接吻环稳定性的物理基础将是有用的，以便为未来设计靶向抑制剂的尝试提供信息。突变分析表明，环残基两侧的几个碱基对高复杂稳定性至关重要，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的氢键，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，不能用部分碱基配对或间隙水分子的存在来解释。我们假设侧翼残基通过调节局部离子环境影响邻近碱基对离解动力学。我们还预测，在过渡态部分脱水离子的释放构成了吻环解离的限速步骤。通过显式离子、隐式溶剂蒙特卡罗模拟，确定了亲环解离的动力学途径。我们将建立一个主要解离途径的马尔可夫状态模型，然后利用大量短时间、全原子的解离中间体过渡分子动力学模拟进行详细研究。这些模拟将利用施加的外力来增强解离，类似于单分子拉扯实验。模拟的准确性将通过预测的力-延伸曲线、过渡态分离、临界力和解离率与实际实验测量值的比较来确定。通过这种方式，将确定通过离子介导的环环界面上碱基配对动力学的调制，未配对的侧翼残基间接影响复合物整体机械稳定性的程度。了解离子介导的复合物形成驱动力可以更好地预测稳定和不稳定突变，以及识别特定的离子介导的相互作用，这可能在小分子抑制剂的设计中被利用。

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.