Retroviral Genomic RNA Dimer Structure and Function

描述(申请人提供)：逆转录病毒是一种严重的病原体，可导致人类免疫缺陷综合征、癌症和神经系统疾病。相反，逆转录病毒在将有益基因导入人类细胞方面非常有用。具有良好特性的逆转录病毒蛋白通常可以被药物靶向或通过基因操纵来实现重要的生物医学进步。与之形成鲜明对比的是，逆转录病毒具有RNA基因组，对在逆转录病毒感染和发病中起关键作用的RNA结构知之甚少。一个引人注目的例子是，逆转录病毒RNA基因组作为稳定的二聚体进入和离开目标细胞。逆转录病毒RNA基因组的两条链在其5‘端通过一种精确的、但鲜为人知的三维结构相连。因此，在分子水平上了解逆转录病毒二聚化的机制，既有机会破坏致病病毒的传染性，也有机会增强治疗载体的功能。因此，这项工作从基本的机械洞察力延伸到潜在的临床应用。我们的病毒学家和化学生物学家团队将致力于以下目标：(1)完善Moloney鼠肉瘤病毒二聚结构域的三维模型。对该模型的分析将提供有关伴随二聚化的RNA构象变化的重要新信息，并将有助于识别抗逆转录病毒药物的结构靶点。(2)确定在存在和不存在病毒Gag蛋白的情况下，Moloney小鼠白血病病毒(MuLV)RNA在核苷酸分辨下的折叠途径。我们将探索这样的假设，即二聚化是通过涉及不同RNA结构域的高度特异性的顺序相互作用来进行的，并且这种逐步组装对于病毒以如此非凡的特异性包装其基因组RNA的能力是至关重要的。(3)使用本实验室发明的高通量RNA结构分析技术，分析(I)二聚化结构域的结构和(Ii)正品MuLV病毒粒子内蛋白质-RNA的相互作用。然后，这些信息将被用来定义并在单核苷酸分辨率下在体内对MuLV的包装信号进行遗传验证。(4)启动一项程序，以确定HTLV-1的二聚化结构域和特异性病毒蛋白结合位点。这一目标进一步扩大了我们与逆转录病毒学家合作成功发明的直接方法，用于分析逆转录病毒的病毒外和病毒内的RNA结构，从而绕过了对HTLV-1进行费力和众所周知的低效操作和转基因的要求。在这一目标上开发的实验创新将对病毒学社区分析任何RNA病毒的任何RNA结构广泛有用。公共卫生相关性：这项工作将极大地促进我们对逆转录病毒RNA基因组结构和功能的理解。该项目利用了多种创新的技术进步，并涉及化学生物学家和病毒学家之间独特而密切的合作。从长远来看，随着分子质量的提高，有关逆转录病毒基因组结构的信息具有巨大的潜力，可以为设计新的抗逆转录病毒疗法和设计更好的基因治疗载体，将纠正基因导入人类细胞提供一个框架。

DESCRIPTION (provided by applicant): Retroviruses are serious pathogens that cause immune deficiency syndromes, cancer, and neurological disease in humans. Conversely, retroviruses are highly useful for directing beneficial genes into human cells. Well characterized retroviral proteins can often be targeted by drugs or be manipulated genetically to achieve important biomedical advances. In strong contrast, retroviruses have RNA genomes and very little is known regarding RNA structures that play critical roles in retroviral infection and morbidity. One compelling example is that retroviral RNA genomes both enter and leave a targeted cell as a stable dimer. The two strands of a retroviral RNA genome are linked at their 5' ends by a precise, but poorly understood, three-dimensional structure. Understanding the mechanism of retroviral dimerization at a molecular level therefore represents opportunities both to disrupt the infectivity of pathogenic viruses and to enhance the function of therapeutic vectors. This work thus extends from basic mechanistic insights to potential clinical applications. Our team of virologists and chemical biologists will tackle the following Aims: (1) Refine a three-dimensional model for the dimerization domain of Moloney murine sarcoma virus. Analysis of this model will provide important new information regarding the RNA conformational changes that accompany dimerization and will facilitate identification of structural targets for anti-retroviral agents. (2) Determine the folding pathway for the Moloney murine leukemia virus (MuLV) RNA dimerization at nucleotide resolution in the presence and absence of the viral Gag protein. We will explore the hypothesis that dimerization progresses by highly specific sequential interactions involving distinct RNA domains and that this step-wise assembly is crucial for the ability of the virus to package its genomic RNA with such extraordinary specificity. (3) Use high-throughput RNA structure analysis technology, invented in our laboratory, to analyze (i) the structure of the dimerization domain and (ii) protein-RNA interactions inside authentic MuLV virions. This information will then be used to define, and genetically validate, the packaging signal of MuLV in vivo at single nucleotide resolution. (4) Initiate a program to determine the structure of the dimerization domain and sites of specific viral protein binding in HTLV-1. This Aim further extends our successful invention, in collaboration with retrovirologists, of direct approaches for analyzing retroviral RNA structure ex virio and in virio that circumvent the requirement for laborious and notoriously inefficient manipulation and transfection of HTLV-1. Experimental innovations developed in this Aim will be broadly useful to the virology community for analyzing any RNA structure in any RNA virus. PUBLIC HEALTH RELEVANCE: This work will advance significantly our understanding of the structure and function of the RNA genomes of retroviruses. This project makes use of multiple, innovative, advances in technology and involves a distinctive and close collaboration between chemical biologists and virologists. Over the long term, improved, molecular quality, information about retroviral genome structure has significant potential to provide a framework for designing new anti-retroviral therapeutics and for engineering better gene therapy vectors for introducing corrective genes into human cells.