Regulation of AAV Rep Protein Function

AAV Rep 蛋白功能的调节

• 批准号: 6623086
• 负责人: JAMES P TREMPE
• 金额: $ 27.66万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623086
• 关键词: adeno associated virus group; enzyme activity; phosphoprotein phosphatase; phosphorylation; posttranslational modifications; protein kinase; protein structure function; tissue /cell culture; virus protein

DESCRIPTION (provided by applicant): Adeno-associated Virus (AAV) is a nonpathogenic, defective human parvovirus. AAV requires that a helper virus co-infect the host cell for efficient virus replication. In the absence of helper virus infection, AAV readily integrates its genome into a specific region in the long arm of chromosome 19. AAV's natural defectiveness, nonpathogenic nature and ability to integrate site-specifically have prompted numerous studies on its potential as a human gene therapy vector. Virus replication and site-specific integration is dependent upon two replication proteins, Rep78 and Rep68, that are encoded by the virus. These proteins mediate virus replication and integration by binding to target sites on the viral DNA and the chromosome 19 locus. These proteins also play important roles in regulation of virus and host cell gene expression. Our laboratory has demonstrated that these proteins are phosphorylated on Ser residues. We propose that differential phosphorylation of Rep78 and Rep68 allow for regulation of these proteins. Preliminary results reveal that the level of phosphorylation varies at different times in the virus replication cycle and that phosphatase inhibition results in an increase in phosphorylation. Experiments proposed here will identify the specific residues that are modified in the Rep proteins and reveal the kinases involved in Rep regulation. We will also determine how specific phosphorylation events modulate the known enzymatic activities of the Rep proteins in in vitro as well as in vivo studies. These investigations will provide essential information required for the understanding of the AAV replication cycle and site-specific integration of AAV gene therapy vectors. These studies may also lead to the development of new and improved AAV vectors.

描述（由申请人提供）：腺相关病毒（AAV）是一种 非致病性缺陷型人类细小病毒AAV需要一个辅助病毒 共感染宿主细胞以进行有效的病毒复制。在没有 辅助病毒感染时，AAV容易将其基因组整合到特定的 19号染色体长臂上的一个区域。AAV的天然缺陷， 非致病性和整合位点特异性的能力促使了 关于其作为人类基因治疗载体的潜力的许多研究。病毒 复制和特定于站点的集成依赖于两个复制 Rep78和Rep68是由病毒编码的蛋白质。这些蛋白质 介导病毒复制和整合的结合上的靶位点 病毒DNA和19号染色体位点。这些蛋白质也发挥重要作用 调节病毒和宿主细胞基因表达。本实验室 证明这些蛋白质在Ser残基上被磷酸化。 我们认为差异磷酸化 Rep78和Rep68之间的相互作用允许这些蛋白的调节。初步结果 揭示了磷酸化水平在病毒中的不同时间变化 复制周期和磷酸酶抑制导致 磷酸化这里提出的实验将确定特定的残留物 在Rep蛋白中被修饰，并揭示了Rep中涉及的激酶 调控我们还将确定特定的磷酸化事件如何调节 已知Rep蛋白在体外以及体内的酶活性， 体内研究。这些调查将提供所需的基本资料 对于理解AAV复制周期和位点特异性 AAV基因治疗载体的整合。这些研究也可能导致 开发新的和改进的AAV载体。