3D Structure of HIV-1 psi-Site

这项研究的目的是解决人类免疫缺陷病毒1型(HIV-1)完整psi位点的三维结构，该psi位点是负责基因组识别、包装和病毒组装的约120个核苷酸片段。完整的psi-复合体包括两个拷贝的psi-RNA和未知数量的核衣壳蛋白p7(NC)，太大了，无法用核磁共振进行结构表征。到目前为止，它表面上的柔韧性阻碍了X射线衍射结构研究的结晶。据信形成psi位的4个单独的RNA茎环及其与NC的络合物的三维结构已被核磁共振解析，可作为完整络合物的分子模拟的起始数据。通过一种基于化学和生化探针与电喷雾电离-离子回旋共振(ESI-ICR)质谱仪相结合的新策略，将获得茎环之间的距离限制、空间方向和接触点。特别是，我们将应用ESI-ICR来获得完整的psi-RNA复合体中psi-RNA与NC结合的化学计量比，并表征通过探测psi-RNA及其与NC的复合体所提供的核酸和蛋白质性质的产物(对于交联物种来说)。将采用大规模测绘和MS/MS测序技术来确定每个探测器攻击点的位置。实验获得的距离约束将用于约束结构计算，以确定在存在和不存在NC的情况下psi-RNA的全局折叠。使用电喷雾电离质谱仪作为经典探针的检测方法，提供了并行检测大量分子接触的可能性，而不需要劳动密集型分离程序或放射性示踪剂。基于可用探针的广泛选择和处理超出核磁共振光谱检测极限的大尺寸RNA的能力，该方法有望对RNA-RNA和RNA-蛋白质相互作用的研究产生重大影响。长期目标是了解psi位点介导的逆转录病毒基因组识别和包装的分子基础。对这一机制的充分理解对于在分子水平上阐明HIV-1和类似逆转录病毒的组装和成熟过程至关重要，并应有助于设计新的艾滋病和癌症治疗策略。

The objective of the proposed studies is to solve the three-dimensional structure of the intact psi-site of Human Immunodeficiency Virus type 1 (HIV-1), the approximately 120 nt segment of RNA responsible for genome recognition, packaging, and virus assembly. The complete psi-complex includes two copies of psi-RNA and an undetermined number of units of nucleocapsid protein p7 (NC) and is too large to be structurally characterized by NMR. Its apparent flexibility has thus far prevented crystallization for structural studies by X-ray diffraction. The 3D structures of the 4 individual RNA stem-loops believed to form the psi-site and those of their complexes with NC have been solved by NMR and could be used as starting data for the molecular modeling of the intact complex. Distance constraints, spatial orientation, and points of contact between the stem-loops will be obtained by a novel strategy based on chemical and biochemical probes in combination with electrospray ionization-ion cyclotron resonance (ESI-ICR) mass spectrometry. In particular, we will apply ESI-ICR to obtain the stoichiometry of binding between psi-RNA and NC in the intact psi-complex and to characterize the products of nucleic acid and proteic nature (or both for crosslinked species) provided by probing psi-RNA and its complex with NC. Mass mapping and MS/MS sequencing techniques will be employed to identify the location of the points of attack of each probe. The distance constraints obtained experimentally will be used in restrained structural calculations to determine the global fold of psi-RNA in presence and absence of NC. The use of electrospray ionization mass spectrometry as the detection method for classical probes offers the possibility of detecting in parallel a large number of molecular contacts with no need for labor-intensive separation procedures or radioactive tracers. Based on the large choice of available probes and the capability of handling large size RNAs, which are beyond the detection limit of NMR spectroscopy, this approach promises to have a major impact on the investigation of RNA-RNA and RNA-protein interactions. The long-term goal is to understand the molecular basis for the recognition and packaging of the retroviral genome mediated by the psi-site. A full understanding of this mechanism is crucial to elucidate at the molecular level the process of assembly and maturation of HIV-1 and similar retro-viruses and should facilitate the design of new therapeutic strategies for AIDS and cancer.