NMR Studies of Retroviral Nucleic Acid Binding Proteins

描述（由申请人提供）：在过去的3 - 1/2年里，我们（i） 鉴定了HIV-1 Psi-RNA包装信号的茎环， 亲和性的核衣壳（NC）蛋白;（ii）确定的结构 NC与HIV-1 Psi位点茎环SL2和SL3的复合物;（iii）建立了 HIV-1CCHC锌指及其保守氨基酸的功能作用 （iv）证明SL4不与NC紧密结合，如 以前被报道，并提出了一个新的作用，这一重要的包装 元素;和（v）发现了一个新的三维折叠的C-末端 小鼠乳腺肿瘤病毒（MMTV）NC蛋白的锌关节。此外，本发明还提供了一种方法， 我们最近（vi）开发了一种过量生产重组NC的方法， 来自莫洛尼鼠白血病病毒（MLV）的蛋白质，其是最广泛的 在人类基因治疗试验中使用载体，和（vii）确定了最小的 高亲和力NC结合所必需的MLV Psi位点部分。具有 完成了NC与孤立茎环结合的研究，我们现在打算研究 NC与MLV和HIV-1的完整Psi位点的相互作用。高质量 初步的NMR谱已经获得了102个核苷酸的NC-结合 MLV Psi站点的域（在提交时分配> 70%）， 在初步的NMR数据中观察到了许多分子间NOES 它与NC的配合物。有希望的初步3D NMR数据也 获得了完整的116个核苷酸的HIV-1 Psi-位点，表明 这种72.4kDa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 比我们以前的研究相对较小的RNA茎环。但 潜在的回报要大得多，并承诺提供第一个 识别复合物的详细结构信息， 逆转录病毒基因组的特殊包装。这样的知识不仅 促进艾滋病和癌症治疗方法的发展， 但也应该有助于MLV作为一种更有效的药物的发展， 人类基因的治疗传递。

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.