HIV-2 Genomic RNA Structure

描述(申请人提供)：逆转录病毒基因组RNA的5‘前导区虽然没有翻译，但充满了其他非编码功能，包括对转录反式激活、引物结合、剪接、封装、病毒RNA的二聚化和启动Gag ORF翻译至关重要的序列。在一个物种中，这些非编码序列的系统发育保守性甚至可以与病毒蛋白的保守编码域相媲美。这一观察结果强调，前导区的序列和结构对病毒复制至关重要。高度的保护也突显了将这一地区作为抗逆转录病毒战略目标的潜力。使用保守的RNA序列作为药物靶标已经得到了很好的证实，因为许多核糖体靶向抗生素识别细菌rRNA的特定片段。 最近的体外实验结果表明，前导RNA的结构设计是可塑性的，其多重构象似乎产生了与复制周期阶段相适应的功能。在上一次资助期间，我们阐明了HIV-2基因组RNA前导区的结构和动态方面，HIV-2是人类艾滋病的病原体之一。特别是，在寻找对基因组RNA二聚化重要的信号时，我们发现这些信号与病毒RNA的封装、剪接和翻译的已知信号点缀和重叠。基于HIV-2前导RNA构象异构体的体外鉴定和所涉及序列的高度系统发育保守性，我们假设HIV-2和其他逆转录病毒的前导区密切参与了几个病毒过程的调控，这种调控表现为在前导区发现的信号结构的差异呈现。 在这个应用中，我们建议通过构建和表征包含替换和缺失突变的病毒突变体来验证这一假设，这些突变旨在干扰在体外被证明在调节RNA结构和行为中重要的长程和短程RNA相互作用。我们将使用细胞培养和生化方法相结合的方法来表征在HIV-2中RNA二聚化、包裹和翻译的大部分未被探索的相互关系。本文提出的研究将有助于我们全面了解HIV-2复制的生物化学和细胞生物学，并验证5‘前导区RNA作为潜在的抗逆转录病毒靶点。

DESCRIPTION (provided by applicant): The 5' leader region of retroviral genomic RNA, while not translated, is replete with other non-coding functionality, including sequences important for transactivation of transcription, primer binding, splicing, encapsidation, dimerization of viral RNA, and initiation of translation of the gag ORF. Within a species, phylogenetic conservation of these non-coding sequences rivals even that of conserved coding domains for the viral proteins. This observation emphasizes that the sequence and structure of the leader region are essential for viral replication. The high degree of conservation also highlights the potential to target this region for antiretroviral strategies. The use of conserved RNA sequences as drug targets have been well established, since many ribosome-targeting antibiotics recognize a specific segment of bacterial rRNA. Recent in vitro results have demonstrated that structure of the leader RNA is plastic by design and its multiple conformations appear to give rise to functions appropriate to the stage of the replication cycle. In the last grant period, we elucidated structural and dynamic aspects of the leader region of the genomic RNA of HIV-2, one of the causative agents of AIDS in humans. In particular, while searching for signals important for genomic RNA dimerization, we discovered that these signals were interspersed with and overlaped with known signals for encapsidation, splicing, and translation of viral RNAs. Based on the in vitro characterization of conformational isomers of HIV-2 leader RNA and upon the high degree of phylogenetic conservation of involved sequences, we hypothesize that the leader region of HIV-2 and other retroviruses is intimately involved in the regulation of several viral processes, and that this regulation is manifested by differential presentation of the signaling structures found in the leader region. In this application we propose to test this hypothesis by constructing and characterizing viral mutants harboring substitution and deletion mutations designed to interfere with long- and short-range RNA interactions that were shown in vitro to be important in the modulation of RNA structure and behavior. We will use a combination cell culture and biochemical approaches to characterize the largely unexplored interrelationships of RNA dimerization, encapsidation, and translation in HIV-2. The studies proposed here will aid in our overall understanding of the biochemistry and cell biology of HIV-2 replication as well as to validate the 5' leader region RNA as a potential antiretroviral target.