NMR Studies of Retroviral Nucleic Acid Binding Proteins

描述(由申请人提供)：在过去3年半期间，我们(I) 识别出HIV-1 Psi-RNA包装信号的茎环具有高 核衣壳(NC)蛋白的亲和力；(Ii)确定了 与HIV-1 Psi位干环SL2和SL3的NC复合体；(Iii)建立了 HIV-1 CCHC锌指关节及其保守氨基酸的功能作用 残基；(Iv)证明SL4不像以前那样与NC紧密结合 以前曾报道过，并提出了这种基本包装的新角色 元素；以及(V)在C-末端发现了一个新的三维折叠 锌指关节的小鼠乳腺肿瘤病毒(MMTV)NC蛋白。此外, 我们最近开发了一种过量生产重组NC的方法 来自Moloney鼠白血病病毒(MLV)的蛋白质是最广泛的 在人类基因治疗试验中使用的载体，以及(Vii)确定了最小 MLV Psi位点的一部分，是高亲和力NC结合所必需的。拥有 已经完成了NC与孤立茎环结合的研究，现在我们打算研究 与MLV和HIV-1完整Psi位点的NC相互作用 初步获得了102个核苷酸NC结合的核磁共振谱 MLV Psi站点的域名(在提交时分配了70%)， 在初步的核磁共振数据中观察到了大量的分子间NO 因其与NC的络合物而获得。有希望的初步3D核磁共振数据也 已经获得了完整的116个核苷酸的HIV-1 Psi位点，表明 这种72.4 kDa对称二聚体的结构研究也是可行的。 研究100个核苷酸或更大的RNA在技术上更具挑战性 与我们之前的研究相比，RNA茎环相对较小。然而， 潜在的回报要大得多，并承诺提供第一个 识别复合体的详细结构信息，这些信息导致 逆转录病毒基因组的特定包装。这样的知识不仅应该 促进制定治疗艾滋病和癌症的方法， 但也应协助将MLV发展为更有效的 人类基因的治疗性传递。

DESCRIPTION (Provided by the applicant): During the past 3-1/2 years we (i) identified the stem loops of the HIV-1 Psi-RNA packaging signal that have high affinities for the nucleocapsid (NC) protein; (ii) determined the structures of NC complexes with HIV-1 Psi-site stem loops SL2 and SL3; (iii) established the functional roles of the HIV-1 CCHC zinc knuckles and their conserved amino acid residues; (iv) demonstrated that SL4 does not bind tightly to NC, as had previously been reported, and proposed a new role for this essential packaging element; and (v) discovered a novel three dimensional fold in the C-terminal zinc knuckle of the Mouse Mammary Tumor Virus (MMTV) NC protein. In addition, we recently (vi) developed a method for overproduction of the recombinant NC protein from the Moloney Murine Leukemia Virus (MLV), which is the most widely used vector in human gene therapy trials, and (vii) identified the minimal portion of MLV Psi-site that is necessary for high-affinity NC binding. Having completed studies of NC binding to isolated stem loops, we now intend to study NC interactions with the intact Psi-sites of MLV and HIV- 1. High quality preliminary NMR spectra have been obtained for the 102 nucleotide NC-binding domain of the MLV Psi-site (>70 percent assigned at the time of submission), and numerous intermolecular NOES have been observed in preliminary NMR data obtained for its complex with NC. Promising preliminary 3D NMR data have also been obtained for the intact, 116 nucleotide HIV-1 Psi- site, indicating that structural studies of this 72.4 kDa symmetrical dimer are also feasible. Studies of RNAs of 100 nucleotides or larger are technically more challenging than our previous studies with relatively small RNA stem loops. However, the potential payoff is substantially greater, and promises to provide the first detailed structural information for the recognition complexes that lead to the specific packaging of retroviral genomes. Such knowledge should not only facilitate the development of approaches for the treatment of AIDS and cancer, but should also assist in the development of MLV as a more effective agent for the therapeutic delivery of human genes.