Retroviral Genomic RNA Dimer Structure and Function

描述（由申请人提供）：逆转录病毒是导致人类免疫缺陷综合征、癌症和神经系统疾病的严重病原体。相反，逆转录病毒在引导有益基因进入人类细胞方面非常有用。具有良好特征的逆转录病毒蛋白通常可以成为药物的靶标，或通过基因操纵来实现重要的生物医学进展。相反，逆转录病毒具有RNA基因组，而对于在逆转录病毒感染和发病中起关键作用的RNA结构，我们所知甚少。一个令人信服的例子是逆转录病毒RNA基因组以稳定二聚体的形式进入和离开目标细胞。逆转录病毒RNA基因组的两条链在它们的5'末端通过一种精确的、但鲜为人知的三维结构连接在一起。因此，在分子水平上理解逆转录病毒二聚化的机制为破坏致病性病毒的传染性和增强治疗载体的功能提供了机会。因此，这项工作从基本的机械见解扩展到潜在的临床应用。我们的病毒学家和化学生物学家团队将解决以下目标：(1)完善Moloney小鼠肉瘤病毒二聚化结构域的三维模型。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，并将有助于确定抗逆转录病毒药物的结构靶点。(2)在存在和不存在病毒Gag蛋白的情况下，确定Moloney小鼠白血病病毒（MuLV） RNA二聚化在核苷酸分辨率上的折叠途径。我们将探索一种假设，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进行的，这种分步组装对于病毒以如此特殊的特异性包装其基因组RNA的能力至关重要。(3)使用我们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）真正的MuLV病毒粒子内蛋白质-RNA相互作用。然后，这些信息将用于在单核苷酸分辨率下定义和遗传验证MuLV在体内的包装信号。(4)启动一个程序来确定HTLV-1中二聚化结构域的结构和特定病毒蛋白的结合位点。这一目标进一步扩展了我们与逆转录病毒学家合作的成功发明，即在体外和体内分析逆转录病毒RNA结构的直接方法，从而避免了对HTLV-1进行费力且臭名昭著的低效操作和转染的要求。本课题的实验创新将广泛应用于病毒学领域，用于分析任何RNA病毒的任何RNA结构。公共卫生相关性：这项工作将显著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先进技术，并涉及化学生物学家和病毒学家之间独特而密切的合作。从长期来看，改良后的逆转录病毒基因组结构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，将纠正性基因引入人类细胞提供框架。

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.