3D Structure of HIV-1 psi-Site

拟议研究的目的是解决人类免疫缺陷病毒1型（HIV-1）的完整psi位点的三维结构，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和数目未定的核衣壳蛋白p7（NC）单元，并且太大而不能通过NMR进行结构表征。 其明显的灵活性，迄今为止，防止结晶的结构研究，通过X射线衍射。 4个单独的RNA茎环的3D结构被认为是形成的PSI网站和他们的复合物与NC已经解决了通过NMR和可以作为起始数据的完整的复合物的分子建模。距离限制，空间方向，和茎环之间的接触点将获得一种新的策略，结合电喷雾电离离子回旋共振（ESI-ICR）质谱的化学和生化探针。 特别是，我们将应用ESI-ICR来获得完整的PSI复合物中PSI-RNA和NC之间结合的化学计量，并表征通过探测PSI-RNA及其与NC的复合物提供的核酸和蛋白质性质（或两者对于交联物质）的产物。 将采用质谱图和MS/MS测序技术来确定每个探针攻击点的位置。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作为经典探针的检测方法提供了平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。基于可用探针的大量选择和处理超出NMR光谱检测限的大尺寸RNA的能力，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。长期的目标是了解由psi位点介导的逆转录病毒基因组识别和包装的分子基础。 充分了解这一机制是至关重要的，阐明在分子水平上的装配和成熟的HIV-1和类似的逆转录病毒的过程，并应促进艾滋病和癌症的新的治疗策略的设计。

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.