HIV-2 Genomic RNA Structure

描述（由申请人提供）：逆转录病毒基因组RNA的5'先导区，虽然没有翻译，但充满了其他非编码功能，包括转录反激活、引物结合、剪接、衣壳化、病毒RNA二聚化和gag ORF翻译起始的重要序列。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的保守编码域相媲美。这一观察结果强调了先导区的序列和结构对病毒复制至关重要。高度的保存也突出了针对这一区域开展抗逆转录病毒战略的潜力。使用保守RNA序列作为药物靶点已经很好地建立，因为许多核糖体靶向抗生素识别细菌rRNA的特定片段。

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.