3D Structure of HIV-1 psi-Site

本研究的目的是解决人类免疫缺陷病毒1型（HIV-1）完整的psi位点的三维结构，这是一个大约120 nt的RNA片段，负责基因组识别、包装和病毒组装。完整的psi-复合物包括两个拷贝的psi-RNA和核衣壳蛋白p7 （NC）的未知单位，并且太大而无法用NMR进行结构表征。其明显的柔韧性迄今为止阻碍了x射线衍射结构研究的结晶。形成psi位点的4个单独的RNA茎环及其与NC的配合物的三维结构已通过核磁共振解析，可作为完整配合物分子建模的起始数据。通过化学和生化探针结合电喷雾电离-离子回旋共振（ESI-ICR）质谱法，将获得茎环之间的距离约束、空间方向和接触点。特别是，我们将应用ESI-ICR来获得完整psi-RNA复合物中psi-RNA和NC之间结合的化学计量学，并表征通过探测psi-RNA及其与NC的复合物所提供的核酸和蛋白质性质（或对于交联物种）的产物。将采用质量测绘和质谱/质谱测序技术来确定每个探针的攻击点的位置。实验获得的距离约束将用于约束结构计算，以确定存在和不存在NC时psi-RNA的全局折叠。使用电喷雾电离质谱法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的分离程序或放射性示踪剂。基于大量可用探针的选择和处理大尺寸rna的能力，这超出了核磁共振波谱的检测极限，这种方法有望对RNA-RNA和rna -蛋白质相互作用的研究产生重大影响。长期目标是了解由psi位点介导的逆转录病毒基因组的识别和包装的分子基础。充分了解这一机制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.