RETROVIRAL PROTEINS INVOLVED IN DNA INTEGRATION/VIRION

参与 DNA 整合的逆转录病毒蛋白/病毒粒子

• 批准号: 3176063
• 负责人: JONATHAN P LEIS
• 金额: $ 21.41万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1994-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3176063
• 关键词: DNA gyrase; Escherichia coli; RNA directed DNA polymerase; Retroviridae; Retroviridae disease; binding proteins; endonuclease; gel electrophoresis; gene deletion mutation; genetic manipulation; genetic transcription; molecular cloning; nucleic acid reconstitution; nucleic acid repetitive sequence; point mutation; protein sequence; protein signal sequence; protein structure function; virus DNA; virus RNA; virus envelope; virus morphology; virus protein; virus replication

The goal of this research is to define biochemical mechanisms of avian retrovirus replication through the study of the interactions of viral proteins with nucleic acids. These multidisciplinary model studies have direct application to development of anti-HIV agents. The first aim of this proposal is to study the interaction of the viral gag nucleocapsid (NC) and matrix (MA) proteins and their Pr76gag precursor with viral RNA and to evaluate their role in virion assembly. Molecular genetic techniques will be used to make amino acid substitutions at highly conserved or biochemically important residues of the NC and MA proteins. The effect these changes have on viral replication in vivo and on the biochemical properties of the mutant proteins purified either from virus or bacteria will be determined. The role of the Pr76gag or NC and MA proteins in the recognition of specific packaging signals in viral RNA will also be studied. The second aim of this proposal is to elucidate the mechanism of action of the DNA endonuclease associated with the viral integration (IN) protein and its role in viral DNA integration. Biochemical and molecular genetic experiments are outlined to relate the sequences at the termini of long terminal repeats (LTR) of viral DNA required for specific cleavage in vitro by the IN protein to those required for integration in vivo. This will be accomplished by analyzing the effect of small U5 LTR deletions on both cleavage and on integration of viral DNA. The requirement for inverted repeat sequences in the LTR in linear and supercoiled DNA substrates for IN protein activity and integration in vivo will be evaluated. Reconstitution of a viral DNA integration system in vitro using purified components will be attempted. In a related project, the role of secondary structure in the U5 region of viral RNA for initiation of reverse transcription will be studied. The observation that such structures potentially exist in other retrovirus RNAs including HIV suggest that this is a general phenomenon of replication.

这项研究的目的是定义 通过研究相互作用的鸟类逆转录病毒复制 用核酸的病毒蛋白。 这些多学科 模型研究直接应用于抗HIV的发展 代理商。 该提议的第一个目的是研究 病毒堵嘴核素（NC）和基质（MA）蛋白及其及其 具有病毒RNA的PR76GAG前体，并评估其在 病毒体组件。 分子遗传技术将用于制造 高度保守或生化的氨基酸取代 NC和MA蛋白的重要残基。 这些效果 体内和生化的病毒复制发生变化 突变蛋白从病毒或 将确定细菌。 PR76GAG或NC和MA的作用 识别病毒特定包装信号的蛋白质 RNA也将被研究。 该提案的第二个目的是阐明 与病毒相关的DNA核酸内切酶的作用 （IN）蛋白质及其在病毒DNA整合中的作用。 概述了生化和分子遗传实验 将长时间重复序列（LTR）的末端的序列相关联 特定裂解在体外所需的病毒DNA 蛋白质与体内整合所需的蛋白质。 这将是 通过分析小U5 LTR缺失对 裂解和病毒DNA的整合。 要求 线性和超螺旋DNA中LTR中的倒重复序列 蛋白质活性的基材和体内整合将是 评估。 重建病毒DNA整合系统 将尝试使用纯化组件的体外。 在相关 项目，二级结构在病毒的U5区域的作用 将研究用于启动逆转录的RNA。 这 观察到这种结构可能存在于其他 包括HIV在内的逆转录病毒RNA表明这是一般 复制现象。