HIV Genomic RNA Structure

描述（由申请方提供）：HIV-1和HIV-2基因组RNA的5'非翻译前导区含有病毒复制所必需的序列，包括转录反式激活、引物结合、剪接、腺苷酸化、RNA二聚化和gag基因翻译起始的信号。在一个物种内，这些非编码序列的系统发育保守性甚至可以与病毒蛋白质的保守编码结构域相媲美，证明了它们对复制的重要性。高度的保护也突出了利用这一地区的抗逆转录病毒战略的潜力。由于许多临床上有用的抗生素识别细菌rRNA的特定片段，因此保守RNA序列作为药物靶标的使用已经得到很好的建立。 HIV-1和HIV-2的前导RNA的生物化学表征表明，它采用多种构象，交替显示或隐藏适合于复制周期不同阶段的RNA信号。基于HIV-2前导RNA构象异构体的体外表征和相关序列的高度保守性，Lodmell博士假设HIV和其他逆转录病毒的前导区密切参与几种病毒复制过程的调节，包括病毒RNA的二聚化、剪接和蛋白质合成，并且这种调节通过在前导区中发现的信号结构的差异呈现来表现。在本申请中，Lodmells博士提出通过构建和表征病毒突变体来验证这一假设，这些病毒突变体含有取代和缺失突变，旨在干扰长距离和短距离RNA相互作用，这些相互作用在体外显示出对RNA结构和行为的调节很重要。细胞培养，遗传和生物化学方法的组合将被用来表征在很大程度上未探索的相互关系的RNA剪接，二聚化，双糖苷化和翻译的HIV 1型和2型。本文提出的研究将有助于我们全面了解人类慢病毒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' untranslated leader region of HIV-1 and HIV-2 genomic RNA contains sequences essential for viral replication, including signals for transactivation of transcription, primer binding, splicing, encapsidation, RNA dimerization, and initiation of translation of the gag gene. Within a species, phylogenetic conservation of these non-coding sequences rivals even that of conserved coding domains for the viral proteins, attesting to their importance for replication. The high degree of conservation also highlights the potential to exploit this region for antiretroviral strategies. The use of conserved RNA sequences as drug targets has been well established, since many clinically useful antibiotics recognize a specific segment of bacterial rRNA. Biochemical characterization of the leader RNA of HIV-1 and HIV-2 suggests that it adopts multiple conformations that alternately display or hide RNA signals appropriate to different stages of the replication cycle. Based on the in vitro characterization of conformational isomers of HIV-2 leader RNA and upon the high degree of conservation of involved sequences, Dr. Lodmell hypothesizes that the leader region of HIV and other retroviruses is intimately involved in the regulation of several viral replicative processes, including dimerization of viral RNA, encapsidation, splicing, and protein synthesis, and that this regulation is manifested by differential presentation of the signaling structures found in the leader region. In this application, Dr. Lodmells proposes 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. A combination of cell culture, genetic, and biochemical approaches will be used to characterize the largely unexplored interrelationships of RNA splicing, dimerization, encapsidation, and translation in HIV types 1 and 2. The studies proposed here will aid in our overall understanding of the biochemistry and cell biology of human lentivirus replication as well as to validate the 5' leader region RNA as a potential antiretroviral target.