Regulation of AAV Rep Protein Function

AAV Rep 蛋白功能的调节

• 批准号: 6712117
• 负责人: JAMES P TREMPE
• 金额: $ 25.73万
• 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/6712117
• 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(申请人提供)：腺相关病毒(AAV)是一种 非致病性的、有缺陷的人类细小病毒。AAV要求辅助病毒 共同感染宿主细胞，以实现有效的病毒复制。在没有的情况下 辅助病毒感染，AAV很容易将其基因组整合到特定的 19号染色体长臂上的区域。AAV的天然缺陷， 非致病性和整合特定部位的能力促使 对其作为人类基因治疗载体的潜力进行了大量研究。病毒 复制和站点特定的集成依赖于两个复制 病毒编码的蛋白质Rep78和Rep68。这些蛋白质 通过绑定到上的目标站点来中介病毒复制和集成 病毒DNA和19号染色体基因座。这些蛋白质也扮演着重要的角色。 在调节病毒和宿主细胞的基因表达方面。我们的实验室有 证明这些蛋白质在丝氨酸残基上是磷酸化的。 我们认为差异磷酸化 Rep78和Rep68的表达允许对这些蛋白的调节。初步结果 揭示病毒中的磷酸化水平在不同的时间是不同的 复制周期和磷酸酶抑制导致增加 磷酸化。这里提出的实验将识别特定的残留物 它们在Rep蛋白中被修饰，并揭示了Rep中涉及的激酶 监管。我们还将确定特定的磷酸化事件如何调节 已知的Rep蛋白在体外和体内的酶活性 活体研究。这些调查将提供所需的基本信息 了解AAV复制周期和特定站点 整合AAV基因治疗载体。这些研究还可能导致 开发新的和改进的AAV载体。