Structural determinants of HIV-1 5'-UTR in virions and infected cells

描述(申请人提供)：根据联合国艾滋病规划署/世卫组织的最新报告，截至2007年底，全球估计有3300万人患有艾滋病，仅在2008年就造成200万人死亡。由高效抗逆转录病毒疗法(HAART)组成的艾滋病毒感染标准治疗的有效性因不断选择耐药毒株而受到破坏。开发新的治疗策略的一个可能的未知靶点是病毒RNA的5‘-非翻译区(5’-UTR)。这个非编码区控制着关键的复制步骤，在这些步骤中，病毒RNA要么作为包装的基因组，要么作为蛋白质表达的mRNA。我们计划继续我们对5‘-UTR结构-功能关系的研究，以了解在两个不同的活动领域协调其多方面功能的调节机制。多年来，我们开发了一种基于结构探测和质谱学检测(MS3D)的方法，以实现对不适用于现有高分辨率技术的底物的结构解析。利用这种方法，我们已经确定了5‘-UTR在体外假设的不同构象的结构，这可能构成调控核糖开关机制的基础。我们现在计划通过直接在病毒粒子和受感染的细胞中进行结构探测来研究这些异构体的生物学意义，这将得到双功能探针的能力的支持，该能力可以永久地使在不同病毒/宿主环境中相互接触的任何RNA或蛋白质物种交联。并列官能团的原位交联将提供实际RNA-RNA和蛋白质-RNA相互作用的详细快照，这些相互作用定义了参与5‘-UTR活性的相关核糖核蛋白复合体(RNP)。将病毒RNA在病毒粒子和选定的亚细胞室中的探测结果进行比较，将使我们能够研究参与基因组或mRNA活性的5‘-UTR的结构。同时检测推定的5‘-UTR结构及其蛋白结合偏好的可能性将为体内不同病毒/细胞因子的识别决定因素提供有价值的信息。此外，研究结构重排如何影响这些因子的选择，将为5‘-UTR作为病毒/细胞环境的函数的结构-功能关系提供独特的见解。对与特定信号相互作用的细胞因子的调查将指导小干扰RNA(SiRNAs)的设计，这些小干扰RNA将以多路复用的方式用于研究涉及特定区域的相互作用。不同环境下分子接触的详细图谱将为涉及多个相关位点的突变研究提供基础，以评估特定相互作用在控制5‘-UTR活性中的作用。拟议的研究将提供一个全面的框架，使目前对5‘-非编码区的结构和功能的认识合理化。一方面，这个框架将有助于验证体外获得的分离结构域的高分辨率结构，将确定它们对全长5‘-UTR假设的替代折叠的贡献，并将证实它们与体内同源蛋白的直接相互作用。另一方面，这些信息将为理解多年来报告的领域之间广泛的协同作用和相互作用提供基础。将高阶结构和分子间相互作用的任何变化与不同的生物学活性联系起来，将为评估病毒/宿主因子在协调5‘-UTR不同功能中的可能作用提供必要的信息。这些信息将有助于制定新的治疗策略，旨在干扰5‘-UTR的功能和调节，这将是对目前因不断选择耐药菌株而受到威胁的治疗的非常可取的补充。 与公共卫生有关：根据艾滋病规划署/世卫组织的最新报告，截至2007年底，全世界估计有3300万人患有艾滋病，仅在2008年就造成200万人死亡。由高效抗逆转录病毒疗法(HAART)组成的艾滋病毒感染标准疗法的有效性正受到不断选择耐药毒株的威胁。大流行的规模和令人望而生畏的抗药性问题是加强寻找替代和更有效的抗病毒治疗的强大动机。我们建议对病毒RNA 5‘-非翻译区(5’-UTR)的结构-功能关系进行体内研究，该区域提供了许多控制病毒复制关键步骤的信号。对5‘-UTR功能的结构决定因素及其调控机制的确切理解对于开发旨在破坏这一尚未探索的病毒靶点的新的治疗策略至关重要。

DESCRIPTION (provided by applicant): According to the most recent UNAIDS/WHO report, an estimated 33 million people worldwide were living with AIDS at the end of 2007, which caused 2 million deaths in 2008 alone. The effectiveness of the standard treatment for HIV infection, which consist of highly active-antiretroviral therapies (HAART), is undermined by the constant selection of drug-resistant strains. A possible unexplored target for the development of new therapeutic strategies is represented by the 5'-untranslated region (5'-UTR) of the viral RNA. This non-coding region governs key replication steps in which viral RNA acts either as genome intended for packaging, or as mRNA meant for protein expression. We plan on continuing our investigation of the structure-function relationships of 5'-UTR to understand the regulatory mechanisms that coordinate its multifaceted functions in the two distinct spheres of activity. Over the years, we have developed an approach based on structural probing and mass spectrometric detection (MS3D) to enable the structure elucidation of substrates that are not amenable to the established high-resolution techniques. Using this approach, we have determined the structure of different conformers assumed in vitro by 5'-UTR, which could constitute the basis for a regulatory riboswitch mechanism. We now plan on investigating the biological significance of these isomeric forms by performing structural probing directly in virions and infected cells, which will be supported by the ability of bifunctional probes to permanently crosslink any RNA or protein species that may come into mutual contact in the different viral/host environments. In situ crosslinking of juxtaposed functional groups will provide detailed snapshots of the actual RNA-RNA and protein-RNA interactions that define pertinent ribonucleoprotein complexes (RNPs) involved in 5'-UTR activities. Comparing the results obtained by probing the viral RNA in virions and selected subcellular compartments will allow us to investigate the architecture of 5'-UTR engaged in genomic or mRNA activities. The possibility of examining simultaneously the putative 5'-UTR structures and their protein binding preferences will provide valuable information about the recognition determinants of different viral/cellular factors in vivo. Further, studying how structural rearrangements affect the selection of such factors will offer unique insights into the structure-function relationships of 5'-UTR as a function of viral/cellular context. The survey of cellular factors interacting with specific signals will guide the design of small interfering RNAs (siRNAs) that will be used in multiplexed fashion to study the interactions involving a specific domain. The detailed maps of molecular contacts in the different contexts will provide the basis for mutagenesis studies involving multiple correlated sites to assess the role of specific interactions in controlling 5'-UTR activities. The proposed research will provide a comprehensive framework for rationalizing current structural and functional knowledge of 5'-UTR. On one hand, this framework will help validate the high-resolution structures obtained in vitro for isolated domains, will determine their contributions to the alternative folds assumed by full length 5'-UTR, and will confirm their direct interactions with cognate proteins in vivo. On the other hand, this information will provide the basis for understanding the extensive synergies and cross-talk between domains, which have been reported over the years. Correlating any variations of higher-order structure and intermolecular association with distinctive biological activities will provide the information necessary to evaluate the possible role of viral/host factors in coordinating the different functions of 5'-UTR. This information will enable the formulation of new therapeutic strategies aimed at interfering with 5'-UTR functions and regulation, which would constitute very desirable complements to current treatments endangered by the constant selection of resistant strains. PUBLIC HEALTH RELEVANCE: According to the most recent UNAIDS/WHO report, an estimated 33 million people worldwide were living with AIDS at the end of 2007, which caused 2 million deaths in 2008 alone. The effectiveness of standard treatments for HIV infection, which consist of highly active-antiretroviral therapies (HAART), is being threatened by the constant selection of drug-resistant strains. The magnitude of the pandemic and the daunting problem of drug resistance are powerful motivations for intensifying the search for alternative and more effective antiviral treatments. We propose in vivo studies of the structure-function relationships of the 5'- untranslated region (5'-UTR) of the viral RNA, which presents numerous signals controlling critical steps of viral replication. A firm understanding of the structural determinants of the 5'-UTR functions and their regulatory mechanisms are essential for the development of new therapeutic strategies aimed at disrupting this yet unexplored viral target.